Delivery of Biomolecules into Mammalian Cells Using Anthrax Toxin
ARCHNES
by
MASSACHUSETTS INSTITUTE
Amy Ellen Rabideau
OF TECHNOLOGY
B.S. Chemistry and Biology
Syracuse University, 2010
NOV 0 9 2015
LIBRARIES
Submitted to the Department of Chemistry
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
at the
Massachusetts Institute of Technology
September 2015
C 2015 Massachusetts Institute of Technology
All rights reserved
Signature of Author:
Signature redacted
1)
Certified by:
Department of Chemistry
ugust 28th, 2015
Signature redacted
Bra ley L. Pentelute
Pfizer-Laubach Career Development Professor of Chemistry
Thesis Supervisor
Signature redacted
Accepted by:
Robert W. Field
Haslam and Dewey Professor of Chemistry
Chairman, Departmental Committee for Graduate Students
2
This doctoral thesis has been examined by a committee of the Department of Chemistry as
follows:
Signature redacted
Alexander M. Klibanov
Novartis Professor of Chemistry and Bioengineering
Thesis Committee Chair
Signature redacted
Bradley L. Pentelute
Pfizer-Laubach Career Development Professor of Chemistry
Thesis Supervisor
Signature redacted
John M. Essigmann
William R. and Betsy P. Leitch Professor of Chemistry and Biological Engineering
Signature redacted
Barbara Imperiali
Class of 1922 Professor of Chemistry and Biology
3
4
Delivery of Biomolecules into Mammalian Cells Using Anthrax Toxin
by
Amy Ellen Rabideau
Submitted to the Department of Chemistry
on August 2 8th, 2015 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
Abstract
The intracellular delivery of biomolecules into mammalian cells is a major challenge due
to the plasma membrane, which acts as a barrier between the extracellular environment and
intracellular components. Recently, a non-toxic delivery platform derived from anthrax lethal
toxin has been developed to overcome this challenge for the delivery of biomolecules into the
cytosol of mammalian cells. The PA/LFN delivery platform has been used to deliver over 30
known biomolecules of diverse sequences, structures, and functionalities. Collectively, these
translocation studies have helped to elucidate the translocation mechanism and to probe
intracellular biological processes.
In this thesis, the PA/LFN delivery platform was used to analyze the delivery of assorted
biomolecules through the PA pore. A facile, modular ligation strategy using sortase A was
developed for the conjugation of biomolecules to LFN. The biomolecules for this analysis
included antibody mimic proteins with defined sizes and secondary structures, mirror image
peptides and proteins, polypeptides containing non-canonical amino acids or small molecule
drugs, and cyclic peptides. Our translocation analyses have led to guidelines for translocation as
well as insight into design parameters for the efficient delivery of new cargos. The PA/LFN
delivery platform has also been used to translocate bioactive cargos for the disruption of
intracellular protein-protein interactions (PPI). The translocation efficiency and bioactivity of a
tandem monobody to Bcr-Abl, an affibody to hRaf- 1, and a mirror image peptide to MDM2 were
analyzed. Efficient translocation and disruption of the intended PPI in each case indicated that
the delivery platform could be used to deliver bioactive cargos into cells for therapeutic utility.
As an application of this technology, the PA/LFN delivery platform was employed to
analyze the intracellular stability of mixed chirality proteins. One major factor that governs a
protein's stability is the N-end rule, which states that the N-terminal residue of a protein impacts
its intracellular stability through the ubiquitin (Ub)/proteasome system. Utilizing the PA/LFN
delivery platform, the stability of proteins containing one N-terminal D-amino acid was
analyzed. In contrast to N-terminal L-amino acids, each N-terminal D-amino acid abrogates
protein degradation by the N-end rule pathway.
Thesis Supervisor: Bradley L. Pentelute
Pfizer-Laubach Career Development Professor of Chemistry
5
6
Acknowledgements
Throughout my upbringing my dad emphasized the philosophy, "Learning is the
journey!" I do not think I entirely understood the true meaning behind what he has been saying
all these years until graduate school, where some of the most important lessons are not taught in
a classroom. My graduate career has been filled with new experiences, troubleshooting,
incredible colleagues, and a lot of really amazing science. I am grateful for everyone who has
supported and guided me throughout this phase of my learning journey.
I am indebted to my advisor, Prof. Brad Pentelute for taking me on as his first graduate
student and exposing me the complexities of biochemistry research. I will never forget walking
into our empty lab in 56-546 on my first day and thinking, "What did I get myself into?"
Fortunately, in true Pentelute lab style (fast), after only a few months of ordering and organizing,
we were up and running-expressing proteins, synthesizing peptides, growing cells, and writing
grants. Since the beginning, Brad has encouraged me to think big and explore new ideas with
confidence. He helped me realize that simple experiments are often the most successful and the
more controls, the better. Under Brad's mentorship, I have developed the intuition to think
critically through challenging questions.
Thank you to Prof. Alex Klibanov, my thesis chair, for his advice and helpful
conversations over the past five years. I also thank Prof. John Essigmann for his encouragement
and support during my transition into the Pentelute lab. Thank you to Prof. Barbara Imperiali for
advising me during my first MIT experience in Summer 2009 and for her constant support
throughout my graduate career.
Completing a Ph.D. would be difficult without the help of incredible lab mates. I am so
grateful to have developed the translocation project with Dr. Xiaoli Liao during the first two
years in the Pentelute lab. Together we expressed the lab's first proteins, worked through
troublesome ligations, and got the project off the ground. I learned so much from Xiaoli-team
translocation's progress would not have been possible without her help. I also thankful for all the
former and current Pentelute lab members for everything they taught me and the fun timesAlex L., Alex M., Alex S., Alex V., Anthony, Chi, Colin, Dale, Dan C., Dan D., Daphne, Ethan,
Faycal, Gizem, Guillaume, Hansol, Jingjing, Jun, Justin, Kyle, Mark, Michael, Mike, Peng,
Richard, Rocco, Surin, Tatiana, Ted, Tessa, Tuang, Yekui, Yuta, Zak, and Zi-Ning. I am
extremely proud of and impressed by the lab's success to date; I am excited to see what the
future holds!
Graduate school would have been completely different had it not been for my colleagues
and classmates. These are the people who shared in the victories, helped troubleshoot the
struggles, and listened to the problems: Ali, Katya, Jingnan, Jon, Nootaree, Vinita, Whitney, and
especially my biological classmates, Austin, Haritha, Kanchana, and Megan. A special thank you
to Austin, a talented scientist and devoted friend, who always had time to help me think through
tough problems, to eat lunch at Cosi, or to take a run around the Charles. His enthusiasm for
science was infectious and I am forever grateful for all his support. My Yorktown and Syracuse
friends have been extremely encouraging and always willing to talk about non-science things
over the past five years as I navigated the ups and downs of graduate school: Anna, Dana, Eileen,
Emily, Heather, Kaitlyn, and Rachel.
My family has been my rock and greatest support system throughout my graduate years.
No matter what time of day it is, someone is always available to listen, laugh, or advise. My best
friend and sister, Erin, for always knowing exactly how to calm me down and make me smile
7
and for buying me my first yoga mat. My mom, for always having time to talk and listen to me
and for teaching me perseverance and dependability. My dad, for the pep talks and wise words
before all my big presentations and meetings and for teaching me to always think about what
comes after what comes next. Indeed, learning is the journey.
8
Table of Contents
Abstract
5
Acknowledgements
7
Table of Contents
9
List of Figures
14
List of Tables
18
Chapter 1: Delivery of Non-Native Cargo into Mammalian Cells Using Anthrax Lethal
Toxin
1.1.
18
Introduction
19
1.2. Anthrax Lethal Toxin
23
1.3.
Fusion and Conjugation Strategies
24
1.4.
Methods to Study Translocation
28
1.5.
Delivery of Non-Native Cargos
31
1.5.1. Natural Proteins
31
1.5.2. Engineered Protein Variants
32
1.5.3. Stabilized Protein Conjugates
34
1.5.4. Mirror Image Polypeptides
35
1.5.5. Non-Natural Peptides
36
1.5.6. Cyclic Peptides
37
1.5.7. Small Molecule Drugs
37
1.5.8. Modified Delivery System
38
1.5.9. Retargeting PA
39
1.6.
Summary and Outlook
42
1.7.
References
43
Chapter 2: Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin
Protective Antigen
48
2.1. Introduction
49
2.2. Results
53
2.2.1. Delivery of antibody mimic proteins
53
9
2.2.2. Translocation requires a functional PA and endocytosis
54
2.2.3. Western blot analysis of cytosolic fraction
57
2.2.4. Comparison to TAT-mediated delivery
58
2.2.5. Delivery of a tandem monobody for SH2 domain of Bcr-Abl
61
2.2.6. Delivery of an affibody for Raf-1
67
2.3. Discussion
69
2.4. Experimental
74
2.4.1. Materials
74
2.4.2. One-pot sortagging reaction using Staphylococcus aureus SrtA*
74
2.4.3. Protein synthesis inhibition assay
75
2.4.4. Uptake of Lvs or TAT-HA-I to -4 in CHO-KI cells
75
2.4.5. Cytosolic protein extraction and whole cell lysate preparation
76
2.4.6. Western blot
76
2.4.7. Co-immunoprecipitation of Lv5 with Abl kinase
76
2.4.8. TUNEL assay with Lv5 in K562 cells
77
2.4.9. Transfection of HEK 293T with pcDNA3-ABRaf
77
2.4.10. Delivery of Lv6 into HEK 293T cells
78
2.4.11. Construction of plasmids for recombinant proteins and transfection
78
2.4.12. Protein expression and purification
79
2.4.13. Synthesis of TAT peptide and native chemical ligation (NCL) of TAT-DTA
80
2.4.14. Synthesis of TAT-HA-LPSTGG peptide and sortagging to G5 -proteins
80
2.5. Acknowledgements
81
2.6. Appendix
82
2.6.1. LC-MS Traces
2.7. References
Chapter 3: Delivery of Mirror Image Polypeptides into Cells
10
101
106
110
3.1. Introduction
111
3.2. Results
113
3.2.1. Delivery of mirror image polypeptides
113
3.2.2. Translocation requires a functional PA and endocytosis
116
3.2.3. Translocation of a D-peptide for MDM2
118
3.2.4. Translocation of mirror image proteins
122
3.3. Discussion
125
3.4. Experimental
126
3.4.1. Materials
126
3.4.2. Solid phase peptide synthesis (Boc)
127
3.4.3. Solid phase peptide synthesis (Fmoc)
127
3.4.4. Analytical LC-MS
128
3.4.5. Preparative, semi-preparative, and analytical RP-HPLC
128
3.4.6. Synthesis of D-affibody
129
3.4.7. Synthesis of D-affibody-alkyne
131
3.4.8. Synthesis of D-GB1
131
3.4.9. Circular dichroism (CD) spectroscopy of folded proteins
133
3.4.10. Synthesis of biotinylated p53/MDM2 Inhibitor D-peptide
133
3.4.11. Construction of plasmids for recombinant proteins
134
3.4.12. Protein expression and purification
134
3.4.13. One-pot sortagging reaction using Staphylococcus aureus SrtA*
135
3.4.14. Translocation of SDvs and protein inhibition assay in CHO-Ki cells
136
3.4.15. Cytosolic protein extraction and whole cell lysate preparation for western blot
137
3.4.16. Western Blot of Svl, 2, 4, and 5 translocated into CHO-KI cells
137
3.4.17. Trypsin digestion of L- and D-Affibody
138
3.4.18. Pull down of Sv4-biotin in U-87 MG cells
138
3.4.19. Translocation of Sv4 and Sv4-biotin in U-87 MG or K562 cells
138
3.4.20. Binding interaction between SUMO-2 5- o 9MDM2 and 4 or Sv4
139
3.5. Acknowledgements
140
3.6. Appendix
141
3.6.1. LC-MS Traces
3.7. References
157
160
Chapter 4: Translocation of Non-Canonical Polypeptides into Cells Using Protective
Antigen
163
11
4.1. Introduction
164
4.2. Results
168
4.2.1. Translocation of non-canonical polypeptide cargos with backbone or side chain
modifications
168
4.2.2. Translocation of cyclic peptides
171
4.2.3. Translocation of complex small molecules
173
4.2.4. Translocation of intact cargo
176
4.3. Discussion
179
4.4. Experimental
181
4.4.1. Materials
181
4.4.2. 'H Nuclear magnetic resonance ('
H NMR)
182
4.4.3. Synthesis of docetaxel-maleimide
182
4.4.4. Synthesis of doxorubicin-maleimide
183
4.4.5. Solid phase peptide synthesis (Boc)
184
4.4.6. Solid phase peptide synthesis (Fmoc)
184
4.4.7. Protein expression and purification
185
4.4.8. Sortase-mediated ligation
186
4.4.9. LC-MS analysis
186
4.4.10. Preparative, semi-preparative, and analytical RP-HPLC
187
4.4.11. Conjugation of docetaxel, doxorubicin, and MMAF to G 5-LRRLRAC
187
4.4.12. Cyclization of linear peptide using native chemical ligation
188
4.4.13. Protein synthesis inhibition assay
189
4.4.14. Translocation of LDn1-1 I and cytosolic and total cell lysate extraction
190
4.4.15. Western blot of extracted material
190
4.5. Acknowledgements
191
4.6. Appendix
192
4.6.1. LC-MS Traces
4.7. References
200
204
Chapter 5: A D-Amino Acid at the N-terminus of a Protein Abrogates its Degradation by
the N-End Rule Pathway
12
206
5.1. Introduction
207
5.2. Results
209
5.2.1. Sortase A Attaches One D-Amino Acid onto the N-Terminus of LFN-DTA
209
5.2.2. One N-Terminal D-Amino Acid Stabilizes LFN-DTA to Proteasomal Degradation 209
5.2.3. Proteasomal Stabilization is Not an Artifact of the Sortag
212
5.2.4. LFN-DTA with One N-terminal D-Amino Acid is Stable In Vitro
214
5.2.5. LFN-DTA with One N-terminal D-Amino Acid is Not Ubiquitinated
214
5.2.6. N-terminal Stabilization is Not Protein-Specific
216
5.2.7. RRSPc 2 is a Ras/Rap 1-Specific Endoprotease
219
5.2.8. EGFR-Targeted Delivery of RRSPc 2 Interrupts the MAPK Pathway
219
5.3. Discussion
222
5.4. Experimental
225
5.4.1. Materials
225
5.4.2. Protein Expression and Purification
225
5.4.3. Fmoc Solid Phase Peptide Synthesis
226
5.4.4. Sortase A-Mediated Ligation of X-LFN-DTAut Constructs
227
5.4.5. Protein Synthesis Inhibition Assay with X-LFN-DTAmut Constructs
228
5.4.6. Translocation and Western Blot Analysis with X-LFN-DTAmut Constructs
228
5.4.7. Native Chemical Ligation of Native X-LFN-DTAmut
229
5.4.8. In Vitro Stability of X-LFN-DTAmut Constructs
230
5.4.9. Streptavidin Pulldown of Ubiquitinated Constructs
231
5.4.10. Stabilization of X-DTAmut or X-DARPin after Translocation
231
5.4.11. EGFR-Targeted Translocation of RRSPc 2
233
5.5. Acknowledgements
234
5.6. Appendix
235
5.6.1. LC-MS Traces
5.7. References
250
265
13
List of Figures
Figure 1.1.1. Anthrax lethal toxin is comprised of two discreet components.
21
Figure 1.1.2. Translocation of biomolecules using PA/LFN delivery platform.
22
Figure 1.3.1. Modular chemistry to modify LFN or LFN-DTA at N- or C-terminus.
27
Figure 1.4.1. Representative assay results from common translocation assays.
30
Figure 1.5.1. Translocation efficiency has been analyzed for various peptides and proteins.
40
Figure 2.1.1. Delivery of antibody mimics into the cytosol by the PA/LFN system.
52
Figure 2.2.1. Control experiments validating the translocation mechanism of LDv1 and Lvl. 56
Figure 2.2.2. TAT peptide mediated translocation of DTA and antibody mimics.
60
Figure 2.2.3. Delivery of Lv5 and binding of Lv5 to Abl kinase in K562 cells.
63
Figure 2.2.4. Monitoring of apoptosis of K562 cells treated with Lv5 and PA by TUNEL assay.
66
Figure 2.2.5. Perturbation of the MAPK signaling pathway by PA mediated delivery of an
affibody (Lv6) that targets Raf.
68
Figure 2.6.1. One-pot sortagging reaction.
82
Figure 2.6.2. Coomassie stained SDS-page gel of LDvs and Lvs obtained after sortagging and
purification.
83
Figure 2.6.3. Thermal stability of 1 OFN3 (4) and HA4 (4mut) was monitored by circular
dichroism (CD) spectroscopy.
84
Figure 2.6.4. Western blot analysis of delivered Lvs.
85
Figure 2.6.5. Coomassie stained SDS-PAGE gel of TAT-HA-I to -4 and -4mut (1 pg).
86
Figure 2.6.6. Cellular uptake of TAT-HA-I to -4 and -4mut. CHO-KI cells.
87
Figure 2.6.7. The level of protein synthesis inhibition in K562 cells.
88
Figure 2.6.8. SPR curves for SUMO-SH2 and varying concentrations of Lv5 or HA4-7c 12.
89
Figure 2.6.9. Linear relationship between signal intensity of each band and the amount of
protein loaded.
90
Figure 2.6.10. Phosphorylation analysis of Bcr-Abl.
91
Figure 2.6.11. Apoptosis measurement of K562 cells.
92
Figure 2.6.12. MTS cell viability assay.
93
Figure 2.6.13. Protein sequences.
95
14
Figure 3.2.1. Delivery of D-cargo sortagged onto LFN and LFN-DTA.
114
Figure 3.2.2. Translocation of mirror peptides using PA/LFN-
117
Figure 3.2.3. Translocation of a D-binder to MDM2.
121
Figure 3.2.4. Synthesis and translocation of mirror image proteins.
124
Figure 3.6.1. Immunoblot of media from CHO-KI cells treated with Svl-3.
146
Figure 3.6.2. Linear relationship between Sv4 band intensity and the amount of protein loaded.
147
Figure 3.6.3. Calibration curve for the interaction between immobilized biotin- -29 p53 and
SUMO-25- 09MDM2.
148
Figure 3.6.4. Binding interaction between SUMO- 2 5- ' 09MDM2 and 4 or Sv4.
149
Figure 3.6.5. Quantification of MDM2, p53, and p21 protein levels for U-87 MG cells.
150
Figure 3.6.6. LC-MS characterization of D-affibody synthesis.
151
Figure 3.6.7. LC-MS characterization of D-GB 1 synthesis.
152
Figure 3.6.8. CD of L- and D-affibody and L- and D-GB 1.
153
Figure 3.6.9. Trypsin digestion of L-affibody, D-affibody, Sv5-L, and Sv5-alkyne over time. 155
Figure 3.6.10. LC-MS characterization of D-affibody-alkyne and D-affibody-biotin.
156
Figure 4.1.1. Delivery of non-canonical polypeptide cargo into the cytosol.
167
Figure 4.2.1. Translocation of non-canonical peptides.
170
Figure 4.2.2. Translocation of cyclic peptides.
172
Figure 4.2.3. Translocation of small molecules.
175
Figure 4.2.4. Translocation of C-terminally biotinylated cargo.
178
Figure 4.6.1. Cyclization of L-linear peptide using native chemical ligation.
196
Figure 4.6.2. Synthesis of doxorubicin-maleimide and docetaxel-maleimide.
197
Figure 4.6.3. Western blot of total extraction of LDn1-8.
198
Figure 4.6.4. Western blot of total extraction of LDn9- 11.
199
Figure 5.2.1. Intracellular stability was monitored for X-LFN-DTA constructs delivered through
protective antigen pore.
210
Figure 5.2.2. One N-terminal D-amino acid on LFN-DTA enhances protein stability.
213
Figure 5.2.3. One N-terminal D-amino acid prevents ubiquitination of LFN-DTA.
215
Figure 5.2.4. N-terminal D-amino acid stabilization is not limited to LFN.
218
15
Figure 5.2.5. Precision delivery of stabilized RRSPc 2 through EGFR into pancreatic cancer cells
interrupts the MAPK pathway.
221
Figure 5.6.1. Translocation of X-LFN-DTAmUt constructs.
235
Figure 5.6.2. Western blot analysis of X-LFN-DTAmut constructs in CHO-KI cells.
237
Figure 5.6.3. Western blot analysis of X-LFN-DTAmut constructs delivered into HEK-293T or
HeLa cells.
238
Figure 5.6.4. Western blot analysis of sortagged and native X-LFN-DTAmut constructs.
239
Figure 5.6.5. In vitro degradation of X-K(bio)-LFN-DTAmut.
240
Figure 5.6.6. Translocation of X-K(bio)-LFN-DTAmut into CHO-Kl cells.
241
Figure 5.6.7. LFN-C* X-cargo-C is the oxidation product of LFN-C* and X-G 5-cargo-C-
Ellman's.
242
Figure 5.6.8. Rate of reduction for 1-X.
243
Figure 5.6.9. Delivery of 1-X.
244
Figure 5.6.10. Translocation of LFN-DTA-RRSPc 2 .
245
Figure 5.6.11. SDS-PAGE analysis of in vitro cleavage of KRas by RRSPC 2 conjugates.
246
Figure 5.6.12. LC-MS analysis of in vitro cleavage of KRas by RRSPc 2 conjugates.
247
Figure 5.6.13. Targeted delivery of LFN-DTA through EGFR.
248
Figure 5.6.14. Quantification of pErkl/2 levels in 3-X-treated AsPC-1 cells.
249
List of Tables
Table 1.5.1. More than 30 different non-native cargos have been delivered into the cytosol of
cells.
Table 2.6.1. PCR primers.
41
96
Table 2.6.2. Observed molecular masses of expressed protein constructs when analyzed by LC-
MS.
97
Table 2.6.3. Isolated yields of sortagging ligations from SrtA* reaction.
98
Table 2.6.4. EC5 0 values of 30-minute protein synthesis inhibition assay.
99
Table 2.6.5. List of variants.
100
Table 3.6.1. Peptides used in this investigation.
141
16
Table 3.6.2. Observed molecular masses of expressed protein constructs when analyzed by LCMS.
142
Table 3.6.3. List of variants.
143
Table 3.6.4. Isolated yields of sortagging ligations from SrtA* reaction.
144
Table 3.6.5. EC50 values of 30-minute protein synthesis inhibition assay.
145
Table 4.6.1. Peptides used in this investigation.
192
Table 4.6.2. List of variants.
193
Table 4.6.3. EC 50 values of 30-minute protein synthesis inhibition assay.
195
Table 5.6.1. EC50 values for X-LFN-DTAut constructs translocated in CHO-Ki cells.
236
17
Chapter 1: Delivery of Non-Native Cargo into Mammalian Cells
Using Anthrax Lethal Toxin
18
1.1.
Introduction
Pathogenic bacteria often express protein toxins capable of delivering cytotoxic payloads
into the cytosol of cells.' The cytotoxic payloads are often referred to as effector proteins and
have diverse functionalities in the host cell-protease activity, modification of intracellular
substrates, or interruption in cell signaling pathways-for the benefit of the bacterium. One
example is anthrax lethal toxin from the gram-positive bacterium, Bacillus anthracis,which has
been extensively studied for the past forty years. Thorough biophysical and biochemical analyses
have led to an increased understanding of each component as a discreet protein and together as a
macromolecular nanomachine. 2 Anthrax lethal toxin has evolved to deliver lethal factor (LF), a
cytotoxic protein payload, into the cytosol of mammalian cells (Figure 1.1.1).3 To accomplish
this transport, anthrax lethal toxin utilizes a second component called protective antigen (PA),
which oligomerizes on the cell surface to form the PA pre-pore. Following endocytosis the PA
pre-pore forms a ~-12 A channel in the endosomal membrane and acts as a conduit for delivery of
LF into the Cytosol.4-6 Once in the cytosol, LF is a Zn
protease that cleaves mitogen activated
protein kinase kinases (MAPKK) and causes cell death.7
While native anthrax lethal toxin expressed by B. anthraciscontinues to be a bioterrorism
threat, in the last two decades the toxin has been modified to serve as a delivery platform for the
transport of biomolecules. The PA/LFN delivery platform consists of PA and the non-toxic, Nterminal PA-binding domain of LF known as LFN (Figure 1.1.2). The delivery of cargos other
than LF (i.e. non-native) such as enzymes, polypeptides comprised of non-natural amino acids,
or small molecule drugs has been explored. Fusions of non-native cargos composed of canonical
amino acids to LFN have been achieved through recombinant expression. Non-recombinant
19
methods of ligation like native chemical ligation (NCL) or enzyme-mediated ligation have been
utilized to study the translocation of cargos containing non-natural functionalities.
This review provides an in-depth analysis of the PA/LFN delivery platform for the
translocation of non-native cargos into the cytosol of cells. Most notably, the PA pore has been
used to deliver more than 30 proteins and polypeptides containing natural and non-natural amino
acids as well as small molecule drugs. Collectively, these analyses provide insight into the
promiscuity of the PA pore and demonstrate its potential for the delivery of bioactive cargos to
disrupt intracellular protein-protein interactions or to study biological processes. Furthermore,
these studies support the current model of protein translocation through the PA pore and provide
insight into design parameters for delivering new cargos efficiently.
20
a.
PA6
LF
PA 8
Anthrax
receptor
Hnosm
cytosol
4
-{MAPKK
b.
catalytic
domain
C.
d-
LFN
PA2
2PAI
3
PA-binding
domain (LFN)
4
e.
90*
PA pore
Phe clamp
Figure 1.1.1. Anthrax lethal toxin is comprised of two discreet components. a. Cytosolic
delivery via anthrax lethal toxin is achieved by anthrax receptor recognition by PA83 then
activation to form PA6 3 by a cell-surface furin family protease (1). Seven or eight PA 63
molecules self-assemble to form the PA pre-pore (2) then LF binds (3) and the entire complex is
endocytosed into the endosome (4). Acidification triggers pore formation and translocation of LF
into the cytosol (5). b. Crystal structure of LF reveals two domains (pdb: 1J7N)-PA binding
domain (green; LFN) and catalytic domain (gray) c. PA83 is composed of 4 domains (blue domain
I initiates pre-pore formation, pink domain 2 and orange domain 3 are involved in forming the
pore itself, and purple domain 4 is the receptor-binding domain) (pdb: 1ACC) d. LFN binds to
two adjacent PA subunits (pdb: 3KWV) e. PA pore structure reveals the restrictive Phe clamp
(within domain 2) at the center of the pore (pdb: 3J9C).
21
cargo
PA 83
Anthrax
receptor
PA 63
cytosol
2LFN
H+
endosome
4
Figure 1.1.2. Translocation of biomolecules using PA/LFN delivery platform. The PA/LFN
delivery platform was developed such that the catalytic domain of LF could be replaced with
various cargo molecules to determine their translocation efficiency through PA pore or to
analyze their biological function in the cytosol of cells.
22
1.2.
Anthrax Lethal Toxin
Anthrax lethal toxin is a two-component system in which lethal factor (LF; 90 kDa) is
transported into the cytosol of a host cell through protective antigen (PA83 ; 83 kDa) pore.2 After
nearly forty years of mechanistic and structural analyses, a model for protein translocation via
anthrax lethal toxin has emerged (Figure 1.1. la). In the presence of a divalent metal ion, PA83 is
recognized by and binds to either of two cell surface receptors, tumor endothelial marker 8 or
capillary morphogenesis protein 2 (TEM8 or CMG2) with nanomolar or picomolar affinity,
respectively. 8-' 0 The 1000-fold disparity in binding affinity has been attributed to non-conserved
residues of CMG2 that interact with domain 2 of PA." While the exact function of each receptor
'
remains unknown, both TEM8 and CMG8 have been shown to regulate angiogenic processes.1 2
13
Furthermore, the anthrax receptors are expressed on most human cells at approximately 2,000
- 50,000 receptors per cell.' 4
Once PA83 (Figure 1.1.1b) is receptor-bound, a furin family protease proteolytically
activates the protein by cleaving the N-terminal 20 kDa portion, leaving PA6 3 to oligomerize into
the PA pre-pore heptamer or octamer.'
15-1"
The PA pre-pore is capable of binding up to three or
'
four molecules of LF (Figure 1.1.1 c) between two adjacent PA 6 3 subunits with 1-2 nM affinity.' 8
19 The entire complex is endocytosed and encapsulated in an endosome. Acidification of the
endosome (pH -5.5) results in a conformational rearrangement of the PA6 3 subunits, which leads
to PA pore formation (~12 A diameter) in the endosomal membrane. 2 0, 21 The pH gradient
generated between the two compartments leads to translocation of protonated LF into the cell
cytosol (pH ~7.0). Translocation through PA pore is considered to be a charge state-dependent
Brownian ratchet motion.22' 23
23
Biochemical and biophysical studies have demonstrated that the structural components of
anthrax lethal toxin relate to protein translocation. Mutagenesis analyses, kinetic studies,
computational models, and a recent crystal structure have revealed how the N-terminal, PA
24-26
4 LF;
LFN) interacts with two adjacent subunits of the PA pre-pore.' 9
'
binding domain of LF (1-1
Specifically, there are key electrostatic and hydrophobic interactions between the first a-
helix and P-sheet of LFN and a deep amphipathic cleft on the surface of PA (alpha clamp) that
bind LF such that the N-terminal region of the protein is partially unfolded and poised for
translocation from N- to C-terminus (Figure 1.1.1d).19,
27
A recent 2.9 A resolution cryogenic
electron microscopy structure was solved for the PA pore that supports the current model for
protein translocation (Figure 1.1.1e).2
The structure confirmed mutagenesis studies that
predicted a narrow ring of solvent-exposed Phe427 residues in the lumen of the channel. 2 9 ,3 0 The
ring of Phe residues, called the Phe clamp, is the most restrictive part of the pore and interacts
with hydrophobic stretches. The structure also revealed negatively charged residues surrounding
the Phe clamp, which are hypothesized to deprotonate the translocated protein and guide
unidirectional translocation.
1.3.
Fusion and Conjugation Strategies
Prior to the elucidation of the LF and PA structures, researchers utilized protein fusions
to explore the translocation mechanism and to analyze the biological function of bioactive
payloads inside the cytosol. The earliest work relied on recombinant expression to create fusions
with the catalytic domains of select protein toxins; however, recombinant expression is not
adequate for probing the delivery of cargos containing non-natural functionalities such as amino
acids with inverted chirality, non-canonical side chain residues, or modified backbone structures.
To work with cargos containing non-natural amino acids, semisynthetic and enzymatic
24
techniques like native chemical ligation (NCL) and enzyme-mediated ligation using sortase A
(SrtA) have been utilized.
For protein fusions comprised of the 20 canonical amino acids, recombinant expression is
often the simplest and most high yielding technique (Figure 1.3.la). However, proteins
containing natural amino acids are susceptible to proteolysis and proteasomal degradation in the
cytosol. Advantages of incorporating non-natural functionalities into protein fusions include
stabilization to intracellular degradation, use of affinity handles, and perturbed binding affinities
to target molecules. Pentelute, et al. developed a semisynthetic approach using NCL to ligate
non-natural peptides onto the N-terminus of LFN in order to explore the specificity of PA with
regard to translocation initiation (Figure 1.3.1 b). 3 ' While NCL facilitates the site-specific ligation
of peptides, oftentimes it is performed under denaturing conditions to achieve optimal substrate
concentrations.
As a result, ligated products must be purified by RP-HPLC, followed by
refolding. While LFN has been found to refold well, some cargos may not refold properly,
resulting in inactivity.3 1
Enzyme-mediated ligation using SrtA has allowed for the specific attachment of nonnatural biomolecules under non-denaturing conditions. The catalytic domain of SrtA from
Staphylococcus aureus has been demonstrated to be useful for in vitro ligations.
As a cysteine
protease and transpeptidase, SrtA ligates substrates containing an N-terminal pentaglycine tag
onto molecules containing the LPXTG motif (Figure 1.3. 1c). Chen, et al. recently evolved SrtA
enzymes to have -50-140-fold increase in LPETG substrate coupling activities such that ligation
reactions can be carried out in less than one hour at physiological conditions with high yields.34
SrtA-mediated ligation has been used to attach antibody mimic proteins and peptides containing
25
non-natural functionalities on the C-terminus of LFN and LFN-DTA as well as mixed chirality
peptides to the N-terminus of LFNRecombinant expression, NCL, and enzyme-mediated ligation each install a natural,
amide linkage between LFN and the cargo of interest. While this is critical for initial analysis of
translocation efficiency, there are a variety of other bioconjugation techniques that can be
employed to fuse cargos onto LFN- Common examples include the genetic code expansion
technique, disulfide linkage, maleimide conjugation, or click chemistry.3 5
Furthermore,
combinations of techniques can be used to produce the desired construct. Recently, Ling, et al.
demonstrated that SrtA can accommodate peptide thioester substrates, which can be
subsequently used for NCL to form protein conjugates separated by non-natural sequences such
as LFN-DTA in which the two proteins were joined together by a D-peptide linker. 3 6
26
a. Recombinant Expression
E. coil
Expression
Expression
E
vector
E Oil
Expression
vectorW
Expression
Eci
vector
W
-
>
b. Native Chemical Ligation
HS
S'R
H
X
L
H 2N
+
0
H2 N
0
I
0
+
H2N
CO 2 H
HS
-
AS'R +
U
SHH
NN
CO
'H 0
S, R
+
* ;
H2N f
c. Enzyme-Mediated Ligation
ORtA
-LPSTGG
-LPSTG 5
-
Ca2
SrtA
-LPSTGG
+
Gso
-LPSTG
+5-GSrtA
+aG24%
tLPSTG 5
-
+
-LPSTGG
SrtA
*LPSTGG
+ G5-
LPSTG A
Figure 1.3.1. Modular chemistry to modify LFN or LFN-DTA at N- or C-terminus. a.
Recombinant expression in bacteria (e.g. E. coli) serves as a straightforward method to obtain
fusion proteins containing canonical amino acids b. A semisynthetic platform using native
chemical ligation (NCL) allows for the ligation of N-terminal cysteine biomolecules with Cterminal thioester biomolecules activated using mercaptophenylacetic acid (MPAA) c. Enzymemediated ligation using sortase A (SrtA) has been developed for the facile ligation of any cargo
to LFN or LFN-DTA under non-denaturing conditions.
27
1.4.
Methods to Study Translocation
The delivery of protein and peptide cargos with non-natural functionalities has provided
insight into the specificity of the PA pore, efficiency of the PA/LFN delivery platform, and a
deeper understanding of the mechanism for protein translocation. Translocation efficiency of
cargos at the C-terminus of LFN (or LFN-DTA) has been analyzed using three common
laboratory assays: planar lipid bilayer,37 protein synthesis inhibition or cytotoxicity analysis
based on enzymatic activity, 38, 39 and western blot analysis of the delivered material 40 . Each
approach has provided new insight into key features of translocation.
As an in vitro assay, planar lipid bilayer has been used to measure the change in ion
current after LFN constructs have been added to a chamber containing PA pore embedded in an
artificial membrane. Electrophysiological measurements from the bilayer experiments have
revealed important features of translocation initiation and the mechanism of delivery through the
PA pore. Cell-based, enzymatic assays have been developed using reporter protein cargos,
providing a sensitive measure of translocation into eukaryotic cells. One specific enzymatic
assay relies on the activity of the A chain of diphtheria toxin (DTA), which inactivates
'
elongation factor 2 (EF-2) through ADP ribosylation and inhibits cytosolic protein synthesis.4
Also known as the protein synthesis inhibition assay, this assay utilizes the activity of DTA to
monitor the delivery of assorted cargos fused to LFN-DTA by 3H-Leu incorporation (Figure
1.4.1 a). The Pseudomonas exotoxin A (PE) catalytic domain, which inhibits protein synthesis by
a similar mechanism as DTA, has also been used as a reporter protein.4 1 Furthermore,
cytotoxicity assays using tetrazolium salts like MTT have been used to measure the translocation
efficiency of toxic enzymatic domains. Western blot analysis has been utilized as a third method
to measure translocation efficiency. After translocation, the cytosolic fraction of cells is lysed
28
using digitonin, a non-ionic detergent used to permeabilize the cytosolic membrane, then
analyzed by western blot (Figure 1.4.1b). 42 Immunostaining for LF, DTA, or biotin (if present)
provides a semi-quantitative measure of translocated material. Further development of more
sensitive assays is underway in order to precisely quantify the amount of material that has been
translocated into the cytosol.
29
a.
c 1.41.21.0.
0.8
-a-LF N-DTA
-.- LF N-DTA, No PA
o- 0.6-
8
-45
0.4-
u- 0.2
0.0-
-14 -13 -12 -1 -1 b -9 -8d -7-Y
Log [Protein Concentration (M)]
b.
PA only
PA + LFN
anti-LF
anti-Erkl/2
anti-Rab
Figure 1.4.1. Representative assay results from common translocation assays. a. Protein
synthesis inhibition assay measures the activity of DTA fused to LFN after translocation. After
incubating cells with LFN-DTA, 3H-Leu is added to monitor the extent of protein synthesis
inhibition caused by DTA. EC50 values of conjugates can be compared with the LFN-DTA
control to determine translocation efficiency. b. Western blot analysis of the cytosolic fraction
measures the amount of material delivered into the cytosol. Cytosolic extraction is achieved
using a lysis buffer containing digitonin.
30
1.5.
Delivery of Non-Native Cargos
1.5.1. Natural Proteins
Early experiments of protein translocation through PA pore monitored the delivery of
protein fusions as a method to characterize the structural requirements of initiating and
sustaining translocation. These studies utilized recombinant expression to generate protein
fusions of LFN with the A chain of diphtheria toxin (DTA), 3 8 , 19,4 Pseudomonas exotoxin A
(PE), 44 A chain of Shiga toxin (STA),
dihydrofolate reductase (DHFR),43 and P lactamase
(Figure 1.5.1a). 4 5 All together these studies demonstrated that regions near the N-terminus of
LFN are important for initiating translocation into eukaryotic cells. 27 The proteins however, must
be unfolded or in an extended conformation for efficient translocation, which was verified by the
impeded translocation of LFN-DTA containing an artificial disulfide (Figure 1.5.1b) and LFN$
DHFR bound to methotrexate.4 3 The efficient translocation of DTA, STA, PE, DHFR, and
lactamase provided the first indication that the PA pore can accommodate non-native protein
cargos. After translocation, the measured enzymatic activity for each protein cargo indicated that
these proteins were folded correctly in the cytosol and recognized their intracellular substrates.
Since the development of the PA/LFN delivery platform, various protein cargos have been
delivered into cells to understand their biological function. Examples include a cytotoxic T
lymphocyte epitope from Listeria monocytogenes listeriolysin 0 (LLO), 46' 47 Legionella
pneumophila flaggellin protein,48 actin cross-linking domain (ACD) of RTX from Vibrio
cholerae,4 9 Rho inactivation domain (RID) from Vibrio cholerae50 and a Ras/Rapl -specific
endopeptidase (RRSP) from Vibrio vulnificus.
Delivery of a cytotoxic T lymphocyte LLO
epitope was analyzed for its immune response activity in mice as a potential method for
developing vaccines against pathogens. 6 The inflammasome plays a major role in the innate
31
immune response. In order to study the biochemical and physiological consequences of
inflammasome stimulation, von Moltke, et al. delivered a Legionella pneumophila flagellin
protein to stimulate the inflammasome and monitored eicosanoid release. 48 Satchell and coworkers have utilized the PA/LFN delivery platform to study the functions of various effector
domains from multifunctional autoprocessing repeats in toxin (RTX) domains (MARTX)
expressed by pathogenic bacteria. The bioactivity of the actin cross-linking domain (ACD) from
Vibrio cholerae was analyzed after translocation into HEp-2 cells. Cordero, et al. demonstrated
that ACD directly catalyzes the covalent cross-linking of actin providing insight into the
mechanism of cell death caused by Vibrio cholerae.49 Sheahan, et al. analyzed the inactivation of
Rho GTPases by the Rho Inactivation Domain (RID) from Vibrio cholera after delivery into
HEp-2 cells using the PA/LFN delivery platform.50 The bioactivity of a toxic domain within
MARTX from Vibrio vulnificus whose function was previously unknown was characterized
using the PA/LFN delivery platform. Antic, et al. delivered the domain into HeLa cells and
demonstrated that the Ras/Rapl -specific endopeptidase (RRSP) effector domain cleaves the
Switch I region of Ras and Rapi proteins and thus interferes with downstream signaling in the
MAPK pathway.5 1 ,5 2
1.5.2. Engineered Protein Variants
Antibodies serve as powerful tools for medical diagnostics and the treatment of disease.
The use of antibodies, however, is limited to outside the cell due to their inability to cross the
plasma membrane into the cytosol and the presence of disulfide crosslinks. Recently, singledomain, cysteine-free scaffold proteins have been developed as antibody mimics. The scaffolds
include monobody from the tenth type III domain of human fibronectin (1OFN3), 5 3 ,5 4 affibody
from immunoglobulin binding protein A,55' 56 designed ankyrin repeats protein (DARPin),
32
and
the B1 domain of protein G (GBI)." Researchers have recently analyzed the translocation
efficiency of these scaffolds, which can be evolved to bind extracellular receptors or intracellular
59
targets with high affinity. '
60
Liao, et al. recently analyzed the delivery and intracellular activity of select affibody and
monobody antibody mimics using the PA/LFN delivery platform. 59 Specifically, the researchers
analyzed the delivery and associated bioactivity of a tandem monobody designed by Koide and
61 62
co-workers to bind the Src homology 2 domain (SH2) of Bcr-Abl (HA4-7cl2; KId 12 nM) ,
and an affibody designed to bind Raf (ABRaf;
Kd
100 nM) developed by Nygren and co-
workers. 63 The HA4-7c12 tandem monobody conjugate was translocated into chronic myeloid
leukemia cells (K562) and intracellular binding to Bcr-Abl
was confirmed by co-
immunoprecipitation. The inhibition of Bcr-Abl kinase activity and induction of apoptosis was
observed by TUNEL staining, which detects DNA fragmentation by labeling the termini. The
ABRaf affibody conjugate was translocated in human embryonic kidney 293T (HEK 293T) cells
and the interruption of the mitogen activated protein kinase (MAPK) pathway was monitored.
ABRaf was found to significantly reduce the phosphorylation levels of Erkl/2 after activation
with epidermal growth factor (EGF). Taken together, the HA4-7c12 and ABRaf delivery data
indicate that antibody mimics can be efficiently delivered into the cell cytosol through the
PA/LFN delivery platform, refold after translocation, and perturb intracellular PPIs.
The GB1 and DARPin antibody mimics have been shown to translocate efficiently into
the cytosol of cells. 59 These scaffolds have yet to be delivered into cells using PA pore to perturb
PPIs; however, the delivery of DARPin constructs with different thermostabilities has been
analyzed. Pl0ckthun and co-workers recently demonstrated that very stable DARPin constructs
33
(i.e. >90'C melting temperatures) cannot translocate efficiently through the PA pore. 60 The
researchers addressed the thermostability of DARPin by engineering constructs with reduced
stability, amenable for delivery via PA pore. A similar observation was made for 1OFN3
(unpublished results) in which wild-type 1OFN3 (Tm ~88C) translocated less efficiently than
HA4, a mutant 1OFN3 construct with Tm~-75'C. Together, these observations indicated that the
PA pore requires destabilization of the cargo for efficient translocation.
1.5.3. Stabilized Protein Conjugates
The ubiquitin (Ub)/proteasome
system is responsible for the majority of protein
degradation in the cell. The N-end rule described by Varshavsky and co-workers states that the
N-terminal amino acid of a protein impacts the protein's intracellular stability with regard to
proteasomal degradation. Since LFN follows the N-end rule, strategies have been developed to
increase cytotoxic activity and decrease immunogenicity of the translocated protein conjugates. 64
Leppla and co-workers demonstrated that reductive methylation to dimethylate the epsilon amino
group of lysine residues improves cytoxicity of LFN-PE conjugates by stabilizing the proteins to
intracellular degradation.65 LFN-PE conjugates including those prone to degradation (i.e. contain
N-terminal His residue) were reductively methylated at all 36 lysine residues using borane
dimethyl amine and formaldehyde. After translocation into several eukaryotic cell lines,
cytotoxicity and western blot assays revealed each methylated conjugate was stabilized to
degradation. A disadvantage of this approach is non-specific methylation, which means that for
substrates requiring lysine for catalysis or structural integrity, reductive methylation cannot be
used for stabilization.
34
Stabilization of proteins using one N-terminal D-amino acid was recently demonstrated
using the PA/LFN system (Chapter 5). Incorporation of N-terminal D-amino acids was achieved
through SrtA ligation or NCL for a native N-terminus. Proteasomal degradation of LFN-DTA
constructs containing L- or D-amino acids was screened using the protein synthesis inhibition
assay as well as western blot analysis. In both assays, while constructs containing N-terminal Lamino acids followed the N-end rule, constructs with N-terminal D-amino acids were stabilized
to degradation. We demonstrated that a protein containing one N-terminal D-amino acid is not
ubiquitinated. In order to prove that this phenomenon was not protein-specific, a hindered
disulfide cleavable linker was incorporated and similar observations of protein stabilization were
made for DTA, DARPin, and RRSPC 2 . This work updated the N-end rule to include D-amino
acids as stabilizing amino acids.
1.5.4. Mirror Image Polypeptides
The biological impact of polypeptides containing amino acids with non-natural side
chains, backbone composition, mirror image chirality and many others remains relatively
unexplored.66 Biomolecules composed of mirror image amino acids are of particular interest for
their unique biological stability and reduced immunogenicity. The translocation efficiency of
mixed chirality fusions to LFN through PA pore was recently analyzed.6 7 Protein synthesis
inhibition assays indicated that mirror image polypeptides and proteins translocate as efficiently
into the cell cytosol as their L-counterparts. Western blot analysis indicated that an unstructured
L-peptide cargo on the C-terminus of LFN resulted in rapid degradation of the protein conjugate
in the cell cytosol; however, a conjugate containing a D-peptide cargo of the same sequence was
found to be stabilized to degradation. Capping the C-terminus with two D-amino acids (D-cap)
35
stabilized the L-peptide cargo. Evidently, the incorporation of a short D-cap at the C-terminus of
an unstructured polypeptide can provide stabilization to intracellular degradation.
Delivery of stable, bioactive cargos into the cell cytosol is an attractive application for
translocation.
Using
the
PA/LFN
delivery
platform,
a
D-peptide
MDM2
antagonist
(TAWYANF*EKLLR, where F* is p-CF 3-D-Phe)68 was recently delivered into the glioblastoma
U-87 MG cell line, which overexpresses MDM2.67 A biotinylated form of the LFN conjugate (Kd
12.3
4.3 nM) towards MDM2 was found to interact with MDM2 after delivery into U-87 MG
cells. Moreover, after delivery into U-87 MG cells, the conjugate was found to disrupt the
p53/MDM2 pathway, as evidenced by upregulation of MDM2, p53, and p21 protein levels. For
the first time, the PA/LFN delivery platform was utilized to deliver a bioactive D-peptide into the
cytosol of a eukaryotic cell, where it subsequently bound the target protein and disrupted a
critical PPI.
1.5.5. Non-Natural Peptides
The translocation of proteins containing non-natural moieties is of particular interest for
their bioactivity, stability, or affinity properties. Conjugates containing peptide cargo with
p-
alanine, N-methyl alanine, propargylglycine, or 2,4,5-trifluorophenylalanine modifications were
found to translocate efficiently into CHO-Ki cells by western blot and protein synthesis
inhibition assays.69 Thus, subtle non-natural modifications to the peptide cargo do not affect
translocation efficiency through PA pore. The same conjugates with biotinylated C-termini were
also found to translocate efficiently, as indicated by co-staining of DTA and streptavidin on the
western blot. These results demonstrated efficient translocation of the biotin moiety and provided
further support for the delivery of intact conjugates into the cytosol. There are several other
36
examples of translocating biotinylated cargo, which collectively
confirm the efficient
translocation of biotinylated protein conjugates and the accessibility of biotin as an affinity
60
handle. , 67,
70
1.5.6. Cyclic Peptides
The cyclization of peptides is a useful approach to develop proteolytically stable
therapeutics with large surface areas for protein binding. 69 The delivery of L and D-forms of a
cyclic peptide comprised of 11 amino acids was recently explored using the PA/LFN delivery
platform. According to the protein synthesis inhibition assay and western blot analysis, the cyclic
peptide conjugates were unable to translocate through the PA pore. Rabideau, et al. hypothesized
that the cyclic peptides' constrained conformation and inability to unfold contributed to their
inefficient translocation (Figure 1.5.1b). Further investigation is required to fully understand the
potential of cyclic peptides as cargo of the PA/LFN delivery platform.
1.5.7. Small Molecule Drugs
To further probe translocation through PA pore, the delivery of small molecule drugs has
been explored. Small molecule drugs have diverse properties, functionalities, and threedimensional structures. The translocation of three common chemotherapeutics, doxorubicin,
docetaxel, and monomethyl auristatin F (MMAF) was analyzed by Rabideau, et al. 69 Analysis by
protein synthesis inhibition assay as well as western blot of the cytosolic fraction indicated that
small molecule drugs can translocate through the PA pore, but with limitations. Of the three
molecules tested, docetaxel was unable to translocate into the cytosol through PA pore. We
hypothesized that constrained or rigid molecules such as docetaxel cannot unfold or adopt a
conformation amenable to translocation through the -12 A pore (Figure 1.5. 1b).
37
1.5.8. Modified Delivery System
The PA/LFN delivery platform has been modified in some cases for the delivery of
fluorescent cargos such as small molecule probes or proteins. Using the genetic code expansion
technique, Zheng et al. installed an alkynyl-pyrrolysine residue within LF at position K581 then
used click chemistry to site-selectively label the protein with AlexaFluor545. 7 1 The researchers
monitored endocytic trafficking of the labeled protein in BHK fibroblast cells. Zornetta, et al.
demonstrated the PA-mediated delivery of GFP into the cytosol fused to LF.7 The delivery of
mCherry, however, proved inefficient. The researchers hypothesized that the difference in
translocation was due to a higher resistance of mCherry to unfolding. The delivery of fluorescent
proteins requires further investigation with respect to the cargo protein's melting temperature and
the fate of the chromophore during translocation.
The PA pore is cation-selective, thus favoring the passage of protonated or neutral
species. While the N-terminal 28 residues (18 are charged) are critical for initiating translocation,
the incorporation of cysteic acid (pKa -1.9) in this region halts translocation (Figure 1.5.1b). 73 Nterminal fusions of polycationic stretches (Lys, Arg, His) have been investigated for the delivery
of DTA using PA only. Blanke, et al. and Sharma, et al. showed that DTA fused to polycationic
residues rather than LFN can be delivered into cells through PA pore. 74' 75 Wright, et al. recently
utilized PA-mediated delivery to investigate the delivery of peptide nucleic acids (PNA).76
Reporter cells containing luciferase transgenes with mutant splice sites were treated with PA and
antisense PNA(Lys) 8 oligomers, which bind to the mutant splice sites. The researchers
demonstrated that the PNA oligomers were delivered into cells, corrected the splice defect, and
induced luciferase expression.
38
1.5.9. Retargeting PA
Recently, the PA/LFN delivery platform has been modified for the translocation of
material into specific cells such as those that overexpress specific receptors. Collier and coworkers retargeted PA to recognize non-native receptors such as HER2 and EGFR by mutating
two key residues responsible for binding the anthrax receptors and adding a new targeting
domain to the C-terminus of PA. 77~79 Retargeting PA to cells that overexpress specific receptors
is a method to increase the amount of material delivered into the cell. Investigations are
underway to study the delivery efficacy and bioactivity of delivered non-native cargos using
retargeted PA proteins.
39
a. Efficient translocating cargos
LFN
L
LFNAffibody
LFN
D-Affibody
DARPin
DTALFL
LI
FJ
LN
LFN
O
LFN
a
LFN
D-GB1
GB1
PE
®
1OFN3 (HA4)
LFN-QOjK
JONH,
(-CONH,
S
k
f
r
p
d
s n
v
r
N
G
ONH2
~
o01y
HN-NH
H
N
LFN
Q = L-amino acid
- = D-amino acid
HO
b. Inefficient translocating cargos
LF N-Gi(S3(D(PCONH,
LFN.
S
F
S
LFN
7q,
V
GR
IK
fl-c.
E~~S5
0K
0
0
0
~YONO
.,
K
*0&
NK I
H
yz
,
DTA with N58C+S146C disulfide
OH
Figure 1.5.1. Translocation efficiency has been analyzed for various peptides and proteins.
a. Representative protein and peptide cargos that translocated efficiently through the PA
pore
include DTA, PE, affibody (L and D forms), GB1 (L and D
forms), DARPin, HA4,
AKFRPDSNVRG peptide (L and D forms), biotinylated AKFRPDSNVRG peptide,
and a
doxorubicin-peptide conjugate b. Representative protein and peptide cargos that did
not
translocate efficiently (gray box) through the PA pore include DTA containing N58C+S146S
disulfide, AKFRPDSNVRG cyclic peptide, docetaxel-peptide conjugate, and peptide containing
three cysteic acid residues.
40
Table 1.5.1. More than 30 different non-native cargos have been delivered into the cytosol
of cells. Cargos range from proteins comprised of natural or non-natural amino acids, peptides
with different functionalities, and small molecules. The cargos that could not be efficiently
translocated by the PA/LFN delivery platform are highlighted in gray.
Cell Type
Fusion and
Pseudomonas exotoxin A (PE)
CHO
Recombinant
Diphtheria toxin, A chain (DTA)
CHO-KI, RAW264.7,
MC3T3, RBL-1, VERO,
L6
CHO-KI
Mouse, P815 (H-2d)
Mouse spleen, CHO, HeLa
CHO-KI, L6
HEp-2
Recombinant or
enzymatic or NCL
Recombinant
Recombinant
Recombinant, chemical
Recombinant
NCL
Recombinant
AlexaFluor545 (conjugated to K581 of LF)
Affibody
B 1domain of protein G (GB 1)
Tenth human fibronectin type three domain (IOFN3)
Designed ankyrin repeats protein (DARPin)
Mouse
HEp-2
HN6
BHK
CHO-KI
HeLa, HCT 116, MDAMB-231, HEK 293T
RAW264.7, CHO-KI,
HeLa, HN6
J774A.1, BHK-21
CHO-KI, HEK 293T
CHO-KI
CHO-KI
CHO-KI
HA4-7c12 tandem monobody
AKFRPDSNVRG peptide
akfrpdsnvrG (all D) peptide
AKFRPDSNvrG (Dcap) peptide
tawyanf*ekllr (all D; P is p-CF 3-D-Phe) peptide
Affibody (all D)
GBl (all D)
Biotinylated affibody (all D)
[D-AlaKFRPDSNVRG peptide
[N-Me-Ala]KFRPDSNVRG peptide
[Prop-Gly]KFRPDSNVRG peptide
AK[F 3-Phe]RPDSNVRG peptide
AK(Cys)FRPDSNVRG peptide
LRRLRAC(Doxorubicin) peptide-drug conjugate
K562
CHO-KI
CHO-KI
CHO-KI
U-87 MG
CHO-KI
CHO-KI
CHO-KI
CHO-KI
CHO-KI
CHO-KI
CHO-Ki
CHO-KI
CHO-KI
A chain of Shiga toxin (STA)
Listeriolysin 0 epitope (LLO)
0 lactamase
Dihydrofolate reductase (DHFR)
Actin cross-linking domain (ACD) of RTX from Vibrio
cholerae
Legionella pneumophila flagellin protein
Rho Inactivation Domain (RID)
Reductively methylated PE
Green fluorescent protein (GFP) (at C-terminus of LF)
KEKEKNKDENKRKDEER (ligated to N-terminus of LFN)
Ras/Rap I-specific endopeptidase (RRSP)
Cytolethal distending toxin B (CdtB)
Citation
conjugation strategies
Non-Native Cargo
Wn-,47
Recombinant
Recombinant
46,47
Recombinant
Recombinant
Recombinant
49
Recombinant, NHS
bioconjugation
Site specific click
Enzymatic
Enzymatic
Enzymatic
Enzymatic or
Recombinant
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic, click
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
Enzymatic
8
_
_
59
__
59
67
69
69
69
69
41
1.6.
Summary and Outlook
The PA/LFN delivery platform permits the facile delivery of biomolecules with diverse
structures and functionalities into the cytosol of eukaryotic cells (Figure 1.5.1 and Table 1.5.1).
The PA pore is relatively promiscuous for the delivery of non-native cargo on the C-terminus of
LFN.
Analyses by several groups have demonstrated that once translocation is initiated by LFN,
there are a few guidelines that cargos must follow in order to gain efficient cytosolic entry
through the PA pore. First, the cargo must be able to adopt an unfolded or extended
conformation in the endosome. Second, non-natural moieties such as non-natural backbone
structures and side chain modifications like mirror image or modified amino acids do not disrupt
the translocation process. Third, cargos containing moieties with low pKa values that cannot be
protonated in the endosome may inhibit translocation. Taken together, these design principles
can be employed for the delivery of previously unexplored cargos such as oligonucleotides or
post-translationally modified proteins.
There are several questions that remain unanswered regarding the PA/LFN delivery
platform. The amount of material delivered into the cytosol varies based on the cell type (i.e.
number of anthrax receptors expressed), incubation time (i.e. rate of endocytosis and receptor
recycling), concentration of PA and LFN constructs, and translocation efficiency of the LFN
construct. While western blot analysis provides a semi-quantitative analysis of the amount of
material delivered into the cytosol, a more accurate and sensitive method will provide
researchers with a better glimpse of translocation efficiency and a more precise measure of the
amount of material delivered into the cytosol. Furthermore, exploration into the in vivo effects of
the PA/LFN delivery platform will provide insight into the platform's efficacy and possible
immunogenicity in multicellular organisms.
42
1.7.
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47
Chapter 2: Delivery of Antibody Mimics into Mammalian Cells via
Anthrax Toxin Protective Antigen
Portions of the work presented in this chapter were published in the following manuscript and is
reproduced with permission from John Wiley & Sons:
Liao, X.,* Rabideau, A.E.* & Pentelute, B.L. Delivery of Antibody Mimics into Mammalian
Cells via Anthrax Toxin Protective Antigen. ChemBioChem 15, 2458-2466 (2014).
*: co-first author
48
2.1. Introduction
Antibodies have proven to be powerful tools in science and therapeutics by facilitating
the elucidation of disease mechanisms and generating novel and effective therapeutics. However,
the use of antibodies has been limited to outside of the cell because of two major factors:
antibodies containing disulfide bonds may be unstable in the reducing environment of cytosol,
and antibodies are unable to cross the cell plasma membrane to reach the cytosol. There are
numerous intracellular targets and protein-protein interactions with large flat contact areas that
are considered difficult to perturb by small molecules. We believe it would be of immediate
interest to use antibody mimics to target the intracellular protein-protein interactions provided
that they can be transported into the cytosol using a straightforward delivery platform.
In recent years, certain scaffold proteins that are robust, single-domain, and cysteine-free
have emerged as antibody mimics. These antibody mimics include monobodies derived from the
tenth
type III domain of human fibronectin (1OFN3),
affibodies derived
from the
immunoglobulin binding protein A, DARPins based on ankyrin repeat modules, and B 1 domain
of protein G (GB 1).1~6 These antibody mimics have not only been engineered to bind
extracellular receptors such as EGFR, HER2 receptor, VEGFR and integrin, but also various
intracellular targets including caspases, Raf, Erk, c-Jun N-Terminal Kinases (JNK), Abl-SH2,
and c-Jun.7'1 4 Advances in directed evolution and molecular display technologies such as phage
display, yeast display, and ribosome display make it possible to routinely generate a wide variety
of high affinity binders for specific protein targets.15-17
Some research efforts are now focused on applying these antibody mimics inside the cell
(intrabodies) to target cytosolic proteins.' 8 To achieve this goal, strategies are needed to allow for
49
the facile and reliable delivery of these bioactive antibody mimics into the cytosol of various cell
types. Delivery methods based on lipid-derived compounds,' 9 polymeric nanoparticles,2 0 , 21
inorganic nanocarriers, 2 2 supercharged proteins,2 3'
24
and most commonly, cell-penetrating
peptides (CPPs) including the transactivator of transcription (TAT) of HIV-1, oligoarginine, and
penetratin peptide derived from the Drosophilaantennapedia protein,25-28 have been extensively
developed to deliver proteins of interest to the cytosol of mammalian cells. In most cases, high
concentrations of these agents are required to achieve even modest effects often due to inefficient
cargo escape from the endosome to the cytosol.
Nature has evolved a variety of mechanisms to transport proteins across lipid membranes
into the cytosol of mammalian cells.2 9 One protein transport nanomachine from bacteria is
protective antigen (PA; 83 kDa), a component of anthrax toxin. PA is a receptor-binding, poreforming transporter, which has evolved to convey the enzymatic moieties of the toxin (lethal
factor and edema factor) from the external milieu to the cytosol of mammalian cells. PA binds to
)
host cell receptors and is cleaved by a furin-family protease to yield a 63 kDa species (PA 63
(Figure 2.1.1 a; step 1)30 that self-assembles to form ring-shaped heptamers 3 1 and octamers.3 2
These oligomers then form complexes with the cargo proteins (K
1I nM) and are endocytosed
to the endosome (Figure 2.1.la; steps 2-4), where the acidification triggers conformational
change of the PA 6 3 oligomers to form a transmembrane pore that unfolds and translocates the
bound cargo proteins to the cytosol (Figure 2.1.1a; step 5).33 The N-terminal domain of lethal
factor (LFN; ~30 kDa) is sufficient to bind PA oligomers and initiate translocation. 34 Prior studies
have shown fusions of cargo to the C-terminus of LFN can be transported to the cytosol via PA;
however, most efforts have focused on the delivery of peptides for vaccine development,3 5 or
enzymes including P-lactamase, 36' 37 or enzymatic domains from diphtheria toxin (DTA), Shiga
50
toxin, Pseudomonas exotoxin A (PEIII), and RTX toxin (ACD).38-41 More recently, the PA/LFN
system was shown to deliver Legionellapneumophilaflagellin into macrophages. 42 Despite these
prior efforts, no study has investigated the ability of PA to translocate the above-mentioned
antibody mimics for perturbation of intracellular protein-protein interactions, and the possibility
of using the PA/LFN system and its active endosomal escape mechanism as another delivery tool
to expand antibody therapeutics inside the cell.
Herewith we use the transpeptidase sortase (SrtA) 43 to conjugate several commonly used
antibody mimics to the C-terminus of LFN and find that PA can mediate their transport into the
cytosol of several different cell lines. We confirm the refolding and binding of a tandem
monobody to the protein target Bcr-Abl inside cell by co-immunoprecipitation. We observe
inhibition of Abl kinase activity and subsequent cell death caused by the PA-delivered
monobody. In a separate case, we show that the PA system delivers an hRaf-1 binder based on
the affibody scaffold that disrupts MAPK signaling pathway.
51
b)
PA 3
PA
PA
63
LDv:
LF N
2
Lv: W
LPSTG
LPSTG
5
*
a)
H+
c)
Antibody mimics
d)
1.2
U)
M*44C-term
~LFN-DTA
1.0
OD
-A-LDv2
1: Affibody
-w- LDv3
LDv4
0.8
'c-err
4: 1OFN3
-+-LDv4
0.
mr-m
4C:eHAm
0.2
.
2: GB1
LL
4rrl HA4
0.2
AA
-14
-13
-12
-11
-10
-9
-8
-7
Log [Protein Concentralon (M)]
-6
-5
3: DARPin
Figure 2.1.1. Delivery of antibody mimics into the cytosol by the PA/LFN system. a)
Mechanism of entry into cells. The star represents the antibody mimic to be delivered. b)
Antibody mimics 1-4 and 4mut: affibody (PDB: 1Q2N), GB1 (PDB: 1PGB), DARPin (adapted
from PDB: 3ZU7), 1OFN3 (PDB: lTTF), and HA4 (PDB: 3K2M). c) Variants of antibody
mimics attached onto the C-terminus of LFN (Lv) or LFN-DTA (LDv). d) Protein synthesis
inhibition assay using CHO-Ki cells treated with varying concentrations of LDvl-4 and 4 mut in
the presence of 20 nM PA for 30 minutes. The cells were then washed 3 times with PBS and
incubated with 1 pCi mL-1 3H-Leucine for 1 h. Subsequently, the cells were washed three times
with PBS and scintillation fluid was directly added to each well and 3H incorporation into the
cellular proteome was measured to determine the level of protein synthesis inhibition by LDvs (n
= 3). The radioactive counts were normalized to that of cells treated with only PA (set to 1).
52
2.2. Results
2.2.1. Delivery of antibody mimic proteins
In this study, our antibody mimics consisted of scaffolds widely used to generate highly
specific and potent binders: an affibody, protein GB1, DARPin, and monobody (Figure 2.1. 1b).
These scaffolds are disulfide-free, avoiding possible interference with passage through the PA
translocase and potential stability problems in the reducing environment of the cytosol. Our
chemoenzymatic bioconjugation route based on an evolved SrtA (SrtA*) is shown in Figure
2.6.1.
SrtA* catalyzed the formation of covalent conjugates between LFN containing C-
terminal LPXTG recognition motif and antibody mimics containing N-terminal oligoglycine,
designated Lv (Figure 2.1.1c).
We also prepared a series of conjugates between LFN-DTA and each antibody mimic,
(designated LDv, Figure 2.1.1c) to measure their PA-mediated translocation into the cytosol. In
anthrax toxin translocation studies, the A chain of diphtheria toxin (DTA), which catalyzes the
ADP-ribosylation of EF-2 and inhibits protein synthesis, has been frequently used as a
straightforward measure and a gold standard assay of PA-mediated translocation into the cytosol.
Therefore, the LFN-DTA variants (LDv) allow us to compare our findings to existing reports that
used the same assay.38'40'46'47 For each antibody mimic, after confirming the translocation of the
LDv conjugate, we then carried out additional studies using Lvs, which lack the toxic DTA
protein, thus avoiding interference with further characterization of antibody mimic function and
delivery into the cytosol.
To begin our study, each purified LFN-DTA variant (LDvl-4 and 4mut, Figure 2.6.2) was
added to CHO-Ki cells in the presence of 20 nM PA. After 30 minutes of treatment, the cells
53
were washed and incubated with medium supplemented with 3H-Leu. The efficiency of antibody
mimic translocation was measured by the incorporation of 3H-Leu in the cellular proteome. This
indicates the level of protein synthesis inhibition and is determined by the amount of LFN-DTAcontaining variants that reach the cytosol. As shown in Figure 2.1.1d, despite their structural
differences, all four variants, LDvl-3 and
4 mut
translocated efficiently into cell cytosol at levels
comparable to the positive control, LFN-DTA. Only LDv4 containing the highly stable 1OFN3
(Tm ~-88'C) translocated ~ 0-fold less efficiently, while the variant, LDv4mut containing a mutant
form of 10FN3 (HA4) with reduced stability (Tm -75'C) (Figure 2.6.3) translocated as
efficiently as LFN-DTA, indicating that the cargo thermal stability can affect translocation
efficiency. To confirm that the full-length protein was required for translocation to the cytosol,
we performed control experiments by treating cells with DTA plus PA, or DTA together with
LFN-affibody (Lvi) plus PA, and observed no protein synthesis inhibition in either case (Figure
2.1.1 d).
2.2.2. Translocation requires a functional PA and endocytosis
To further investigate if the LFN-DTA-antibody mimics (LDv) translocated into the
cytosol by the same mechanism as LFN-DTA, we carried out a series of control experiments with
LDv1 containing affibody as the cargo (Figure 2.2.la). We investigated the role of LFN in LDvl
during the translocation process using an excess amount of LFN (1 gM) to outcompete LDvI
from binding to PA. We found that 1 pM of LFN significantly abolished translocation of LDvl,
confirming the importance of LFN-mediated binding to PA (step 3, Figure 2.1. 1a). We then
studied the role of endocytosis in LDvI translocation by incubating cells with PA and LFN-DTA
or LDvl at 4'C instead of 37'C to arrest endocytosis. The translocation of both LFN-DTA and
LDvl were abolished under this condition, indicating that endocytosis is a necessary step in the
54
translocation process (step 4, Figure 2.1.1 a). To test the role of endosome acidification and active
translocation (step 5, Figure 2.1.1 a), we performed two additional control experiments. First we
treated the cells with PA and LFN-DTA or LDvl in the presence of 200 nM Bafilomycin Al, a
specific inhibitor of the vacuolar H+-ATPase that blocks endosome acidification. We found that
inhibition of endosome acidification abolished translocation for both LFN-DTA and LDvl,
confirming the importance of the pH gradient for translocation. An additional control experiment
was performed with a PA mutant (PA[F427H]) that binds and delivers the cargo to endosomes
but arrests translocation through PA pore.48 ' 4 We found that PA[F427H] completely arrested
translocation of LDvl, indicating that the functional PA was required for LDv1 to reach the
cytosol. These controls indicate that the translocation of LDvI follows the same mechanism as
LFN-DTA.
55
a), .2L 1.0-
0.8
0.LFN-DTA
0.4-
o
-e
- LDv1
-LDv1 + LFN
-4 - LFN-DTA 40C
-4 -LDv1 40C
r -LFN-DTA + BA
-I Lvl.RA
LDv1 + PA[F427H]
,u
-15
b)
-14 -13 -12 -11 -10 -9
-8
Log [Protein Concentration (M)]
Digitonin extracted
-
+
+
-
Total cell lysate
-
-
+
-6
-.
--
+
-
+
PA[F427H]
PA
-7
+
+
-
+
BA
4r g
LF 1 LFN Lvi Lv1 Lv1 LFN Lv1
Lv1
-
0.0 --
+
0.2
BA 40C
Lv1 Lv1
anti-LF i
anti-Erki /2 Oo -w6em6
.mw %.o..* -. 4
anti-Rab5
Figure 2.2.1. Control experiments validating the translocation mechanism of LDv1 and Lv1.
a) Protein synthesis inhibition assay of CHO-KI cells treated with varying concentrations of
variants in the presence of 20 nM PA for 30 minutes. The treatment conditions of control
experiments were modified as follows: BA = addition of 200 nM Bafilomycin Al, 4*C =
incubation at 4'C instead of 37*C, or PA[F427H] = mutant PA instead of PA. b) CHO-Ki cells
were treated with 250 nM Lvi in the presence of 40 nM PA or PA[F427H] for 12 hours. The
treatment conditions of control experiments were modified as described above. The total lysate
and digitonin extracted cytosolic proteins were prepared separately (~300,000 cells per lane).
56
2.2.3. Western blot analysis of cytosolic fraction
After confirming the key steps responsible for the translocation of antibody mimics, we
investigated the delivery of Lvs into the cytosol by digitonin extraction and western blot.
Digitonin is a weak, nonionic detergent and at low concentrations selectively permeabilizes the
plasma membrane, thereby releasing cytosolic components from cells while the nuclear envelope
and other major membrane organelles remain intact.5 0 CHO-Ki cells were incubated with Lvl
and PA overnight to allow for multiple rounds of receptor-mediated endocytosis and
translocation, washed, and trypsin digested to remove cell surface receptors and bound proteins.
In order to obtain the cytosolic fraction, cells were incubated with a phosphate buffer containing
50 pg mUl digitonin and 250 mM sucrose. Immunoblot analysis with antibodies against Rab5,
an early endosome marker, and Erkl/2, a cytosolic marker protein, was carried out to confirm
only the cytosolic fraction was present. Minor amounts of Rab5 protein were detected in the
digitonin-extracted cytosolic fraction, indicating little contamination by early endosomes (Figure
2.2.1b). We observed a significant amount of LvI in the cytosolic extraction, similar to that in
the total cell lysate obtained by lysis buffer containing 1% NP-40. Again, we performed control
experiments to further validate translocation of Lvl into cytosol. No Lvl was observed even in
the total cell lysate when the cells were incubated at 4'C in which endocytosis is arrested. The
absence of detectable Lvl at 4'C also validated that the wash and trypsin digestion protocol was
sufficient to eliminate potential contamination from surface bound proteins. When the cells were
treated with Bafilomycin Al, a significant amount of Lvl was observed in the total cell lysate
while the band was not detectable in the cytosolic fraction, indicating that Lvi was endocytosed
but trapped in the endosome. In addition, the absence of Lvl in the cytosolic fractions for the
Bafilomycin Al treated cells served as further evidence that our digitonin extraction protocol
57
was sufficient to separate the cytosolic proteins from the rest of the cell lysate, including
endosomes. Finally, when the cells were treated with LvI and PA[F427H] instead of Lvl and
PA, some amount of Lvl was observed in the total cell lysate but no Lvi was detected in the
cytosolic fraction (Figure 2.2.1b). Under this condition, the endocytosed LvI was trapped in the
endosome due the non-functional pore formed by the mutant PA, and subsequently sorted to the
lysosome for degradation, leaving only a small amount of LvI detectable in the total cell lysate,
but not detectable in the cytosolic fraction. The western blot results further corroborated the
protein synthesis inhibition results based on LFN-DTA (vide supra, Figure 2.1.1 d) and confirmed
the translocation mechanism of the antibody mimic cargos. The PA-mediated translocation of
other analogues (Lv2, 3, 4,
4 mt)
was also studied by digitonin extraction and western blot
(Figure 2.6.4). The detection of bands at different molecular weight by anti-LF antibody
confirmed the cytosolic presence of these antibody mimic conjugates.
2.2.4. Comparison to TAT-mediated delivery
To compare the efficiency of PA/LFN system with the CPP system, we first used the
protein synthesis inhibition assay. We prepared DTA with a TAT peptide covalently attached at
the N-terminus. This construct showed at least 1000 times lower efficiency in protein synthesis
inhibition than LFN-DTA and PA (Figure 2.2.2a), which is consistent with a previous report 5
and indicates the PA/LFN system is more efficient in delivering DTA into cytosol. Next, we
compared the efficiency of the PA system and CPP system in delivering antibody mimics. We
conjugated the oligoglycine-containing antibody mimics 1-4 and 4mut to a TAT peptide
containing an HA tag and LPSTGG motif to make the TAT-HA-antibody mimic constructs
(Figure 2.6.5).52 We studied their translocation again by digitonin extraction and western blot
analysis using the anti-HA antibody. We used a concentration of 2.5 pM for the TAT-HA58
antibody mimic constructs, 10-fold more than the amount of Lv used, to treat the cells for 4
hours in serum free media and observed minor amounts of material in total cell lysate (Figure
2.6.6a). Further, using the trypsin digestion and digitonin extraction protocol described above,
we detected none of the TAT-HA-antibody mimic proteins by anti-HA immunoblot in the
cytosolic fractions, while a significant amount of Lvi with HA tag (Lvl-HA) was detected on
the same blot for cells treated with 250 nM Lvl-HA and 40 nM PA for 16 hours (Figure 2.2.2b).
To test if a different incubation time would improve translocation by a TAT peptide, we treated
cells with 2.5 jM TAT-HA-antibody mimics for 16 hours, but again observed no material in the
total cell lysate (Figure 2.6.6b). These results demonstrate the higher efficiency of the PA/LFN
system in delivering antibody mimics over the TAT peptide. Additionally, the amount of LvlHA detected by anti-HA immunoblot was similar to that of LvI detected by anti-LF immunoblot.
The presence of a C-terminal HA tag further validated the presence of full-length Lvi -HA in the
cytosol.
59
a)
1.2D'
1.0
90.8-
0.40.2.
-M-LFN-DTA + PA
-+-TAT-DTA
0.-15
b)
-14
-13 -12 -11 -10 -9
-8
Log [Protein Concentration (M)]
-6
-7
Digitonin extracted
cc)
31
9'
V
V-1*
-t7
J~-I
kDa
49
38
anti-HA
28
17
14
6
anti-Erki /2
anti-Rab5
Figure 2.2.2. TAT peptide mediated translocation of DTA and antibody mimics. a) Protein
synthesis inhibition assay of CHO-Ki cells treated with varying concentrations of TAT-DTA for
30 minutes, in comparison to LFN-DTA plus 20 nM PA. The assay conditions were described in
Figure 2.1.1. b) Western blot analysis of cytosolic fractions extracted by digitonin from CHOKi treated with 2.5 pM TAT-HA-i to -3 and -4mut (1-3 and 4mut see Figure 2.1.1b) for 4 h (4'C
= incubation at 4*C instead of 37*C), in comparison to treatment with 250 nM Lvl-HA and 40
nM PA for 16 h. The cytosolic fraction was extracted using digitonin (~280,000 cells per lane).
The corresponding immunoblots of total lysates from cells treated by TAT-HA-i to -3 and - 4 mut
for 4 hours and 16 hours are shown in Figure 2.6.6.
60
2.2.5. Delivery of a tandem monobody for SH2 domain of Bcr-Abl
Our next question was to ask whether the delivered antibody mimics can refold and bind
to their targets inside the cytosol. Based on the protein synthesis inhibition assay, we confirmed
that DTA could correctly refold in the cytosolic environment after translocation. For the antibody
mimics, we chose to study the tandem 1OFN3 monobody (HA4-7c12), which binds with
nanomolar affinity to the Src homology 2 (SH2) domain of the oncoprotein Bcr-Abl.
First, we
sortagged this tandem monobody to LFN-DTA (LDv5) and used the protein synthesis inhibition
assay as a first measure to study the variant's translocation efficiency in chronic myeloid
leukemia (CML) K562 cells. The protein synthesis inhibition assay showed that PA translocated
LDv5 as efficiently as the LFN-DTA control (Figure 2.6.7). After confirming that the PA system
efficiently translocated the tandem monobody HA4-7c12, we sortagged it to LFN (Lv5, Figure
2.2.3a), which lacks the cytotoxic DTA protein, and studied if Lv5 could bind to Bcr-Abl in
K562 cells after translocation into the cytosol. To ensure that the binding affinity of the
monobody was not affected by the presence of LFN, which apart from initiating translocation
provided an important epitope for detection in the cytosol by western blot, we first measured the
binding affinity of Lv5 toward the Abl SH2 domain. We obtained a Kd value of 12 nM, similar to
that of the monobody by itself (6 nM, Figure 2.6.8). Next, we investigated if the delivered Lv5
could refold and bind to Abl kinase inside the cell. K562 cells were treated with Lv5 and PA, and
subjected to immunoprecipitation with an anti-Abl antibody linked to agarose beads. Proteins
eluted from the beads were subjected to immunoblot analysis with an anti-LF antibody. As
shown in Figure 2.2.3b, we observed a pull-down band corresponding to Lv5 when cells were
treated with Lv5 and PA, confirming that at least some of the monobody could properly fold
after reaching the cytosol and was functional to bind to intracellular Abl kinase. We also carried
61
out control experiments where PA[F427H] was used instead of PA to arrest translocation from
the endosome to cytosol. No band was observed under this condition, confirming cytosolic
access of Lv5 was critical for the binding. Finally, the cells were treated with the binding mutant
Lv5mut (HA4: Y87A; 7c62: Y62E/F87K) instead of Lv5. Similar amounts of Lv5 and Lv5mut
were delivered to the cell; however, the pull-down band was absent in the Lv 5 mut condition
(Figure 2.2.3b), indicating the binding interaction only occurred when the LFN variant contained
the functional binder. These results demonstrated that PA-delivered tandem monobody could
refold and bind to its target inside the cell.
62
a)
W
LPSTG
7c12
HA4
-
PA[F427H]
Lv5
Lv5mut
-
+
+
-
Input
IP: anti-Abi
+
+
PA
-
-
+
-
-
+
-
-
+
Lv5mut
+
+
-
-
-
PA[F427H]
Lv5
anti-Abi
anti-Abi
anti-LF
anti-LF
anti-GAPDH **mto
+
-
-
PA
+
b)
+
Lv5
MWMw
Figure 2.2.3. Delivery of Lv5 and binding of Lv5 to Ab kinase in K562 cells. a) Construct of
Lv5 containing HA4 (PDB: 3K2M), GS-rich linker, and 7c12 (PDB: 3T04). b) Western blot
analysis (left) of total cell lysate from K562 cells treated for 24 hours with 50 nM Lv5 or Lv5mut
in the presence of 20 nM PA or PA[F427H]. The lysate was then subjected to coimmunoprecipitation (right) by anti-Abl agarose beads. Cells treated with Lv5mut/PA or
Lv5/PA[F427H] served as negative controls for the co-IP.
63
Our next goal was to investigate the possibility of using the delivered tandem monobody
to perturb protein function and related signaling pathways in cancer cells. Overexpression of
HA4-7c 12 in K562 cells was reported to strongly inhibit kinase activity and induce apoptosis by
disrupting a critical intramolecular SH2-kinase domain-domain interaction. Despite the high
affinity and specificity of the monobody in targeting the allosteric module in Bcr-Abl and its
utility in fighting the Bcr-Abl drug resistance, Hantschel et aL indicated the intracellular delivery
was the biggest hurdle to push its practical application. 53 In order to test if PA-mediated delivery
could overcome this challenge, we used PA to translocate Lv5 into K562 cells. Based on the
linear relationship between the amount of protein loaded and the signal intensity of each band
detected by anti-LF antibody (Figure 2.6.9), we estimated a total of I ng Lv5 delivered into
~100,000 cells (Figure 2.6.10), that was ~10 fg or 110,000 molecules of Lv5 in each cell, giving
a cytosolic concentration of -80 nM. Although this concentration was above the Kd towards Abl
SH2 domain, we would not expect strong inhibition of Bcr-Abl kinase due to the high
concentration of Bcr-Abl inside K562 cells. As detected by western blot, monobody binding
resulted in modest reduction of activation loop (Tyr412) phosphorylation of Bcr-Abl (Figure
2.6.10). We then investigated the effect of this activity inhibition in inducing apoptosis of K562
cells. Using TUNEL staining that detects DNA fragmentation by labeling the terminal ends, we
observed apoptosis after K562 cells were treated with Lv5 and PA (Figure 2.2.4a). This effect
was not observed when PA[F427H] or Lv5mut were used. The amount of apoptotic cells caused
by delivered Lv5 was 20% of that caused by the small molecule imatinib (I pM, Figure 2.6.11),
which inhibits kinase activity by binding close to the ATP binding site of Bcr-Abl. The different
inhibition mechanism and the much higher concentration of imatinib may explain the difference
in the amount of apoptotic cells as compared to the Lv5 and PA treatment. In addition, the extent
64
of apoptosis caused by delivered Lv5 was also lower than that induced by overexpression of
HA4-7c12 as shown by Grebien et al. The high protein level of the monobody achieved by
overexpression and selection for only transfection positive cells overcame the challenge of
requiring high local concentration to interfere with the intramolecular interactions between the
SH2 domain and kinase domain in Bcr-Abl. With the modest inhibition of kinase activity and
induction of apoptosis by PA mediated delivery of Lv5, it serves as a proof-of-concept example
showing delivered monobodies can perturb the activity of an oncoprotein.
65
a)0._
8.54
PA
P+m597
PA + Lv5mut
PA + Lv5
0
IIi-
102
0.
lo,
0
PA[F427H]
P911
063PA[F427H]
PA[F427H + Lv5
+
Lv5mut
104-
C)
Lj_
10 3
0,
0 1&
10
0i 0S 2 103 101 101
PefCP-A
b)
0
ftrCP-A
102
1&~
PerCP-A
10.
0
24-
I
20
:
8 16
L
.J
J 12
D8
4-
T
A-
PA
+
-
+
-
+
-
-
+
Lv5
Lv5mut
-
0-
PA[F427H]
Figure 2.2.4. Monitoring of apoptosis of K562 cells treated with Lv5 and PA by TUNEL
assay. a) K562 cells treated with the indicated analogs (500 nM Lv5 or Lv5mut in the presence
of 80 nM PA or PA[F427H]) for 3 days and analyzed for apoptosis. 1 pM imatinib served as a
positive control. Representative dot plots from flow cytometry showed terminal
deoxynucleotidyl transferase (TdT) catalyzed BrdUTP incorporation into the DNA strand breaks
of apoptotic cells, which was detected by Alexa Fluor 488-labeled anti-BrdU antibody (FITC).
PerCP indicates the DNA fraction. b) Quantification of TUNEL-positive cells, where the
intensities were normalized to K562 cells treated with imatinib (set to 100%) and non-treated
cells (set to 0%). The dot-plots of imatinib-treated and non-treated cells are shown in Figure
2.6.11. Each data point represents an average of three independent experiments.
66
2.2.6. Delivery of an affibody for Raf-1
In a separate case, we investigated PA-mediated delivery of another binder based on
affibody. The affibody (ABRaf) was evolved to bind to human Raf- 1 (hRaf-1, Kd = 100 nM), 9 a
protein kinase of central importance in the MAPK/ERK proliferation pathway. Although ABRaf
was shown to inhibit the Ras/Raf interaction in vitro, it has not been tested for direct inhibition of
signaling through the MAPK pathway in cells. We transfected human embryonic kidney 293 T
(HEK293T) cells with ABRaf, and observed on average 37% reduction in phosphorylation levels
of MAPK (p-Erkl/2, downstream of Ras/Raf pathway) upon EGF activation (Figure 2.2.5a),
validating the cellular function of this binder in blocking MAPK pathway. We then conjugated
ABRaf to LFN (Lv6), and found that PA effectively delivered Lv6 to an estimated concentration
of 240 nM (~5 fg or 79,000 molecules per cell) in HEK293T cells (Figure 2.2.5b). We measured
the phosphorylation level of MAPK (p-Erkl/2) upon EGF activation in cells treated with Lv6
and PA or PA[F427H]. As shown in Figure 2.2.5c, the cells treated with Lv6 and PA showed
-25% reduction of p-Erkl/2 when comparing to EGF-activated untreated cells (P < 0.01, Figure
2.2.5c). PA[F427H] was used as a negative control and showed similar level of p-Erkl/2 as the
EGF-activated untreated cells (P = 0.27, Figure 2.2.5c). This result provides another example of
using PA to translocate functional binders to disrupt protein-protein interactions and related
signaling pathway in cells.
67
+
PA[F427H]
EGF
anti-pErki /2
+
-
+
+
+
-
+
anti-pErk1 /2
W
anti-Erki /2
-
-
Lv6
PA
-
+
+
+
+
a )pcDNA3-ABRaf
EGF
anti-Erki/2
Lv6
PA
PA[F427H]
anti-LF
rn
P= 0.27
1.2.
LPSTG
+
+
4 ng
w
-
-
IP< 0.01
1.0.
0.8.
- o+
0.6-
+
b)
anti-Erki /2
U
anti-Rab5
c
0.2.
n.n
.
I
W
Figure 2.2.5. Perturbation of the MAPK signaling pathway by PA mediated delivery
of an
affibody (Lv6) that targets Raf. a) HEK293T cells were transfected with pcDNA3-ABRaf
for
24 hours, starved overnight, treated with 5 ng mL~ 1 EGF for 7 minutes, lysed with
buffer
containing 1% NP-40, and subjected to anti-pErkl/2 immunoblotting analysis. The membrane
was stripped and re-blotted with anti-Erk1/2 antibody to serve as loading control. b)
Western blot
analysis of cytosolic fractions extracted by digitonin (as described above) from HEK293T
cells
treated with 500 nM Lv6 and 80 nM PA or PA[F427H] in serum-free medium for 12
hours
(-400,000 cells per lane). c) HEK293T cells were treated with 500 nM Lv6 and 80 nM
PA or
PA[F427H] in serum-free medium for 12 hours, treated with 5 ng mL- 1 EGF for 7 minutes,
lysed
and analyzed as described in (a). Bar graph represents the quantification of level
of
phosphorylation of Erkl/2 (pErkl/2) (n = 3), where each bar is corresponding to the lane
in the
western blot (top). The pErk1/2 band intensities were normalized to that of Erkl/2,
and
compared to cells treated with EGF (set to 1). Each data point represents an average
of three
experiments. P values were calculated from Student's t-Test.
68
2.3. Discussion
In this paper, we demonstrate the ability of PA/LFN to deliver antibody mimics to the
cytosol of cells and also the possibility of using the delivered binders to perturb critical proteinprotein interactions in cancer cells. Based on prior work with PA/LFN mediated delivery, the use
of this system to perturb protein-protein interactions was not precedented. Efforts have mainly
focused on the delivery of enzymes that can exert a strong biological effect at very low
concentrations, which is demonstrated by the LFN-DTA activity in the cell assays presented here.
However, for an antibody mimic binder to exert inhibitory effects on protein-protein interactions
inside cells, the concentration of the antibody mimic binder needs to reach a sufficient level that
is above the K4 as well as close to the concentration of competing endogenous binding proteins.
We aimed to answer the question of whether PA could not only efficiently deliver the antibody
mimics, but also transport enough cargo to perturb protein-protein interactions.
We first systematically investigated the translocation mediated by PA of four different
antibody mimics: all a-helical (affibody, DARPin), all P-sheet (monobody), or a-helical and
p-
sheet proteins (GB1). We did not assume these scaffolds would translocate efficiently because
prior investigations have indicated that certain C-terminal modifications of LFN or LFN-DTA can
abrogate translocation through PA. In particular, imaging studies have indicated that fluorescent
proteins fused to the C-terminus of LF significantly attenuate the efficiency of translocation.
Additional studies have shown DTA with an artificial disulfide or DHFR complexed with
methotrexate can arrest translocation when attached to the C-terminus of LFN. 56 Using the DTAbased protein synthesis inhibition assay, we found all four antibody mimics translocated as
efficiently as the positive control LFN-DTA. We are unaware of any system that permits the
facile delivery of antibody mimics ranging in structural diversity as shown here.
69
Our translocation studies began with the protein synthesis inhibition assay because it is
commonly used to probe anthrax toxin entry into cell cytosol thereby allowing us to compare our
findings to other reports. However, in order to use antibody mimics to perturb protein-protein
interactions, we needed to eliminate the interference of DTA. Therefore, we also investigated the
translocation of LFN-antibody mimics (Lvs) in the presence of PA. To facilitate the analysis of
Lv delivery, we made extensive use of a reliable western blot approach to monitor full-length
cargos in the cytosolic fraction. The cytosolic fraction was obtained using digitonin that
selectively permeabilizes the plasma membrane. For all experiments, we confirmed successful
extraction of cytosolic proteins from the rest of the cellular components by staining for the
presence of Erkl/2 and absence of Rab5. The successful extraction of cytosolic proteins was
further validated by the absence of Lvl in the digitonin-extracted fractions from cells treated
with Bafilomycin Al or PA[F427H]. Additionally, we observed no Lvl in the total cell lysate
under 4'C treatment condition, validating the effectiveness of trypsin digestion in removing
surface bound Lv, leaving only intracellular proteins for detection. We detected the conjugates of
antibody mimics to LFN by the anti-LF antibody, and confirmed the presence of the antibody
mimics at the C-terminus of LFN by the correct molecular weight. Further evidence that fulllength material translocated into the cytosol was the detection of Lvi -HA by the C-terminal HA
tag, which gave a similar amount of material detected by the anti-LF antibody.
We further used this reliable digitonin extraction and western blot method to gauge the
amount of material delivered into the cell cytosol. Based on the linear relationship between the
amount of loaded protein and the signal intensity from the anti-LF immunoblot, we were able to
estimate the amount of material delivered by comparing the immunoblot signal to a known
amount of protein loaded on the same blot. Since the antibody mimics do not interfere with
70
translocation through PA pore, the amount delivered to the cytosol is dependent on the number
of PA receptors on cells, which are present on most human cells. Such receptors have been
reported to range in copy number from 2,000 to 50,000 and are boosted in certain cancer cells.5 7
Theoretically, one round of translocation for cells harboring 50,000 receptors would give
~20,000 molecules in one cell assuming every seven receptors deliver three copies of LFN-CargO
variant. Therefore we estimate, from the use of western blot quantification, that after multiple
rounds of translocation we achieve mid-nanomolar concentrations of cargo in the cytosol when
accounting for the cell volume.
With knowledge of the amount of material delivered to the cytosol, we next studied
whether a functional antibody mimic binder can properly refold after cytosolic delivery since
translocation through PA pore requires protein unfolding. Based on the co-immunoprecipitation
of the monobody with the anti-Abl antibody, we confirmed that the monobody correctly refolds
in the cytosolic environment after translocation. The efficiency of refolding and the portion of
functional protein is presumably dependent on the cytosolic stability or degradation rates of these
variants. For the binders based on monobody and affibody, we observed the functional inhibition
of Bcr-Abl kinase and disruption of the MAPK pathway, respectively. Both results again
provided evidence that the antibody mimics properly fold and function after translocation into
the cytosol. Due to the fact that the amount of tandem monobody delivered was not significantly
higher than the Kd for the Bcr-Abl target, the extent of apoptosis caused by Lv5 was not as
robust as that achieved by overexpression of the tandem monobody, where its cytosolic
concentrations reach a much higher level. The mild biological effect caused by Lv5 is probably
due to the high concentration of AbI kinase and also the difficulty to interfere with the
intramolecular domain-domain interaction within Abi kinase. In contrast, even though the
71
amount of affibody binder for hRaf- 1 delivered was not much higher than the Kd values for its
target, the inhibition of the MAPK pathway by Lv6 was close to that achieved by overexpression
of the affibody binder. This effect could be attributed to the lower target concentration and the
higher efficiency to disrupt Ras/Raf intermolecular interaction, as compared to the Bcr-Abl
target. The efforts to deliver more material or to increase the potency of the delivered cargo for
its target are additional challenges in biomolecular delivery.
The high adaptability and promiscuity of the PA transporter enabled delivery without the
need for protein engineering or screening, unlike delivery methods such as CPPs where a range
of sequences or linkers need to be screened for efficient delivery of each cargo. In the case of
CPPs, the stability and identity of the peptide transduction sequence may lead to significant
differences in delivery efficiency, and oftentimes strong adherence to the cell surface and
endosomal membranes blocks cargo escape into the cytosol. For example, it was reported that
CPP fusions to DTA were not able to impart delivery into the cell,5 1 even at concentrations a
thousand times more than that required for PA-mediated translocation; we confirmed this result.
Additionally, we investigated the efficiency of the TAT peptide in the delivery of the antibody
mimics. We found that even after treatment of the same cell lines with ten-fold more material,
the TAT peptide was not able to deliver any of the four antibody mimics. These results
demonstrate the significant improvement in delivery efficiency by PA over the TAT peptide.
58
Additionally, due to the highly inefficient endosomal escape for delivery into the cytosol, ' 59
most delivery methods use high concentrations of these components that can give rise to
background cellular toxicity. We studied if the delivery components PA or LFN by themselves or
in combination are toxic to cells. Under the conditions reported here, we did not see any toxicity
72
associated with PA/LFN, based on a TUNEL apoptosis assay (Figure 2.2.4) or MTS assay
(Figure 2.6.12).
In summary, for the first time, we report PA-mediated delivery of antibody mimics into
the cell cytosol and successful disruption of critical protein-protein interactions inside cells. As
one example, we found that an SH2-binding tandem monobody can be delivered and function as
an inhibitor of the oncoprotein Bcr-Abl inside cancer cells. We also demonstrated the inhibition
of the Ras/Raf interaction by an affibody, thereby blocking the MAPK pathway that plays a
central role in the control of cell proliferation, survival, and growth. The delivery of antibody
mimics to the cytosol of cells as indicated by DTA activity and western blot, together with the
functional tandem monobody binder to the oncoprotein Bcr-Abl and affibody binder to hRaf-1,
supports our belief that the PA-mediated protein delivery system designed by nature will
significantly expand the biomolecular delivery toolbox. Our future efforts are to increase the
amount of material delivered or to deliver more potent antibody mimics. This delivery platform
provides new possibilities to apply modern intrabody technology to disrupt processes in cells.
73
2.4. Experimental
2.4.1. Materials
All reagents were purchased from Sigma-Aldrich and Life Technologies, or as otherwise
indicated. The following primary and secondary antibodies were used goat anti-LF (bD-17, Santa
Cruz Biotechnology), rabbit anti-HA (Sigma), rabbit anti-Abi and agarose beads (K-12, Santa
Cruz Biotechnology), rabbit anti-pAbl (pTyr412, Cell Signaling), rabbit anti-Erkl/2, rabbit antiphospho-Erkl/2 (Thr202/Tyr2O4, Cell Signaling), rabbit anti-Rab5 (Cell Signaling), goat antirabbit IRdye 800CW (LI-COR Biosciences), donkey anti-goat IRdye 680LT (LI-COR
Biosciences).
2.4.2. One-pot sortagging reaction using Staphylococcus aureus SrtA*
Enzyme-mediated ligation using SrtA* was utilized to ligate the proteins to His 6 -SUMO-
LFN-DTA-LPSTGG-His
5
or His 6 -SUMO-LFN-LPSTGG-His
6
as previously described (Figure
2.6.1).45 For all ligations, 50 jtM LFN-DTA-LPSTGG-His 5 or LFN-LPSTGG-HiS 6 , 5 iM SrtA*,
and 100 to 500 pM G 5-protein were incubated with Ni-NTA beads in sortase buffer (10 mM
CaCl 2 , 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 30 min at RT nutating. Immediately after 30
min, the Ni-NTA beads, which bound any unreacted starting material, SrtA*-His 6 , His 6 -SUMO,
and GG-His 6 , were spun down at 4 'C to minimize the formation of the hydrolyzed side product.
The supernatant containing the sortagged product was collected. The beads were washed three
times. The supernatant and three washes were followed by an extra step of gel-filtration to
separate excess oligoglycine protein reactants. SDS-PAGE gel and LC-MS were used to confirm
the purity of the products (Figure 2.6.2, Table 2.6.2, and LC-MS traces). The isolated yields of
sortagging ligations were listed in Table 2.6.3. A list of all the protein variants is supplied in
Table 2.6.5.
74
2.4.3. Protein synthesis inhibition assay
CHO-K1 cells were maintained in F-12K media supplemented with 10% (v/v) fetal
bovine serum at 37 'C and 5% CO 2. The cells were plated in 96-well plate at a density of
approximately 30,000 per well 16 hours prior to the assay. The LDvs were prepared in ten-fold
serial dilutions in 50 ptL, and added 50 tL of F-12K media containing 20 nM protective antigen
.
(PA8 3). The 100 p.L samples were added to CHO-Ki cells for 30 minutes at 37 'C and 5% CO 2
The cells were washed three times with PBS and 100 [LL leucine-free F-12K medium
supplemented with 1 pCi mL)' 3H-leucine (Perkin Elmer, MA) was added for 1 h at 37 'C 5%
CO 2 . The cells were washed three times with PBS and suspended in 150 tL of scintillation fluid.
3H-Leu
incorporation into cellular proteins was measured to determine the inhibition of protein
synthesis by LFN-DTA. The scintillation counts from cells treated with only PA were used as
control value for normalization. Each experiment was done in triplicate. The data were fitted
using Origin8 using Sigmoidal Boltzmann Fit using equation:
Y=
A2+
A, -A.
where xO represents the logEC5o values (Table 2.6.4).
2.4.4. Uptake of Lvs or TAT-HA-1 to -4 in CHO-K1 cells
CHO-Ki cells were plated in 12-well plates 16 hours prior to the treatment. For Lvs, cells
were treated with 250 nM Lv in the presence of 40 nM PA in complete medium overnight at
37 'C and 5% CO 2 . For TAT-HA-antibody mimic constructs, cells treated with 2.5 pM TATHA-i to -4 and - 4mut in serum free medium for 4 h at 37 'C and 5% CO 2 . Translocation controls
included PA[F427H] instead of PA, addition of 200 nM Bafilomycin Al, or incubation at 4 'C
instead of 37 'C.
75
2.4.5. Cytosolic protein extraction and whole cell lysate preparation
After uptake of the antibody mimics, cells were washed with PBS, detached and digested
with 0.25% Trypsin-EDTA for 5 minutes at 37 'C to remove surface bound proteins, then
washed twice with PBS. The cell pellets were then subjected to cytosolic extraction or whole cell
lysate preparation. For cytosolic protein extraction, 1 x 106 cells were resuspended in 100 uL of
50 pg mL 1 digitonin in 75 mM NaCl, 1 mM NaH 2 PO 4, 8 mM Na 2HPO 4, 250 mM sucrose
supplemented with Roche protease inhibitor cocktail for 10 min on ice, and centrifuged for 5
minutes at 13,000 rpm. For whole cell lysate, cells were lysed in IP lysis buffer (25 mM Tris,
150 mM NaCl, 1% v/v NP-40, pH 7.5) supplemented with Roche protease inhibitor cocktail on
ice for 30 minutes and centrifuged for 10 minutes at 13,000 rpm. Both supernatants were
collected for blotting and analysis.
2.4.6. Western blot
Transfer was done with TE 70 Semi-Dry Transfer Unit (GE) onto nitrocellulose
membrane (Whatman) using buffer containing 48 mM Tris, 39 mM glycine, 0.0375% SDS, 20%
methanol. The membrane was blocked at room temperature for 2 hours with LI-COR blocking
buffer and was then incubated with goat anti-LF antibody or rabbit anti-HA in LI-COR blocking
buffer overnight at 4 'C. The membranes were washed with TBST (50 mM Tris, 150 mM NaCl,
0.1% Tween-20) and blotted with secondary antibody conjugated with IRdye from LI-COR and
imaged with LI-COR Odyssey Infrared Imaging System. The western blot images were analyzed
and quantified using the Image Studio Lite program (LI-COR).
2.4.7. Co-immunoprecipitation of Lv5 with Abl kinase
K562 cells were treated with 40 nM PA or PA[F427H] and 50 nM LFN-antibody mimic
for 24 hours. Approximately I x106 cells were trypsinized, washed with PBS, frozen at -80 'C,
76
and then lysed in 500 gL IP lysis buffer supplemented with Roche protease inhibitor cocktail on
ice for 30 min. After centrifugation at 13,000 rpm for 15 min, 450 pL lysates were incubated
with 12.5 pg anti-Abi agarose beads for 4 hours. The resulting immune complexes were washed
three times with lysis buffer and once with lysis buffer without NP-40. The bound proteins were
eluted with 0.2% SDS and 0.1% Tween 20 and subjected to SDS-PAGE separation. Standard
immnuoblotting was performed using anti-Abl or anti-LF antibody.
2.4.8. TUNEL assay with Lv5 in K562 cells
K562 cells were plated in a 24-well plate at a density of 150,000 cells mUl and treated
with 500 nM LFN-antibody mimic and 60 nM PA or PA[F427H] in serum-free medium for 1 day.
FBS (10%) was added to the medium and the cells were treated for additional two days. Cells
treated with 1 pM imatinib served as the positive control. The cells were fixed with 3.2%
paraformaldehyde, treated with in 90% methanol on ice or stored at -20 'C, followed by TUNEL
staining according to the manufacturer's instructions (Life Technologies).
2.4.9. Transfection of HEK 293T with pcDNA3-ABRaf
HEK 293T cells were plated in 24 well plates overnight to reach ~90% confluency. The
cells were then transfected with the plasmid pcDNA3-ABRaf-GFP using Lipofectamine 2000
(Life Technologies). The medium was changed after 5 hours of transfection. The total
transfection time was 24 hours. For the pErkl/2 analysis, the cells were starved for 12 hours
before they were treated with 5 ng mL-' EGF for 7 minutes, washed with cold PBS, and lysed in
plate with IP lysis buffer containing 1% NP-40, Roche protease inhibitor cocktail, and Roche
phosSTOP. The lysates were subjected to SDS-PAGE separation, transferred onto nitrocellulose
membrane, and blocked with 5% BSA in the presence of Na 3VO 4 and NaF. The immunoblotting
with anti-pErkl/2 antibody (Cell Signaling) was performed in 3% BSA in the presence of
77
Na 3VO 4 and NaF overnight. After stripping the membrane with Restore Plus Western Blot
Stripping Buffer (Thermo Scientific), the membrane was blocked again and immunoblotted with
anti-Erkl/2 antibody (Cell Signaling).
2.4.10. Delivery of Lv6 into HEK 293T cells
HEK 293T cells were plated in 24 well plates overnight to reach ~80% confluency. The
cells were then treated with 80 nM PA and 500 nM Lv6 in serum-free medium for 12 hours. For
detection of cytosolic Lv6, the cells were detached with trypsin, washed with PBS, and
resuspended in 100 ptg mLU digitonin for 10 minutes as described above. For detection of
pErkl/2, the cells were treated with 5 ng mL- EGF for 7 minutes, washed with cold PBS, lysed
in plate with IP lysis buffer containing 1% NP-40, Roche protease inhibitor cocktail, and Roche
phosSTOP. The immunoblotting with anti-pErkl/2 and anti-Erkl/2 were as described above.
2.4.11. Construction of plasmids for recombinant proteins and transfection
The gene for Abl-SH2 was purchased from Addgene (pDONR223-ABL1, 23939).0 The
genes for affibody," ABRaf, DARPin," and 7cl212 were purchased from DNA2.0 (Menlo Park,
CA). The gene for 1OFN3 was kindly provided by K. Dane Wittrup (MIT, Department of
Biological Engineering). The 1OFN3 mutant, HA463 was generated by mutagenesis. The amino
acid sequences are shown in Figure 2.6.13. The constructs, pET SUMO-G 5-affibody, pET
SUMO-G 5 -lOFN3, and pET SUMO-SH2, were prepared using the Champion pET SUMO
protein expression system (Life Technologies). AccuPrime Taq DNA polymerase (Life
Technologies) was used to PCR amplify the DNA using the primers listed in Table 2.6.1. pET
SUMO-G 5 -HA4 with two restriction sites after the stop codon (BamHI and XhoI) was generated
by mutagenesis based on pET SUMO-G 5 -1OFN3. 7c12 was PCR amplified, digested with
BamHI and XhoI, and ligated in pET SUMO-G 5 -HA4 vector with a 20-amino acid linker
78
GGSG(GGSGG) 3G between HA4 and 7c12. For pET SUMO-LFN-DTA (C186S)-LPSTGG-His 5
and pET SUMO-LFN-LPSTGG-His 6, a G2S mutation was introduced to minimize undesired loss
of Methionine. The plasmid for transfection of eGFP-ABRaf was generated by a two-step
ligation. eGFP was inserted into pcDNA3 using NotI and BamHI restriction sites and ABRaf
was then inserted using BamHI and XhoI restriction sites, with a GSGGGGSGGGGG between
eGFP and ABRaf.
2.4.12. Protein expression and purification
His6-SUMO-Gs-affibody, His 6-SUMO-G 5-GB 1, His 6-SUMO-G 5-DARPin, His 6 -SUMOG 5-HA4, His 6 -SUMO-G 5 -HA4-7c 12, His6-SUMO-G 5 -HA4-7c 1 2mut and His6 -SUMO-SH2 were
expressed in F. coli BL21 (DE3) cells in 1 L LB culture and approximately 5 g of cell pellet was
obtained for each. HiS 6-SUMO-LFN-DTA (C186S)-LPSTGG-His 5 , His 6 -SUMO-LFN-LPSTGGHis 6 , His 6-SUMO-LFN-DTA (C186S), SrtA*-His 6 , WT anthrax protective antigen (PA), and
PA[F427H] were expressed in . coli BL21 (DE3) cells at New England Regional Center of
Excellence/Biodefense and Emerging Infectious Diseases (NERCE). For each purification,
approximately 40 g of cell pellet was lysed by sonication in 100 ml of 50 mM Tris-HCl, 150 mM
NaCl, pH 7.5 buffer containing 200 mg lysozyme, 4 mg Roche DNAase I, and 2 tablet of Roche
protease inhibitor cocktail. The suspension was centrifuged at 17,000 rpm for 40 minutes. The
supernatant was loaded onto three 5 ml HisTrap FF crude Ni-NTA column (GE Healthcare, UK)
and washed with 100 mL of 20 mM Tris-HCl, 150 mM NaCl, at pH 8.5 and 100 mL of 40 mM
imidazole in 20 mM Tris-HCl, 500 mM NaCl, at pH 8.5. The protein was eluted from the
column with buffer containing 500 mM imidazole in 20 mM Tris-HCl, 500 mM NaCl, pH 8.5,
and buffer exchanged into 20 mM Tris-HCI, 150 mM NaCl, pH 8.5 using a HiPrep 26/10
79
Desalting column (GE Healthcare). WT PA and PA[F427H] was overexpressed in the periplasm
of E. coli BL21 (DE3) cells and purified by anion exchange chromatography."
2.4.13. Synthesis of TAT peptide and native chemical ligation (NCL) of TAT-DTA
The TAT peptide (YGRKKRRQRRRLLG)
2
was synthesized by Fmoc solid phase
peptide synthesis on hydrazide resin (calculated mass: 1856.1 Da; observed monoisotopic mass:
1856.1
0.1 ). The crude peptides were purified by preparative RP-HPLC on Agilent Zorbax
300SB C18 column (9.4 x 250 mm, 5 jim). The hydrazide C-terminus was converted to an MPAA
thioester according to the previously published method by Fang, et al.65 TAT-MPAA was ligated
to 'C-DTA by NCL using 1.4 mM TAT-MPAA and 270 ptM 1 C-DTA in 100 mM phosphate
buffer pH 7.0, 150 mM NaCl, 20 mM TCEP in 4 h. The reaction was quenched with 200 mM
sodium 2-mercaptoethane sulfonate and desalted to 20 mM Tris buffer pH 7.5, 150 mM NaCl.
To alkylate the cysteine, 50 mM bromoacetamide was added to react for 10 minutes. The
reaction was again quenched with 200 mM sodium 2-mercaptoethane sulfonate and buffer
exchanged into 20 mM Tris buffer pH 7.5, 150 mM NaCl to obtain TAT-DTA protein.
2.4.14. Synthesis of TAT-HA-LPSTGG peptide and sortagging to G 5 -proteins
The TAT-HA-LPSTGG peptide (AGYGRKKRRQRRRGYPYDVPDYAGLPSTGG) was
synthesized by Fmoc solid phase peptide synthesis on aminomethyl resin (calculated mass:
3397.8 Da; observed average mass: 3397.8
0.1). The crude peptides were purified by
-
preparative RP-HPLC on Agilent Zorbax 300SB C18 column (9.4 x 250 mm, 5 pm). The G5
proteins (1 - 4 and 4mut) were conjugated to TAT-HA-LPSTGG using 10 [tM SrtA* in sortase
buffer (10 mM CaCl 2, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 1 h at RT. For each reaction,
-
the TAT-HA-LPSTGG peptide (230 - 600 jM) was used in two-fold excess compared to the G5
protein (115 - 300 pM). After 1 h, all of ligation reactions were diluted approximately 20-fold
80
with 20 mM Tris-HCl pH 8.5. The samples were purified by anion exchange chromatography
(GE HiTrap
Q HP)
at 0-30% 1 M NaCl in 20 mM Tris-HCl pH 8.5 over 100 mL. The fractions
containing the ligated product were concentrated using a 3 kDa or 10 kDa concentrator. For the
TAT-HA-I (affibody) ligation, the product eluted with TAT-HA-LPSTGG peptide in the flow
through, which was subsequently concentrated and buffer exchanged three times to remove
excess peptide. Purity of proteins was analyzed by LC-MS and SDS-PAGE gel (Figure 2.6.5).
2.5. Acknowledgements
This research was generously sponsored by MIT start-up funds, MIT Reed Fund, and
Damon Runyon Cancer Research Foundation award for B.L.P, and National Science Foundation
Graduate Research Fellowship for A.E.R. We would like to thank R. J. Collier (Harvard) for his
encouragement, support, and some of the laboratory equipment. We are indebted to the NERCE
facility (grant: U54 A1057159) for expressing the toxin proteins. Provisional patent applications
covering material reported in this manuscript were filed by MIT-TLO. We thank Jingjing Ling,
Mike Lu, Dan Chinnapen, and Daphne van Scheppingen for their helpful discussions. We also
thank Dane Wittrup for providing the fibronectin clone.
81
2.6. Appendix
H6-SUMO-
LPSTGG-H 5
D@i)
30 min, RT
H-SUMO
+
LPSTGG-H5
G
30 min, RT
NI-NTA
SrtA*
40 _(
His tagged reagents
LPSTG5-
Figure 2.6.1. One-pot sortagging reaction. N-terminal small ubiquitin-like modifier (SUMO)
was first cleaved from the protein substrate, His6-SUMO-LFN-DTA-LPSTGG-His , using 1
g
5
SUMO protease per mg of protein substrate at RT for 30 minutes to expose the native
Nterminus of LFN-DTA-LPSTGG-His 5 , then the sortagging reaction was carried out as described.
82
i nv 1 1nval
1 Dv4
Lnvs LnvR
Lv1
Lv2 Lv3
Lv4,Ut Lv5
Lv6
Figure 2.6.2. Coomassie stained SDS-page gel of LDvs and Lvs obtained after sortagging and
purification.
83
-1000-2000
1OFN3
-4-HA4
-30002 -400
-5000-6000
z -7000-8000
20
30
40
50 60 70 80
Temperature (*C)
90
100
Figure 2.6.3. Thermal stability of I0FN3 (4) and HA4 (4mut) was monitored by circular
dichroism (CD) spectroscopy. CD spectra were recorded on an Aviv model 202 instrument at 25
'C. A 1 mm path length cell was used. The proteins were prepared by dissolving 0.06 mg in 50
mM sodium sulfate and 5 mM Tris-HCl at pH 8.5. The molar ellipticity (0 in deg cm 2 dmol-')
was calculated by [N]x = Oobs x 1/(10 lcn), where Oobs = observed ellipticity at k, 1 = path length
(cm), c = concentration of protein (M), n = # of amino acids. To monitor the thermal
stability,
the ellipticity at 216 nm was measured from 25 'C to 95 'C.
84
3
4
4t
+
+
+
2
+
Lv 2 (4 ng)
PA
anti-LF
anti-Erki/2
anti-Rab5
Figure 2.6.4. Western blot analysis of delivered Lvs. CHO-Ki cells were treated with 250 nM
Lv2-4 and 4 mut in the presence of 40 nM PA for 12 hours. After treatment, cells were detached
with trypsin and the cytosolic proteins were extracted with digitonin buffer and analyzed by
immunoblotting. Lane 1 contains 4 ng of Lv2.
85
TAT-HA
1
2
3
4 4mut
kDa
62
49
38
14
-
28
6
Figure 2.6.5. Coomassie stained SDS-PAGE gel of TAT-HA-I to -4 and -4mut (1 pg).
86
Total cell lysate
a)
1
TAT-HA4
3
2
4,U1
b)
2 Lv1 -HA
4*C + PA
Ud
Total cell lysate
4 ng
Lvl-HA
TAT-HA-
-
1
2
3
4,t
4*C
2
4 ng
2
38
anti-HA28
17
14
anti-HA
(increase
intensity)
17j
14
anti-HA
I ng
TAT-HAanti-Erk1/2
%004
anti-Rab5
anti-Erkl/2
anti-Rab5
Figure 2.6.6. Cellular uptake of TAT-HA-I to -4 and -4mut. CHO-KI cells were treated with 2.5
pM TAT-HA-1 to -3 and -4mut in serum-free medium for 4 hours (a) or 16 hours (b) then lifted
with trypsin and washed with PBS. The total lysates were analyzed by immunoblotting. Inset
showed even with increased anti-HA intensity there was little amount of TAT-HA-2 and -3
detectable (a).
87
LDv5
1.2-
LPSTG
HA4
7c12
1.00-8-
0.8LF.-DTA
-4LDv5
5 0.2
U-
0.0
-16
-14
-12
-10
-8
Log [Protein Concentration (M)I
-6
Figure 2.6.7. The level of protein synthesis inhibition in K562 cells treated with varying
concentrations of LFN-DTA or LDv5 in the presence of 20 nM PA for 4 hours.
88
a
b
Lv5
2500- -100
2000-
180C
nM
-50 nM
-25 nM
- 12.5 nM
1400
1500-
HA4-7c12
-100 nM
-5o nM
-
1000-
.
CE
1000-
600-
500-
2000-
0-
-100
0
100
Time (s)
200
300
40
-100
110
200
300
400
Time (s'
Figure 2.6.8. SPR curves for SUMO-SH2 and varying concentrations of Lv5 (a) or HA4-7cl2
(b). SPR measurements were taken in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20, and
3 mM EDTA with a BIAcore3000 instrument. The SUMO fusion of Abl SH2 domain was
immobilized to an ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide activated
CM5 sensor chip. Purified HA4-7c 12 or Lv5 was then flowed over the sensor chip at a rate of 20
pl min-' and the association and dissociation kinetics were monitored. Surface was regenerated
by 10 mM glycine pH 2.0 between different runs. Data was fit to a 1:1 Langmuir binding model
using multiple kinetic traces with the BlAevaluation software. The Kd was calculated as 12 nM
for Lv5 (a) and 6 nM for HA4-7c 12 (b).
89
Lv5 (ng)
0.1
0.25
1.0
2.5
5.0
7.5
anti-LF
5000000.
Lv5
40000003000000
8 200000010000000
0
1
2
3
4
5
Lv (ng)
6
7
8
Figure 2.6.9. Linear relationship between signal intensity of each band and the amount of
protein loaded (Lv5: y = 378000x - 137000 R2 = 0.989). The western blot images were analyzed
in Image Studio Lite program (LI-COR Biosciences). The signal intensity of each band
was
compared to that of 0.1-7.5 ng of pure protein loaded on the same blot. The amount of protein
in
each lane was calculated based on the linear relationship between the signal intensity
and the
amount of protein.
90
b
a
4 ng Lv5
PA
PA[F427H]
Lv5
+
1.2
Lv5mut
-
+
0 U5
-
*Lv5mut
-
0.8
anti-pAbI
(pY4l 2)
anti-LF
0.4
anti-GAPDH
Z
+ PA
+ PA[F427H]
Figure 2.6.10. Phosphorylation analysis of Bcr-Abl. a) Western blot analysis of Lv5 and BcrAbl pY421. b) The Bcr-Abl pY412 intensity was normalized to GAPDH and was set to 1 in the
non-treated condition. K562 cells were treated with 500 nM Lv5 and 60 nM PA or PA[F427H]
in serum-free medium for 1 day and FBS (10% v/v) was added to treat the cells for an additional
day. Cells were lysed and immunoblotted with antibodies recognizing Abl phosphorylated on
Tyr412 or LF. The amount of Lv5 on the blot was quantified as 1 ng (a). The estimation of the
molecules per cell and concentration of Lv5 in the cell was based on cell number of ~ 100,000
cells and cell volume of- 2 pL.
91
I-
0652
Cell
45 2..
Imatinib
101054
104
10 3
103
-
-
104
-
103
10
10 2
-
IL
102
03
0,2
3
0
104
102
PerCP-A
105
0
102
103
PerCP-A
104
105
Figure 2.6.11. Apoptosis measurement of K562 cells that were not treated or treated with 1 RM
imatinib for 3 days. PerCP indicates the DNA fraction and FITC reports the TUNEL positive
fraction by detecting the AlexaFluor 488 dye-labeled anti-BrdU antibody.
92
120-
120-
100.
100.
-8080
60.
40
-11-LFN-DTA only
-4-LFN-DTA + 20 nM PA
2
>
60
2
40-
-0-Lv1 +20 nM PA
-4-Lv only
orgy
-PA
2
20
20-
0
1E-4
1E-3
0.01
0.1
Protein Concentration (nM)
10
100.1000
Protein Concenration (nM)
Figure 2.6.12. MTS cell viability assay. CHO-Ki cells were plated in a 96-well plate at 2,500
cells per well and treated with LFN-DTA or Lvi with or without PA, or with PA at the indicated
concentration for 48 hours. CellTiter 96@ Aqueous One Solution Cell Proliferation Assay
(Promega, 10 ptL) was added to each well and incubated for 1 hour according the manufacturer's
instructions. The absorbance at 490 nm was normalized to 1 nM LFN-DTA + PA (set as 0) and
untreated cells (set as 100).
93
Affibody
Val 'AspAsnLysPheAsnLysGuGnGln 0 AsnAlaPheTyrGluleLeuHisLeuPro 2O
Asn LeuAsnGluGluGlnArgAsnAlaPhe
Ser 4 1AlaAsnLeuLeuAlaGluAlaLysLys
30 lleGlnSerLeuLysAspAspProSerGln 40
50LeuAsnAspAlaGlnAlaProLys
GB1
Met]ThrTyrLysLeulleLeuAsnGlyLys 0 ThrLeuLysGIyGluThrThrThrGluAla
20
44
Val AspAlaAlaThrAlaGluLysValPhe 30 LysGlnTyrAlaAsnAspAsnGlyValAsp
Gly 4'GluTrpThrTyrAspAspAlaThrLys
50 ThrPheThrValThrGlu
DAPRin
Ser'AspLeuGlyLysLysLeuLeuGluAlal0 AlaArgAlaGlyGlnAspAspGluValArg
21
Ile LeuMetAlaAsnGlyAlaAspValAsn
30
His 4LeuAlaAlaPheLeuGlyHisLeuGlu
50IleVatGIuValLeuLeuLysTyrGlyAla 60
AlaTyrAspAspAsnGlyValThrProLeu
Asp 61ValAsnAlaAlaAspSerTrpGyThr 70 ThrProLeuHisLeuAlaAlaThrTrpGy
20
40
80
His 81LeuGlulteVaIGluValLeuLeuLys oHisGlyAlaAspValAsnAlaGtnAspLysloo
Phe 10 1GlyLysThrAlaPheAspIIeSerIle' ""AspAsnGlyAsnGluAspLeuAlaGlulle
Leu1 GnLysLeuAsn
1OFN3
Val SerAspValProArgAspLeuGluVallValAlaAlaThrProThrSerLeuLeulle
20
Ser TrpAspAlaProAlaValThrValArg 0 TyrTyrArgleThrTyrGlyGluThrGly
40
Gly 4 1AsnSerProValGlnGluPheThrVal 50 ProGlySerLysSerThrAlaThrIleSer6 0
Gly 6 LeuLysProGlyValAspTyrThrlle 0 ThrValTyrAlaValThrGlyArgGlyAsp
94
20
Ser8 1ProAlaSerSerLysProlleSerl le 9 AsnTyrArgThrGlulleAspLysProSer
00
Gin' 01'
HA4
Val' SerSerValProThrLysLeuGluVal '0 ValAlaAlaThrProThrSerLeuLeule
20
Ser2 1TrpAspAlaProMetSerSerSerSer 30ValTyrTyrTyrArgleThrTyrGlyGlu
40
Thr 41GlyGlyAsnSerProValGlnGluPhe
50
ThrValProTyrSerSerSerThrAlaThr60
Ile 6 1SerGlyLeuSerProGlyValAspTyr 7 0 ThrIleThrValTyrAlaTrpGlyGluAsp 80
Ser 8 AlaGlyTyrMetPheMetTyrSerPro 9 lteSerIleAsnTyrArgThr
7c12
Gly' GlySerGlySerSerValSerSerVal 0 ProThrAsnLeuGluValValAspAlaThr 20
Pro 2 1ThrSerLeuLys1leSerTrpAspAla 3 0TyrTyrSerSerTrpGlnAsnValLysTyr40
Tyr 4 t ArgIleThrTyrGlyGluThrGlyGly 5 "AspSerProValGlnGluPheThrValProO
Gly 61TyrTyrSerThrAlaThrIeSerG ly 7 LeuSerProGlyValAspTyrThrIleThr 80
Val 8 'TyrAlaTyrAspThrPhePheProGly 90 TyrGluProAsnSerProlleSerIleAsn
00
Tyr'0 1ArgThr
ABRaf
Val' AspAsnLysPheAsnLysGluValAsn 0 LeuValAlaAspG lu1leTrpLeuLeuPro 20
Asn2 ' LeuAsnAsnGlnGlnValTrpAlaPhe 30 IleThrSerLeuLysAspAspProSerG In40
Ser4' AlaAsnLeuLeuAlaGluAlaLysLys 50 LeuAsnAspAlaGlnGluProLys
Figure 2.6.13. Protein sequences
95
Table 2.6.1. PCR primers
1 OFN3 fwd
5'-GGCGGTGGCGGTGGCGTTTCTGATGTTCCGAGGGAC-3'
1 OFN3 rev
5'-TAACTGGGATGGTTTGTCAATTTCTGTTC-3'
Affibody fwd
5'-GGCGGTGGCGGTGGC
GTCGACAATAAGTTCAATAAAGAACAACAG-3'
Affibody rev
5'-TTACTTCGGCGCTTGTGCG-3'
GB 1 fwd
5'-GGCGGTGGCGGTGGCATGACGTACAAACTGATCCT
GAACGG-3'
GB1 rev
5' ttaTTCGGTTACCGTGAAGGTTTTGG-3'
DARPin fwd
5'-GGCGGTGGCGGTGGATCCGATCTGGGCAAGAAAC-3'
DARPin rev
5'- ttaGTTCAGTTTCTGCAGAATCTCC-3'
SH2 fwd
5'-TCCCTGGAGAAACACTCCTGGTACC-3'
SH2 rev
5'-TTAGTTGCGCTTTGGGGCTG-3'
7c 12 fwd
5'-TTTTTGGATCCGGCGGCGGTTCTGGTGGCGGTGGTAGCG-3'
7c12 Rv
5'- TTTTTCTCGAGTTAGGTGCGATAATTGATGCTAATCGG-3'
96
Table 2.6.2. Observed molecular masses of expressed protein constructs when analyzed by LCMS
Calculated MW
Observed MW (Da)
Protein
(Da; average)
SrtA*-His 6
19214.6
0.4
19214.5
LFN-DTA
52047.0
0.4
52046.2
66700.0
0.4
66700.1
45289.8
0.4
45289.8
WT PA
83754.1
0.4
83751.6
PA[F427H]
83742.4
0.4
83738.3
Affibody
6925.8
0.2
6925.6
GB1
6481.4
0.2
6481.0
DARPin
13701.5
0.2
13701.3
10FN3
11022.4
0.2
11022.2
HA4
10977.1
0.2
10977.1
HA4-7c12
23244.1
0.4
23244.2
HA4-7c12mut
23099.1
0.4
23099.1
SUMO-SH2
24626.6
0.4
24626.4
ABRaf
6864.9
His 6-SUMO-LFN-DTA-LPSTGG-His
His 6 -SUMO-LFN-LPSTGG-His
C-DTA
6
5
20840.60
DTA
20897.5
TAT-DTA
22722.84
0.2
0.4
0.4
0.4
6864.6
20840.1
20898.2
22723.4
97
Table 2.6.3. Isolated yields of sortagging ligations from SrtA* reaction.
Protein
Isolated Yield (%)
LDv1
21
LDv2
33
LDv3
34
LDv4
44
LDv4mut
28
LDv5
22
Lvi
42
Lv2
47
Lv3
38
Lv4
44
Lv4mut
35
Lv5
37
Lv5mut
38
Lv6
42
98
Table 2.6.4. EC50 values of 30-minute protein synthesis inhibition assay (Figure 2.1.1d). The
errors represent fitting errors from Sigmoidal Boltzmann Fit.
Protein
EC50 (pM)
LFN-DTA
41
8
LDvl
33
11
LDv2
56
29
LDv3
62
9
LDv4
532
LDv4mut
38
209
4
99
Table 2.6.5. List of variants
Abbreviation
LDvIPT
Variant
Ctr
C-term
LDv2
(affibody)
C-tr
LPSTG
__ _ _
LDv3
(GB1)
PS*G"
(DARPin)
LDv4
_____
____(FN
_____
41V~t
PSTG,
C-term
LDv4mut
F33)
t
OD-( TALPSTG
Cer
(HA4)
LDv5
4___""
Lvi
PSTG
C
term
Lv2
(HA4-7c12)
(affibody)
C
LPSTG(B
(DARPin)
Lv4
PSTG,
~LPSTG>
C term
(IOFN3)
LV4mut
G
Cterm
(HA4)
Lv5
_____
(HA4-7cl2)
Lv5mut
LV~ m t
~
-term
(HA4(Y87A)-7c12(Y62E/F87K))
Lv6
W
LPSTG
.1 ""(ABRaf)
100
2.6.1. LC-MS Traces
LDvl
obs.59353.1
ca 593522
"'4-T)-PSTG5--
.
C-term
62500
57600
2
1
6
5
4
3
8
7
9
0.4
C
11
10
Tlme
12
(mini
13
16
15
14
19
16
17
21
20
LDv2
obs.58907.7 t OA4
ca.
C
LP"TG
GW(
58907.7
-lerm
57500 60000
2
1
3
4
6
5
7
8
9
10
f3
12
11
rime [min)
15
14
16
17
19
18
20
21
LDv3
obs.66128.8
66127.9
0.4
ca.
S
S
PSTG
;~:W0 L7vsw
1
Time
11
10
9
8
7
6
5
4
3
2
lmn
LDv4
0.4
obs.63449 7
ca. 63448.8
DA
-WtAC-term
PSTG,,
62560
1
2
3
4
5
6
7
8
9
10
12 13
11
ime [mini
14
15
16
17
S000
18
19
20
21
101
LDv4mut
obs.64112.2 0.4
64111.5
ca.
MD-CDT ALPSTG
--
C-term
ca. 7567059
72500 77500
1
2
3
4
5
6
7
8
9
10
11
ime (fln]
Lvl
obs.3780b 5
ca 37805 4
PSTG
C-term
35000
1
2
3
0.4
4
5
6
7
8
9
10
11
Time
13
12
14
15
16
17
40000
18
19
20
21
[minj
Lv2
obs.37361.6
ca. 37360.8
C-term
PSTG,""0
] 30040000
1
2
3
4
5
6
Time [min)
102
7
8
9
10
11
*OA.
Lv3
obs 44581.4 20.4
ca. 44581 1
PSTG,
4:0e.4
sA 4
A4
5
4
3
2
1
9
8
7
6
Time [min]
11
10
Lv4
obs.41902.5 * 0.4
ca. 41902.0
C-term
LPSTG
45000
40000
1
3
2
4
5
6
8
7
10
9
12
11
13
14
15
17
16
18
19
21
20
Time [min
Lv4mut
0.4
obs.418571
ca. 41856.9
LPSTG
Cterm
4000
1
3
2
4
5
6
8
7
10
9
11
12
Time
[min]
13
14
15
17
16
18
45000
19
21
20
Lv5
obs.54124.9 t. 0.4
ca
541240
52589 96089
1
2
3
4
5
6
7
8
9
11
10
Time [min]
Lv 5 mut
obs
ca,
97,9 * A4
3978,9
62SW0 48000
1
2
3
4
5
6
7
9
1
11
ime (minj
103
Lv6
obs.37745.0
37744.4
ca.
LPSTG
1
0.4
*
JA pO
2
3
4
5
6
Time [minj
7
8
9
11
10
Lvl-HA
obs23
39.1 T
ca. 388W95
W
,
PSTG,-$
C-term
3750040000
TTH
i
1
2
3
4
5
r
6
7
Time [min)
8
9
10
11
TAT-HA-1
obs.10192.5
Ctermca.
LPST~'-
10192.2
9000
1
2
3
4
5
6
7
Time [min)
8
9
10000 1low
10
11
obs.9747.9
02
TAT-HA-2
-termca.
LPSTG
1
104
2
3
4
5
6
ime [min]
9747.6
7
8
9
10
11
*0,2
0.4
TAT-HA-3
obs.16965.0
ca. 16967 9
0.2
LPSTG-
16"0 17000 lebo0
1
6
5
4
3
2
1
7
Time
9
10
1l
(min)
TAT-HA-4
obs. 14289.0 i0 2
ca
-term
LWPSTG,
1L3000
3
1
14289.2
4
5
6
Time (min1
7
8
9
14000 15000
10
11
TAT-HA-4mut
obs 13950.7 0.2
ca. 13950.4
L
-
, PSTG
"C-term
13060 14000 15000
2
3
4
5
6
7
9
9
10
11
Time [min)
105
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109
Chapter 3: Delivery of Mirror Image Polypeptides into Cells
The work presented in this chapter was published in the following manuscript and is open access
from the Royal Society of Chemistry:
Rabideau, A.E.,* Liao, X.* & Pentelute, B.L. Delivery of mirror image polypeptides into cells.
Chemical Science 6, 648-653 (2015).
*: co-first author
110
3.1. Introduction
The chirality of biomolecules is critical for their structural integrity and function; 1
proteins produced by the ribosome are primarily composed of L-amino acids and achiral glycine.
Polypeptides that contain mirror image or D-amino acids therefore provide a few key advantages
over their natural counterparts. Mirror image polypeptides are not recognized by natural
proteases and are thus less susceptible to degradation and have been found to cause a low
immunogenic response. 2 Solid phase peptide synthesis from D-amino acids (and in some cases
native chemical ligation) is used to synthesize mirror image polypeptides.3 ,4 Mirror image phage
display was developed to generate functional D-polypeptide binders for L-target proteins. 5
The evolution of bioactive mirror image binders toward intracellular targets has been
largely unexplored in part due to the challenge of delivery into the cell cytosol. There are only a
few bioactive mirror image peptides that have been delivered inside cells to perturb a biological
function, including a D-peptide evolved from mirror image phage display to bind to MDM2
protein6, 7 or a retro-inverso 8 peptide that activates the p53 protein. In addition, there are no
examples of mirror image proteins delivered into the cell cytosol. Despite extensive efforts in
developing intracellular delivery techniques based on cell penetrating peptides (CPPs), 9' 10
cationic lipids,1 ' nanoparticles,
2
or liposomes,"3 limited success has been achieved for D-
polypeptides.1 4 In the case of D-peptide binders towards MDM2, the fusions to CPPs were
nonspecifically cytotoxic in a p53-independent manner, and the most active binders could not be
packaged into liposomes for delivery. ' 7 A system capable of efficient delivery of Dpolypeptides into the cell cytosol is of immediate interest since it allows us to not only
investigate the fundamental properties of these modem agents in the cell, but also to use them for
perturbation of intracellular processes.
111
Nature has evolved machineries to transport various biomolecules into cells.1 5 One
example is Bacillus anthracis, which produces anthrax toxin. Anthrax toxin is a three-component
system containing protective antigen (PA), a receptor-binding, pore-forming protein, lethal factor
(LF)1 6 and edema factor (EF)' 7 as the enzymatic moieties. PA binds to receptors on host cells
and is cleaved by furin-family proteases (Figure 3.2.1 a, step 1).18 The resulting fragment PA6 3
self-assembles into the ring-shaped heptameric1 9 and octameric 2 0 pre-pore (Figure 3.2.1 a, step 2),
forming complexes with LF and EF with high affinity (Figure 3.2.1 a, step 3). The complexes are
then endocytosed (Figure 3.2.1 a, step 4) and acidification triggers conformational rearrangement
of the pre-pore to form an ion-conductive P-barrel transmembrane pore. 2 ' The pore then
translocates LF and EF into the cytosol to act on their selective target. LF binds
stereospecifically to PA pre-pore through the 263-residue N-terminal domain (LFN), which
initiates the unfolding and translocation of the protein in an N- to C-terminal direction through
the narrow P-barrel channel (Figure 3.2.1a, step
5).22
Prior work has shown this system can
transport cargo into cells but never for mirror image cargo.
Here we combined synthetic chemistry with enzyme-mediated bioconjugation to attach
mirror image polypeptides as cargo to the C-terminus of LFN- We found that the PA pore can
translocate mirror image polypeptide cargos as efficiently as the all L constructs. For the first
time, we demonstrated the delivery of intact mirror image protein cargo into the cytosol of
eukaryotic cells. We further demonstrated that a mirror image peptide binder delivered by PA
was able to disrupt the intracellular p53/MDM2 interaction in cancer cells.
112
3.2. Results
3.2.1. Delivery of mirror image polypeptides
We used an evolved sortase A (SrtA*) 2 3 for the facile attachment of different D-cargo
with an N-terminal oligoglycine motif to the C-terminus of LFN or LFN-DTA containing the
LPXTG recognition site to give sortagged variants (Sv) or sortagged DTA variants (SDv)
(Figure 3.2.1b). LFN-DTA contains DTA, which is the A-chain of diphtheria toxin and is
extensively used as a reporter of translocation based on its enzymatic activity to ADP ribosylate
elongation factor-2 and block protein synthesis once in the cytosol.2 4
25
After confirming the
translocation of the LFN-DTA variants (SDv), we investigated delivery into the cytosolic
compartment via western blot using LFN variants that lack DTA (Sv).
113
a)
D-cargo
PA63
PA83
*
2
13
LFN
__3
4
[D-cargo
b)
-LPSTGG
or
G 5-D-ro
SV:
or
->
LPSTGG
SrtA
-LPSTG,-Ei
SDv:
LPSTG,-c
Figure 3.2.1. Delivery of D-cargo sortagged onto LFN and LFN-DTA. a) Translocation of Dcargo conjugated to LFN is mediated by protective antigen (PA) from anthrax toxin. b) G5 -Dcargo are conjugated to LFN and LFN-DTA using SrtA to yield sortagged variants (Sv) and
sortagged DTA variants (SDv).
114
-
To begin our studies, we synthesized (Table 3.6.1) and sortagged two peptides, (G5
AKFRPDSNVRG) one containing all L-amino acids and the other with all D-amino acids, to
LFN-DTA (Table 3.6.2) to give SDv1 and SDv2 (Table 3.6.3 and Table 3.6.4), respectively
(Figure 3.2.2a). The SDv1 and SDv2 conjugates were added to CHO-Ki cells in the presence of
20 nM PA for 30 minutes, which was sufficient time to allow entry of DTA cargo enzyme into
cytosol. After treatment, the cells were washed and incubated with medium supplemented with
3 H-Leu
to determine the efficiency of cargo delivery by measuring protein synthesis (Table
3.6.5). As shown in Figure 3.2.2b, both protein conjugates translocated efficiently, relative to the
positive control (LFN-DTA), indicating that the PA/LFN system is adaptable for the intracellular
delivery of mirror image peptides. Once unfolding and translocation is initiated by LFN the cargo
can 'piggy-back' through PA pore regardless of the stereochemistry.
We further analyzed the cytosolic delivery of the LFN variants Svl and Sv2 by western
blot. CHO-Ki cells were incubated with Svl and Sv2 in the presence of PA overnight to allow
for multiple rounds of receptor mediated endocytosis and translocation. Cells were washed,
trypsin digested to remove cell surface receptors and bound proteins, and then extracted with
buffer containing 50 pg mL-' digitonin. Digitonin is a mild detergent used to permeabilize
plasma membrane and extract only the cytosolic fraction.2 6 Immunoblotting with antibodies
against Rab5 (an early endosome marker) and Erkl/2 (a cytosolic protein) is used to validate the
extraction of cytosolic proteins. Immunoblot analysis using anti-LF antibody revealed that both
conjugates translocated into cells and Sv2 reached a similar steady state concentration to LFN;
however for SvI, we observed a significantly less intense band than Sv2 (Figure 3.2.2c). Since
the delivery efficiencies were the same for all three constructs from the protein synthesis
inhibition assay, the steady state levels in the cell could reflect the difference in extracellular or
115
intracellular stability. We observed similar stability in serum for Svl, Sv2, and LFN (Figure
3.6.1), we therefore hypothesize that the peptides used here acted as unstructured cargo on the Cterminus of LFN, and facilitated degradation. In this case, the L-peptide promoted degradation
while the D-peptide did not. We investigated this observation with a variant containing the Lpeptide capped with two D-amino acids at the C-terminus (Sv3). We observed a similar
concentration of Sv3 in the cytosol as wild-type LFN, indicating the C-terminal D-amino acids
played an essential role in the variant's intracellular stability. These observations suggest that a
D-peptide cap can possibly promote stabilization inside the cell.
3.2.2. Translocation requires a functional PA and endocytosis
In order to confirm the translocation mechanism of Svi and Sv2, we performed three
control experiments and monitored their translocation by western blot (Figure 3.2.2d). We first
incubated the cells at 4 'C instead of 37 'C, which inhibited translocation of both Svl and Sv2,
indicating that endocytosis is necessary for translocation. Next, we found that treatment with 200
nM Bafilomycin Al, a vacuolar H+-ATPase inhibitor, prevented translocation, indicating the
importance of an acidic endosome. Finally, we used a PA mutant, PA[F427H], that binds LFN
and is endocytosed but arrests translocation. We observed no cargo in the cytosol when cells
were incubated with PA[F427H] instead of PA, indicating that Svl and Sv2 must pass through
the translocase for entry into the cytosol. These results confirmed that the translocation of Svl
and Sv2 followed the same mechanism as LFN-
116
a) 1
QQ
2
a k
Q
QQ
f I r I pId I s In
Q
b)
S
C
r
r
I
NH
G
CONH 2
[~~ = D-amino acid
=L-amino acid
1.4
_1.2.
1.0
0.8
a.
-a-LF N-DT A
I
-*-SDv-1
0.6
-+-SDv-2
-*-LF N-DTA, No PA
-&-SDv-1, No PA
0.41. 0.2'
0.0
-6 4 -6
-14 -1'3 -1'2 -1'1 -0
Log [Protein Concentration (M)]
C) 3
-6
G
4 ng
+
+
2
-
LFN
+
2
+
1
+
PA
Sv-
NH 2
3
100i000
anti-LF wAli
WWON
anti-Erkl/2
" Ow Owf
PA
PA[F427H]
Sv-
+
+
-
-
4 ng
-
-
+
+
2
1
2
1
2
+
-
+
-
+
-
1
2
1
4*C 4*C Ba
-
d)
+
anti-Rab5
2
Ba
anti-LF 4*
anti-Erki /2
."n
- *am
anti-Rab5
Figure 3.2.2. Translocation of mirror peptides using PA/LFN. a) Peptide cargo 1 and 2
contain either L or D-amino acids, respectively. b) Translocation efficiency of SDv1 and SDv2
were analysed using the protein synthesis inhibition assay in CHO-Ki cells treated with varying
concentrations of each variant in the presence of 20 nM PA for 30 minutes and protein synthesis
was measured using 3H-Leu. c) Peptide cargo 3 and western blot for CHO-Ki treated with 250
nM wild-type LFN or Svl-3 in the presence of 40 nM PA overnight. d) CHO-Kl cells were
treated with 250 nM Svl or Sv2 in the presence of 40 nM PA overnight. The treatment
conditions for the control experiments included mutant PA (PA[F427H]), the addition of 200 nM
Bafilomycin Al (Ba), and 4*C instead of 37'C incubation (4*C).
117
3.2.3. Translocation of a D-peptide for MDM2
We next explored PA-mediated delivery of a bioactive D-peptide to disrupt an
intracellular protein-protein interaction. We chose a D-peptide (TAWYANF*EKLLR, where F*
is p-CF 3 -D-Phe) that has a Kd of 0.45 nM towards MDM2. 7 Prior work with variants of this
peptide proved challenging as fusions to CPPs were toxic and the most active binders could not
be packaged into liposomes for delivery. To study if PA-mediated delivery of the D-peptide
binder into the cytosol of human glioblastoma (U-87 MG) cells, we sortagged the peptide and its
biotinylated form onto LFN-DTA to give SDv4 and SDv4-biotin, respectively (Figure 3.2.3a).
Both variants translocated equally well based on the protein synthesis inhibition assay (Figure
3.2.3b). We then studied the cellular function of the D-peptide binder by sortagging the peptide
and its biotinylated form onto LFN to give Sv4 and Sv4-biotin, avoiding the interference of DTA
toxicity to the cell. Both constructs were delivered and detected by western blot in U-87 MG
cells. Based on the linear relationship between the amount of protein loaded and the band
intensity detected by anti-LF antibody, we estimated a total of 0.9 ng Sv4 delivered into 76,000
cells, giving 250,000 molecules per cell or 110 nM of cytosolic concentration (Figure 3.2.3d and
Figure 3.6.2).
We investigated whether the attachment of LFN would affect the interaction between the
peptide and MDM2. We expressed
-' 0 9MDM2 as a SUMO fusion (SUMO- 2 5- 09MDM2) and
25
used this construct to measure the binding affinity for cargo 4 and its LFN conjugate Sv4 using a
bilayer interferometry system (Figure 3.6.3 and Figure 3.6.4). Both cargo 4 and Sv4 effectively
competed with immobilized biotin- 15 -2 9p53 for SUMO- 2 51- 0 9MDM2 binding, yielding Kd values
of 1.0
0.7 nM and 12.3
4.3 nM, respectively. Despite the reduction in binding affinity as
compared to the cargo 4 by itself, Sv4 still had low nanomolar affinity for MDM2.
118
We analyzed whether the delivered peptide could interact with MDM2 in the cell cytosol.
Streptavidin agarose beads were used to capture Sv4-biotin from the lysate of cells treated with
Sv4-biotin and PA. We then eluted the bound proteins from the beads and analyzed by
immunoblotting with anti-LF and anti-MDM2 antibody. As shown in Figure 3.2.3c, we observed
bands corresponding to Sv4-biotin and the bound MDM2 from the lysate of cells treated with
Sv4-biotin and PA, whereas no MDM2 was detected in the control experiments when
PA[F427H] was used instead of PA, or a MDM2 non-binding control (Sv5-biotin, Figure 3.6.5)
was used instead of Sv4-biotin. These results show the specific binding between delivered Sv4biotin and intracellular MDM2. To confirm that the binding event occurred inside the cell and
not post-lysis, we added similar amount of Sv4-biotin directly into the U-87 MG cell lysate, and
no MDM2 was detected in the pull-down fraction. The absence of MDM2 pulldown in this
control experiment confirmed that the binding between delivered peptide and MDM2 occurred in
the cytosol.
Next, we studied the biological effect of the binding between Sv4 and MDM2 inside U87 MG cells. Binding an antagonist in the p53-binding domain of MDM2 disrupts this
interaction and results in activation and expression of MDM2, p53, and p21 .27 Therefore, we
quantified the relative change in protein levels of p53, MDM2, and p21 in U-87 MG cells under
different treatment conditions, and used the small molecule MDM2 antagonist nutlin-3 as a
positive control. According to the western blot, we observed increased levels of MDM2, p53,
and p21 for U-87 MG cells treated with Sv4 and PA, similar to the cells treated with 1 jM
nutlin-3, in comparison to the PA only or PA[F427H] conditions (Figure 3.2.3d). In order to
confirm that this upregulation is dependent on the disruption of p53/MDM2 interaction, we
performed the same analysis on the K562 leukemia cell line, which lacks p53.28 Despite the
119
delivery of Sv4 into K562 cells, we observed no perturbation of MDM2 and p21 protein levels.
These findings strongly support Sv4 regulated protein levels by specifically inhibiting the
p53/MDM2 interaction. Taken together, our results indicate a D-peptide can be delivered to the
cytosol of U-87 MG cancer cells and reach a concentration sufficient to bind the target protein
and disrupt a critical protein-protein interaction.
120
4
-4iFIyEEJLv1nT
blotin
l(G2'
Pull down: streptavidin
+
+
PA
PACAF47U
|-ONH,
hT2
s)3-f Frat I wyI a I n y I kIl I
C)
t.
j
4-biotin
(doped in)
4-biotin
4-biotin
Sv 5-biotin
anti-LF
-CONH
anti-MDM2
,
SDv4
PA
+
d)
PA[F427H] 5 ng
Sv 4
1 sM
1.0-
-0.8-
anti-LF
h. 0.6
anti-MDM2
anti-p53
* 0.4u-
4
nutlin-3
4
U-87 MG
-0000
6660
K562 (
a
-
1.2.
+
-- LF N-DTA
SDv4-biotin
1.4-
+
b)
anti-p21
0.2-
anti-GAPDH
0.0.
-15 -14 -13 -12 -11 -10 -9
-8
-7
Log [Protein concentration (M)l
-
+-
= D-amino acid
-
-
-
a)
K562 (p53-null)
-6
anti-LF
M
anti-MDM2
anti-p53
4
anti-p21
anti-GAPDH
""W-OM
Figure 3.2.3. Translocation of a D-binder to MDM2. a) Structure of a D-peptide binder to
MDM2 (4) and its biotinylated form (4-biotin). b) Translocation efficiency of SDv4 and SDv4biotin were analysed using the protein synthesis inhibition assay in CHO-KI cells treated with
varying concentrations of each variant in the presence of 20 nM PA for 2 hours. c) U-87 MG
cells were treated with 150 nM Sv4-biotin in the presence of 20 nM PA for 24 h. The cell lysates
were then treated with streptavidin beads to co-precipitate Sv4-biotin and MDM2. Sv5-biotin
with PA and Sv4-biotin with PA[F427H] served as negative controls. An additional negative
control was run where 5 nM Sv4-biotin was doped into cell lysate then treated to the same pull
down procedure. d) Western blot analysis of U-87 MG cells treated with 150 nM Sv4 in the
presence of 20 nM PA or PA[F427H] or 1 gM nutlin-3 for 24 h. As a negative control, K562
(p53-null) cells were analysed under the same conditions.
121
3.2.4. Translocation of mirror image proteins
After demonstrating that mirror image peptides can be efficiently delivered via the PA
pore, we next investigated the possibility of translocating mirror image proteins-a feat never
accomplished before. We chose to study two mirror image proteins, D-affibody containing three
a-helices 29 and D-protein G B1 containing a-helices and a P-sheet.30 We chemically synthesized
these proteins from three fragments (Figure 3.6.6 and Figure 3.6.8) using in situ neutralization
Boc SPPS. 3 1After cleavage and purification of each fragment, the peptide segments were ligated
together using native chemical ligation, according to the synthetic strategies in Figure 3.2.4a and
Figure 3.2.4b. 4 The cysteine residues of D-affibody were alkylated to give pseudo-glutamine
within the full-length protein, while the cysteine residues of D-GB1 were desulfurized 32 to give
the native sequence. The mirror image conformation of the folded proteins was evidenced by
circular dichroism (CD) spectra (Figure 3.6.8). An in vitro tryptic digest of L- and D-affibody
was performed to measure the stability of the proteins over time (Figure 3.6.9). Based on LC-MS
analysis, we observed complete degradation of the L-affibody and no degradation of the Daffibody. This demonstrated that mirror image polypeptides are less susceptible to proteolytic
degradation. When conjugated to LFN, we found that LFN-D-affibody (Sv5) was more stable than
the all L-protein LFN-L-affibody (Sv5-L) towards trypsin digestion, indicating a unique property
of the hybrid protein.
To study translocation efficiency, the D-proteins were sortagged onto LFN-DTA to give
SDv5 and SDv6, respectively. As evidenced by the DTA-mediated inhibition of protein
synthesis, both mirror image variants translocated as efficiently as LFN-DTA (Figure 3.2.4c). In
order to confirm that D-affibody was delivered to the cytosol as an intact protein, we studied the
translocation of LFN conjugates of D-affibody (Sv5) and installed an alkyne functional group at
122
the C-terminus for functionalization with azide-biotin to yield Sv5-biotin (Figure 3.6.10). After
translocation into CHO-KI cells, the digitonin-extracted cytosolic fraction was immunoblotted
with anti-LF antibody and showed Sv5-biotin delivery into the cytosol (Figure 3.2.4d). Delivery
of Sv5-biotin was further confirmed by staining with streptavidin conjugated to an IR680 dye,
which revealed a similar amount to that detected by anti-LF antibody (Figure 3.2.4d). For the
first time we show the delivery of intact mirror image proteins into the cytosol by PA, thereby
facilitating the investigation of their biological properties inside the cell.
123
GGGGGVDNKF'NKEQQNAFYE20ILHLPNLNEE
*
a)
QRNAF IQSLK 4*DDPS.QSANLL'OAEAKKLNDAQ60APK
NH
-
COSR
Thz-
4 -COSR CyO-4-63 1-CONH,
1) NCL 2) -ThZ
1) NCL 2) Akylation
NH- (
33-44 -Cys'-
Cys
45-63 -CONH,
Fold
b)
*
D-affibody
5
GGGGGMTYKL'ILNGKTLKGE20TTTEAVDAAT30
AEKVFKQYAN40DNGVDGEWTY5*DDATKTFTVT6*E
NH
-2
COSR
Thz-29-38C-COSR Cys- 39-81
-CONH,
1) NCL 2) -ThZ
1) NCL 2) Desollugization
NH
1-61
-CONH,
Fold
6
D-GB1
1.4
C)
1.2
1.0
--- LF -DTA
-- SDv5
S0.8
0,6-
0-
-*--SDv6
-*-SDv5-L
-A-SDV6-L
0.4
U-
0.20.
+
PA
Sv5-biotin
4 ng
+
d) Sv5-biotin
-5
+
-14 -13 -12 -11 -10
9 -8
-6
Log [Probin Concentration (M)]
anti-LF
-LPSTG"
Strep-680
anti-Erkl/2
anti-Rab5
Figure 3.2.4. Synthesis and translocation of mirror image proteins. a) Synthetic scheme for
D-affibody (5) and LC-MS analysis of D-affibody (TIC with deconvoluted mass inset). b)
Synthetic scheme for D-GB1 (6) and LC-MS analysis of D-GB1 (TIC with deconvoluted mass
inset). c) Translocation efficiency of SDv5 and SDv6 were analyzed using the protein synthesis
assay in CHO-Ki cells treated with varying concentrations of each variant in the presence of 20
nM PA for 30 min and compared to the efficiencies of L-affibody (SDv5-L) and L-GB 1 (SDv6L) were also analyzed. d) CHO-KI cells were treated with 100 nM Sv5-biotin in the presence of
40 nM PA for 24 hours and analyzed after digitonin extraction with anti-LF antibody and
streptavidin-IRDye 680.
124
3.3. Discussion
In this work, we showed that once translocation is initiated by LFN, the interaction
between the PA pore and cargo attached at the C-terminus of LFN is independent of the
stereochemistry. It was not clear that the PA/LFN system would allow for the delivery since most
protein-protein interactions are stereospecific and disruption of chirality commonly results in
loss of function. The proper function of PA/LFN is dependent on stereospecific interactions that
are required for binding and initiation of translocation; however, we demonstrated that the
interaction between the PA translocase and cargo is not necessarily stereospecific. In this study,
we used two assays: a protein synthesis inhibition assay based on DTA activity and western blot
analysis of cytosolic proteins. Collectively, these assays showed that PA can efficiently
translocate mirror cargos into the cell cytosol.
We found that translocation of mirror cargo is not limited to the length or fold of the
polypeptide. We demonstrated the translocation of two mirror image proteins: affibody and GBI.
We further confirmed delivery of an intact mirror image protein by staining the translocated
biotinylated D-affibody with anti-LF antibody and fluorescently labeled streptavidin. However,
we currently have no tools to assess their folding or activity inside the cytosol. Based on the
protein synthesis inhibition data, the foreign protein DTA correctly refold in the cytosolic
environment. Our future efforts to generate bioactive mirror image proteins will give us the
capability to assay their intracellular function and provide insight into refolding after
translocation into the cell.
We further demonstrated that PA/LFN system efficiently delivers functional D-peptides
for the disruption of intracellular protein-protein interaction in cancer cells. We focused on a
mirror image peptide that was previously reported a challenge to deliver into cells. 6, 7 Our own
125
efforts to load this peptide into liposomes for delivery failed. In contrast, for the first time we
found that the PA/LFN efficiently delivered this D-peptide. We found it bound to its target after
reaching the cytosol as confirmed by capture assays and western blot. Furthermore, the D-binder
perturbed the p53/MDM2 and activated this pathway. The delivery problem has been the major
obstacle to place mirror image polypeptides in the cytosol of cells. Using SrtA-mediated ligation
and the PA/LFN delivery platform, we overcame this challenge.
3.4. Experimental
3.4.1. Materials
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU), N-
a-Boc, and N-a-Fmoc protected L- and D-amino acids were purchased from Chem-Impex
International,
IL,
Peptide
Methylbenzhydrylamine
Institute,
(MBHA)
Japan,
resin
was
and
Midwest
obtained
Bio-Tech,
from
Anaspec,
Inc.,
CA.
IN.
4N,N-
dimethylformamide (DMF), dichloromethane (DCM), methanol (MeOH), diethyl ether, HPLCgrade acetonitrile (MeCN), and guanidine hydrochloride (guanidine-HCl) were from VWR, PA.
Trifluoroacetic acid (TFA) was purchased from NuGenTec, CA and Halocarbon, NJ. 2,2'Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) was purchased from Wako Pure
Chemical Industries, Ltd., Japan. All other reagents were purchased from Sigma-Aldrich, MO
and Life Technologies, CA.
The following primary and secondary antibodies were used goat anti-LF (bD-17, Santa
Cruz Biotechnology, TX), rabbit anti-MDM2 (N-20, Santa Cruz Biotechnology, TX), rabbit antip21 (C-21, Santa Cruz Biotechnology, TX), mouse anti-p53 (DO-1, Santa Cruz Biotechnology,
TX), rabbit anti-GAPDH (Sigma Aldrich, MO), goat anti-mouse IRdye 680RD (LI-COR
126
Biosciences, NE), goat anti-rabbit IRdye 800CW (LI-COR Biosciences, NE), and donkey antigoat IRdye 680LT (LI-COR Biosciences, NE).
3.4.2. Solid phase peptide synthesis (Boc)
Select peptides were synthesized on a 0.2 mmol scale on MBHA resin using in situ
neutralization Boc chemistry protocols.3 1 Peptide thioesters were prepared using a S-trityl
mercaptopropionic acid (MPA) strategy.33 Side-chain protection for L- and D-amino acids
included: Arg(Tos), Asn(Xan), Asp(OcHex), Cys(4-MeBzl), Glu(OcHex), His(Bom), Lys(2ClZ), Lys(Alloc), Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(2-BrZ). After completion of stepwise
SPPS, peptides containing Trp were subjected to 10% piperidine and 5% H 2 0 (v/v) in DMF for 2
h at RT to remove the formyl protecting group prior to final deprotection of Boc group.
The allyloxycarbonyl
(Alloc) protecting group was removed using 4.85 mmol
phenylsilane and 39.5 pmol tetrakis(triphenylphosphine)palladium(0) in DCM for 20 min at
with DCM
DMF.
RTE 32 34 The resinn was
a washed
ahdwt
C then
hnDF
The peptides were simultaneously cleaved from the resin and side-chains were
deprotected by treatment with 10% (v/v) p-thiocresol and 10% (v/v) p-cresol in anhydrous HF
for 1 h at 0 'C. Peptides were triturated with cold diethyl ether, dissolved in 50:50 (v/v) H 2 0:
MeCN containing 0.1% TFA (v/v) and lyophilized. The solvent compositions used in the
experiments will be referred to as A: 0.1 % TFA in H 20 (v/v), B: 0.1 % TFA in MeCN (v/v).
3.4.3. Solid phase peptide synthesis (Fmoc)
Select peptides were synthesized on a 0.1 mmol scale on aminomethyl resin with a Rink
amide linker using fast flow Fmoc synthesis. 35'
36
Side-chain protection for the D-amino acids
(Chem-Impex International) included: Arg(Pbf), Asn(Trt), Glu(OtBu), Lys(Boc), Lys(Alloc),
127
Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). After synthesis, peptides were cleaved from resin
with 94% TFA containing 2.5% EDT, 2.5% H2 0, and 1% TIPS (v/v) for 2 h at RT. After
cleavage, TFA was dried under N 2 (g) and triturated three times with cold ether then dissolved in
50:50 A:B and lyophilized. Purification of the crude peptides was achieved using RP-HPLC.
3.4.4. Analytical LC-MS
All peptides, proteins and semi-synthetic products were analyzed on an Agilent 6520
Accurate-Mass quadrupole time-of-flight (Q-TOF) liquid chromatography-mass spectrometry
(LC-MS) system. Solvent A' 0.1% formic acid in H2 0 and solvent B': 0.1% formic acid in
MeCN was used. LC-MS used Agilent Zorbax 300SB C 3 column (2.1 x 150 mm, 5 ptm) using a
linear gradient of 5-65% B' over 15 min at a flow rate of 0.4 mL/min. The observed mass was
generated by averaging the major peak in the total ion current (TIC). The charge-state series of
the species were deconvoluted using Agilent MassHunter Bioconfirm using maximum entropy
setting.
3.4.5. Preparative, semi-preparative, and analytical RP-HPLC
The crude peptides were dissolved in 99:1 or 95:5 A:B. If the peptide was insoluble under
these solvent conditions, 6M guanidine HCl was added to the solution. Peptides were purified by
preparative RP-HPLC on Agilent Zorbax SB C 18 column (21.2 x 250 mm, 7 pim) at a flow rate of
10 mL/min at 1-41% B, 5-45% B, or 10-50% B over 80 min. For semi-preparative RP-HPLC, a
Agilent Zorbax 300SB C 18 column (9.4 x 250 mm, 5 ptm) was used at a flow rate of 5 mL/min
over the same gradients. HPLC fractions were spotted with MALDI matrix alpha-cyano-4hydroxycinnamic acid (a-CHCA) in 50:50 A:B and checked for the correct molecular masses.
The analytical RP-HPLC Agilent C 18 Zorbax SB column (2.1 x 150 mm, 5 pm) was used to
128
confirm the purity of fractions at a flow rate of 0.5 mL/min over a linear gradient of 1-51% B
over 12 min. Analytical HPLC UV absorbance traces were measured at 214 nm.
3.4.6. Synthesis of D-affibody
The synthesis of D-affibody containing G 5 at the N-terminus (63-mer) was performed by
native chemical ligation (NCL) of three peptide segments (Figure 3.2.4a).
The peptide segments (and the corresponding masses) used in this synthesis were as
follows (X: -S-CH 2-CH 2-CO-, Z: thiazolidine):
[Gly -Arg 32 ]-"thioester: GGGGGVDNKFNKEQQNAFYEILHLPNLNEEQRXA
(calc. average: 3773.8 Da, obs. 3773.8
0.1)
[Thz 3 3-Ser 4 4] thioester: ZAFIQSLKDDPSXA
(calc. monoisotopic: 1493.7, obs: 1493.7
[Cys 45 -Lys
63]-CONH
2:
0.1)
CSANLLAEAKKLNDAQAPK
(calc. monoisotopic: 1983.1 Da, obs: 1983.1
0.1)
Purification of peptide segments All three segments were dissolved in 99:1 A:B and
purified by preparative RP-HPLC. Fractions containing the purified fragments were combined
and lyophilized: [Glyl-Arg32 ]-athioester 43.1 mg (10.2 ptmol), [Thz 3 -Ser
44]-"thioester
118.7 mg
(68.9 pmol) and [Cys 45 --Lys 6 3]-CONH 2 152.1 mg (62.3 pLmol).
NCL of three peptide segments Peptide fragments [Thz 33 Ser 44]-"thioester (11.0 mg, 6.4
gmol) and [Cys 4 5-Lys 6 3]-CONH2 (14.0 mg, 5.8 pmol) were dissolved to a concentration of 4
129
mM in NCL buffer (6 M guanidine-HC, 20 mM TCEP-HCl, and 40 mM MPAA in 0.2 M
sodium phosphate buffer) (Figure 3.6.6A) at pH 6.95 at RT for 7 h. MeONH 2 HCl was then
added to the crude reaction mixture at a final concentration of 0.2 M and pH 4.0 at RT overnight
to give [Cys3 3 Ser4 4 ]-[Cys 4 5-Lys
athioester
63 ]-CONH2
32
1(Figure 3.6.6B). In the same pot, [Gly -Arg]
(26.6 mg, 6.3 pmol) was added (Figure 3.6.6C) and incubated at pH 7.0 at RT for 7 h
to give [Glyl-Arg 3 2]-[Cys -Ser
]-[Cys 4 5-Lys
63]-CONH
2
(Figure 3.6.6D). The product was
purified by semi-preparative RP-HPLC and fractions containing the pure product were combined
and lyophilized to give 11.7 mg, 1.5 pmol (26% yield).
Alkylation [Gly'-Arg 2]-[Cys- Ser4 4 ]-[Cys4-Lys6 3
r2ir-,
33
44-iC
TCONH2The two cysteine positions
45_YS3H
of [Gly'-Arg ]-[C-ys -Ser ]-[Cys -Lys
l,2
]-CONH 2 were alkylated in 6 M guanidine HCl, 20
mM TCEP HCl, and 50 mM 2-bromoacetamide in 0.2 M sodium phosphate buffer. The reaction
was incubated at pH 7.1 for 30 minutes at RT and quenched with MESNa (100 mM final
concentration) to give [Glyl-Lys 63]-CONH 2 . The product was purified by semi-preparative RPHPLC and fractions containing pure product were combined and lyophilized to give 9 mg, 1.1
pmol (76% yield) (Figure 3.6.6E).
Folding of [Gly'-Lys
63 ]-CONH
2
Full-length [Gly' -Lys
63]-CONH
2
was dissolved in 6 M
guanidine-HCl, 20 mM Tris-HCl, and 150 mM NaCl, pH 8.5, and was diluted from 6 M to 2 M
guanidine-HCl using the same buffer without guanidine-HCl. The peptide solution was desalted
into 20 mM Tris-HCl, 150 mM NaCl, pH 8.5 using a HiTrap Desalting column. The folded
protein was concentrated using a 3 kDa concentrator to give 5.5 mg, 0.79 pmol (69% yield)
(Figure 3.6.6F).
130
3.4.7. Synthesis of D-affibody-alkyne
D-affibody-alkyne was synthesized using the same strategy but with an additional
propargyl-glycine incorporated at the C-terminus of the protein. D-affibody-biotin was obtained
by Cu(I)-catalyzed azide-alkynyl click reaction to label the D-affibody-alkyne with biotin-azide.
For the labeling reaction, 50 piL D-affibody-alkyne (332 pig, 46.8 nmol) was mixed with final
concentration of 100 mM Tris pH 8.5, 1 mM biotin-azide (70 nmol), 2 mM CuSO 4 and 200 mM
ascorbic acid in a total 70 ptL reaction. LC-MS analysis showed 80% yield after 4 hours reaction
at room temperature. The reaction mixture was dissolved in 6 M guanidine HCl, 20 mM TrisHCl, and 150 mM NaCl, pH 8.5, and was serially diluted from 6 M to 3 M then to 1 M
guanidine-HCl using the same buffer without guanidine-HCl. The solution was desalted into 20
mM Tris-HCl, 150 mM NaCl, pH 7.5 using a HiTrap Desalting column. The folded protein was
concentrated using a 3 kDa concentrator to give 202 ptg, 28.9 nmol (61% yield).
3.4.8. Synthesis of D-GB1
The synthesis of D-GB1 containing G5 at the N-terminus (61-mer) was performed by
NCL of three peptide segments (Figure 3.2.4b).
The peptide segments (and the corresponding masses) used in this synthesis were as
follows (X: -S-CH 2-CH 2 -CO-, Z: thiazolidine):
[Glyl-Ala 28]thioester: GGGGGMTYKLILNGKTLKGETTTEAVDAXA
(calc. monoisotopic: 2940.4 Da, obs: 2940.5
0.1 Da)
[Thz 29-Tyr 3 s]-thioester: ZTAEKVFKQYXA
(calc. monoisotopic: 1387.6 Da, obs: 1387.6
0.1 Da)
131
[Cys 39-Glu 6 1]-CONH 2 : CNDNGVDGEWTYDDATKTFTVTE
(calc. monoisotopic: 2579.0 Da, obs: 2579.1
0.1 Da)
Purification ofpeptide segments All segments were dissolved in 6 M guanidine-HCl in
95:5 A:B and purified by preparative RP-HPLC. Peptide segments [Glyl-Ala28 ]-"thioester and
[Cys 3 9-Glu 6 1 ] were further purified by semi-preparative RP-HPLC. Fractions containing the
purified segments were combined and lyophilized: [Gly'-Ala 28]-"thioester 34.3 mg (11.65
gmol), [Thz 29-Tyr
38
] -thioester 57.3 mg (41.3 pimol), and [Cys 3 9-Glu 6 1] 40.9 mg (15.8 pmol).
NCL of three peptide segments Peptide segments [Thz2 9-Tyr 3 8]-"thioester (13.2 mg, 9.5
ptmol) and [Cys 3 9-Glu 6 1] (20.5 mg, 7.9 pmol) were dissolved to a concentration of 4 mM in NCL
buffer (Figure 3.6.7A). The reaction was incubated at pH 7.0 at RT for 3 h. MeONH 2 'HCl was
then added to the crude reaction mixture at a final concentration of 0.2 M and pH 4.0 at RT
overnight to give [Cys29-Tyr ]-[Cys 39 -Glu 6 1 ]-CONH 2 (Figure 3.6.7B). The product was purified
by semi-preparative RP-HPLC to give 23.5 mg, 6.21 pmol (78.2 % yield). Peptide segments
[Cys 29 -Tyr]-[Cys 9 -Glu"J-CONH
2
(12.7 mg, 3.4 gmol) and [Glyl--Ala 2 ]8thioester (9.0 mg,
3.1 pmol) were dissolved in NCL buffer (Figure 3.6.7C) and incubated at pH 7.0 for 4.5 h to
give
[Gly'-Ala 28 ]-[Cys 2 9 -Tyr 3 8]-[Cys 39 -Glu 6' ]-CONH 2 (Figure 3.6.7D).
The product
was
purified by semi-preparative RP-HPLC to give 6.0 mg, 0.92 pmol (38.5% yield).
Desulfurization of [Gly1 -Ala_]-[Cys
Tyr ]-[Cys39 -Glu6 1]-CONH2
Desulfurization
was performed according to the protocol by Murakami, M., et al.3 2, 3 4 Full-length peptide [GlylAla2 8 ]-[Cys2 9-Tyr3 8 ]-[Cys 39 -Gu6 1 ]-CONH 2 (6.0 mg) was dissolved to a concentration of 0.25
gM in 6 M guanidine HCl, 0.45 M TCEP-HCl, 0.3 M MESNa and 5 p.M VA-044 in 0.2 M
sodium phosphate buffer at pH 7.0. The reactions were incubated at RT for 8 h and pH was
132
adjusted back to 7.0 every hour. Upon completion of the desulfurization reactions, the products
were purified by semi-preparative RP-HPLC. Fractions containing the desulfurized [GlylGlu 6 l]-CONH 2 were combined and lyophilized to give 2.8 mg, 0.43 tmol (47.1% yield) (Figure
3.6.7E).
Folding of [Gly'-Glu 6 1]-CONH 2 The desulfurized peptide [Glyl-Glu 6 I]-CONH 2 was
dissolved in 6 M guanidine-HCl, 20 mM TCEP-HCl, 20 mM Tris, and 150 mM NaCl, pH 8.5,
and was serially diluted from 6 M to 3 M to 1 M guanidine-HCl using the same buffer without
guanidine-HCl. The peptide solution was desalted into 20 mM Tris-HCl, 150 mM NaCl, pH 8.5
using a HiTrap Desalting column (GE Healthcare, UK). The folded protein was concentrated
using a 3 kDa concentrator to give 1.3 mg, 0.20 tmol (46.4% yield) D-GB1 (Figure 3.6.7F).
3.4.9. Circular dichroism (CD) spectroscopy of folded proteins
Circular dichroism spectra of D-affibody and D-GB1 were recorded on an Aviv model
202 instrument at 25 'C. A 1 mm path length cell was used. The proteins were prepared by
dissolving 0.06 mg in 50 mM sodium sulfate and 5 mM Tris-HCl at pH 8.5. The molar ellipticity
(0 in deg cm 2 dmol-) was calculated by [O]x =
Oobs
x
1/(10 lcn), where Oobs = observed ellipticity
at X, 1 = pathlength (cm), c = concentration of peptide (M), n = # of amino acids. The mirror
image form of the L proteins showed the same optical rotation, but with the opposite sign thus
confirming the mirror image fold of the D-proteins (Figure 3.6.8).
3.4.10. Synthesis of biotinylated p53/MDM2 Inhibitor D-peptide
Biotinylated p53/MDM2 inhibitor D-peptide was synthesized using Fmoc SPPS. The Nterminal glycine residue was protected with Boc. After the peptide was synthesized, the Alloc
(allyloxycarbonyl) protecting group was removed as described above. Biotin (0.5 mmol)
133
dissolved in 0.2 M HBTU (0.95 eq) was activated with 1.4 eq DIEA then coupled for 25 min at
RT. The peptide was cleaved and purified and described above.
3.4.11. Construction of plasmids for recombinant proteins
The gene for
25
-109MDM2
was purchased from Addgene (pGEX-4T MDM2 WT,
16237).37
The pET SUMO-25- 9MDM2 was prepared using the Champion pET SUMO protein
expression system (Invitrogen, CA). AccuPrime Taq DNA polymerase (Invitrogen, CA) was
used to PCR amplify the DNA using the following primers:
5'-GAGACCCTGGTTAGACCAAAGC-3' (forward)
5'-TTATACTACCAAGTTCCTGTAGATCATGG-3' (reverse)
The PCR product was purified by the QlAquick PCR purification kit (Qiagen,
Netherlands), then cloned into pET SUMO by an overnight ligation at 15 'C with 3 ng PCR
product, 50 ng
nET
SIMO vector, and 1 pl T4 DNA ligase. 2 p of the ligation pI-odu' was
transformed into One Shot Machl-Ti competent cells and plated on 30 ptg/mL kanamycin plates
and incubated overnight at 37 'C. Colonies were grown in LB media containing 30 jig/mL
kanamycin. The plasmid DNA was isolated using the Qiaprep spin miniprep kit (Qiagen,
Netherlands).
3.4.12. Protein expression and purification
-
SUMO- 25 - 109 MDM2 was expressed in Rosetta (DE3) pLysS cells in IL LB culture. His 6
SUMO-LFN-DTA (C186S)-LPSTGG-His 5 , His 6-SUMO-LFN-LPSTGG-His 6 , His6 -SUMO-LFNDTA (C186S), SrtA*-His 6 , WT anthrax protective antigen (PA), and PA[F427H] were expressed
134
in E coli BL21 (DE3) cells at New England Regional Center of Excellence/Biodefense and
Emerging Infectious Diseases (NERCE).
Approximately 40 g of cell pellet was resuspended in 100 ml of 50 mM Tris-HCl, 150
mM NaCl, pH 7.5 buffer containing 200 mg lysozyme, 4 mg Roche DNAase I, and 2 tablet of
Roche protease inhibitor cocktail then sonicated for three times for 20 seconds. After sonication,
the suspension was centrifuged at 17,000 rpm for 40 minutes. The lysate was loaded onto three 5
ml HisTrap FF crude Ni-NTA column (GE Healthcare, UK) and washed with 100 mL of 20 mM
Tris-HCl pH 8.5, 150 mM NaCl, at pH 8.5 and 100 mL of 40 mM imidazole in 20 mM Tris-HCl
pH 8.5, 500 mM NaCl. The protein was eluted from the column using 500 mM imidazole in 20
mM Tris-HCl pH 8.5, 500 mM NaCl. The eluted protein was buffer exchanged into 20 mM TrisHCL pH 8.5, 150 mM NaCl using a HiPrep 26/10 Desalting column (GE Healthcare, UK). WT
PA and PA[F427H] was overexpressed in the periplasm of . coli BL21 (DE3) cells and purified
by anion exchange chromatography. 38
3.4.13. One-pot sortagging reaction using Staphylococcus aureus SrtA*
Enzyme-mediated ligation using Sortase A was utilized to ligate the peptide and protein
cargo to LFN-DTA-LPSTGG (for SDvs) or LFN-LPSTGG (for Svs), depending on the assay. We
used Staphylococcus aureus SrtA5 9-206 that was evolved (P94S/D160N/KI96T) by Chen et al.
(SrtA*).3 9 4 1
N-terminal small ubiquitin-like modifier (SUMO) was first cleaved from the protein
substrate, His 6-SUMO-LFN-DTA-LPSTGG-His
5
(or His 6-SUMO-LFN-LPSTGG-His 6 ), using 1
ptg SUMO protease per mg of protein substrate at RT for 30 minutes to give the native Nterminus of LFN-DTA-LPSTGG-His
5
(or LFN-LPSTGG-His 6 ).
135
The ligation reactions were performed on Ni-NTA agarose beads using SrtA*. Ni-NTA
beads were washed three times with SrtA* buffer (10 mM CaCl2 , 50 mM Tris-HCl, 150 mM
NaCl, pH 7.5). In one pot, 50 pM LFN-DTA-LPSTGG-His 5 (or LFN-LPSTGG-His 6 ), 5 pM
SrtA*, and 100 to 500 pM G5 -cargo were incubated with Ni-NTA beads in SrtA* buffer for 30
min at RT nutating. After 30 min, the Ni-NTA beads containing any unreacted LFN-DTALPSTGG-His 5 , SrtA*-His6 , His6 -SUMO, and GG-His 6 were spun down at 4 'C to minimize the
formation of the hydrolyzed LFN-DTA-LPST (or LFN-LPST) side product. The supernatant
containing the sortagged LFN-DTA variant (SDv) or (sortagged LFN; Sv) was collected. The
beads were washed three times with 1 mL of 20 mM Tris-HCI, 150 mM NaCl, pH 7.5. Three
buffer exchanges were performed into 20 mM Tris-HCI, 150 mM NaCl, pH 7.5 to remove the
excess G5 -peptide. Size exclusion chromatography into 20 mM Tris-HCl, 150 mM NaCl, pH 7.5
was used to remove the excess G 5-protein. The purity of the ligated product was analyzed by LCMS.
3.4.14. Translocation of SDvs and protein inhibition assay in CHO-Ki cells
The CHO-Ki cells were maintained in F-12K media supplemented with 10% (v/v) fetal
bovine serum at 37 'C and 5% CO 2. The cells were plated at 3.0 x 104 per well in a 96-well plate
one day prior to the assay. The SDvs were prepared in ten-fold serial dilutions in 50 pL, and
added 50 jtL of F-12K media containing 20 nM PA. The 100 tL samples were added to CHOKI cells and incubated for 30 minutes at 37 'C and 5% CO 2 . (SDv4 and SDv4-biotin was
incubated for 2 hours.) After incubation with SDvs, the cells were washed three times with PBS
and then 100 ptL leucine-free F-12K medium supplemented with 1 tCi/mL 3 H-leucine (Perkin
Elmer, MA) was added and incubated for 1 h at 37 'C 5% CO 2 . The cells were washed three
times with PBS and suspended in 150 ptL of scintillation fluid. 3 H-Leu incorporation into cellular
136
proteins was measured to determine the inhibition of protein synthesis by LFN-DTA. The
scintillation counts from cells treated with only PA were used as control value for normalization.
Each experiment was done in triplicate.
The data were plotted using Origin8 using Sigmoidal Boltzmann Fit using equation
y = A2 +
A1-A2
X-02 and xO represents the logEC5o values.
1+e x-
3.4.15. Cytosolic protein extraction and whole cell lysate preparation for western blot
CHO-KI cells were plated in a 12-well plate. The cells were treated with 250 nM Svl, 2,
4, and 5 in the presence of 40 nM PA overnight at 37 'C and 5% CO 2 . After treatment, the cells
were washed with PBS and lifted with 0.25% trypsin-EDTA for 5 minutes to remove surface
bound proteins then washed twice with PBS. For cytosolic protein extraction, 1 x 106 cells were
resuspended in 100 ptL of 50 pg mL-' digitonin in 75 mM NaCl, 1 mM NaH 2PO 4 , 8 mM
Na2HPO 4, 250 mM sucrose supplemented with Roche protease inhibitor cocktail for 10 min on
ice, and centrifuged for 5 minutes at 13,000 rpm. For whole cell lysate, cells were lysed in IP
lysis buffer (25 mM Tris, 150 mM NaCl, 1% v/v NP-40, pH 7.5) supplemented with Roche
protease inhibitor cocktail on ice for 30 minutes and centrifuged for 10 minutes at 13,000 rpm.
Both supernatants were collected for blotting and analysis.
3.4.16. Western Blot of Sv, 2, 4, and 5 translocated into CHO-Ki cells
Lysates were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane
soaked in 48 mM Tris, 39 mM glycine, 0.0375% SDS, 20% methanol and using a TE 70 SemiDry Transfer Unit (GE). After transferring, the membrane was blocked at room temperature for 2
hours with LI-COR blocking buffer. The membrane was then incubated with anti-LF or anti-
137
Erkl/2, and anti-Rab5 in TBST overnight at 4 'C. The membrane was washed and incubated
with the appropriate secondary antibodies in TBST for 1 h then imaged by the LI-COR Odyssey
infrared imaging system.
3.4.17. Trypsin digestion of L- and D-Affibody
Digestion of 3 pg L- and D-affibody with 3 ng trypsin at 37 'C in 100 gL 20 mM tris,
150 mM NaCl, pH 7.5 buffer were monitored up to 23 hours. The digestion was monitored by
LC-MS.
3.4.18. Pull down of Sv4-biotin in U-87 MG cells
U-87 MG cells were grown in T-75 flasks at a density of 5x10 6 per flask. Cells were
treated with 100 nM Sv3-biotin in the presence of 20 nM PA or PA[F427H] for 24 hours at
37 'C and 5% CO 2. After treatment, the cells were lifted with 0.25% trypsin-EDTA, washed
three times, and lysed with 500 pL IP buffer for 10 min on ice. As a negative control, untreated
cells. Lysates were incubated with 20 tL (42 ptg) streptavidin-agarose beads (Sigma Aldrich) for
4 hours at 4 'C. The beads were washed three times with lvsis huffer and
nnce
with lIs
uIIffer
without NP-40. The bound proteins were eluted in IX Laemmli sample buffer (Bio-Rad), boiled
for 10 min, and then analyzed by SDS-PAGE gel. Standard immunoblotting was performed
using anti-MDM2 antibody and streptavidin-680.
3.4.19. Translocation of Sv4 and Sv4-biotin in U-87 MG or K562 cells
U-87 MG cells were grown in T-25 flasks at a density of lx106 per flask. K562 cells were
treated in 24 well dishes at a density of 8.0 x 10 5 per well. Cells were treated with 150 nM of
Svl, Sv4, or Sv4-biotin in the presence of 20 nM PA or 20 nM PA[F427H] for 24 hours at 37 'C
and 5% CO 2 . As a positive control, cells were treated with 1 pM nutlin-3 for 24 h. After
138
treatment, cells were lifted by trypsin and lysed with IP lysis buffer. The isolated lysates were
subjected SDS-PAGE separation and transferred to a nitrocellulose blot as previously described.
The membrane was immunoblotted with anti-LF, anti-MDM2, anti-p21, anti-p53, and antiGAPDH overnight in TBST. The membrane was washed and incubated with the appropriate
secondary antibody in TBST for 1 h and then imaged.
The western blot images were analyzed and quantified using the Image Studio program
(LI-COR). Each band was normalized to the GAPDH loading control. The band intensity of each
protein was set to one in the PA only control. Percent increase in amount of MDM2, p53, and
p21 present in each lane compared to the PA only control was plotted in Figure 3.6.5.
3.4.20. Binding interaction between SUMO-2s-' 0 9 MDM2 and 4 or Sv4
A competition binding assay was performed using a bilayer interferometry system (Octet
RED96, ForteBio, CA) to determine the binding affinity of 4 (peptide) and Sv4 to SUMO- 2 5
109MDM2. Super streptavidin (SSA) sensors were soaked in IX kinetics buffer (IX PBS
containing 0.02% Tween-20, 0.1% BSA, pH 7.4) for 10 minutes at 30 'C. For immobilization,
the wild-type 15- 2 9p53 peptide was synthesized containing a biotin on the N-terminus (biotin- 15
29p53).
The biotin- 1529p53 peptide (1 RM in IX kinetics buffer) was immobilized on the SSA
sensor surface for 5 minutes at 30 'C and 1000 rpm. The surface was washed for 5 minutes with
IX kinetics buffer to establish a baseline. Two-fold dilutions of SUMO- 2 51- 09 MDM2 in IX
kinetics buffer were analyzed for binding over 5 minutes at 30 'C and 1000 rpm followed by
dissociation with IX kinetics buffer. A calibration curve was generated using GraphPad Prism 6
software using non-linear regression analysis (one-site, specific binding fit). The Kd was found to
be 578
170 nM.
139
For the competition assay, various concentrations of 4 (peptide) or Sv4 were incubated
with 50 nM SUMO- 2 5-'09MDM2 at room temperature for 30 minutes. Meanwhile, SSA sensors
were soaked in IX kinetics buffer for 10 minutes at 30 'C. The biotin- 15-29 p53 peptide (1 gM)
was immobilized on the SSA sensor surface then washed to establish a baseline, as previously
described for the calibration curve. After immobilization, the association and dissociation of
SUMO-25- OMDM2 pre-incubated with 4 or Sv4 samples were analyzed at 30 'C and 1000 rpm
and using IX kinetics buffer. Based on the binding (nm) values, the concentration of unbound
MDM2 was interpolated for each sample using the calibration curve.
Non-linear regression analysis was performed using GraphPad Prism 6 software to
determine the Kd value based on the equation: Kd
following equation to generate fitted curves: y =
=
[peptide][MDM2]/[complex].' We used the
(b-x-K)+(b+X+Kd)
2
2
_4(bX); where y is [MDM2]
in nM, x is [inhibitor] in nM, Kd is the dissociation constant, and b is ymax.
3.5. Acknowledgements
This work was funded by MIT start-up funds, MIT Reed Fund, Damon Runyon
Cancer Research Foundation Innovation Award, National Science Foundation (NSF)
CAREER Award (CHE-1351807) for B.L.P, and NSF Graduate Research Fellowship for
A.E.R. We thank R. J. Collier (Harvard) for his continued support, the NERCE facility
(grant: U54 AI057159) for expression of toxin proteins, and the MIT biophysical
instrument facility (grant: NSF-00703 1). We also thank Prof. Barbara Imperiali, Prof.
Douglas Lauffenburger, Deborah Pheasant, Jingjing Ling, Mike Lu, and Daphne van
Scheppingen for their comments.
140
3.6. Appendix
Table 3.6.1.Peptides used in this investigation
Observed (Da)
Calculated (Da;
monoisotopic)
1529.8
0.1
1529.8
2: (D)-G5 -akfrpdsnvrg-CONH 2
1529.8
0.1
1529.8
3: (D-cap) G 5-AKFRPDSNvrg-CONH 2
1529.8
0.1
1529.8
4: (D) G 5-tawyanf(CF 3)ekllr
1865.0
0.1
1864.9
4-biotin:
(D) G 5-k(biotin)-(GGs) 3-tawyanf(CF 3)ekllr
2820.0
0.1
2820.5
2031.2
0.1
2031.2
Sequence
1: (L)-G 5-AKFRPDSNVRG-CONH
2
biotin- 1s-29p53:
(L) biotin-SQETFSDLWZLLPEN-CONH
2
141
Table 3.6.2. Observed molecular masses of expressed protein constructs when analyzed by LCMS
Calculated MW
Protein
Observed MW (Da)
(Da; average)
SrtA*-His 6
19214.6
0.4
19214.5
LFN-DTA
52047.0
0.4
52046.2
66700.0
0.4
66700.1
45289.8
0.4
45289.8
WT PA
83754.1
0.4
83751.6
PA[F427H]
83742.4
0.4
83738.3
SUMO- 2 5-'0 9MDM2
23297.3
0.4
23296.6
L-affibody
6925.8
0.2
6925.6
L-GBl
6481.4
0.2
6481.0
His6 -SUMO-LFN-DTA-LPSTGG-His
His 6 -SUMO-LFN-LPSTGG-His
142
6
5
Table 3.6.3. List of variants
Sequence
Variant
L PSTG GF OO @@N.
N
S~V1
Sv
-@LPSTG
@
-LPSTGA
S v2
Sv
~-PSTG
SDv4
(
Fp
A
LPSTGF-jIIL
~~~~~LPSTG-EEIELih
Sv4
P
dAFA
G)1DCNEG~
C)K)(DO,
(DT
KP ® XOJ O
Q®J~Ih3
PSTG 5
v
SO
KF
,-
IZIEI-
k
SDv4-biotin
Sv4-biotin
Sv5-alkyne
LSG
WLPSTG
,
Sv5
IIp.I
LSG
SDv5
N
LPSTGA-,, A
G
Sv5-biotin
(~'$
LPTAb-'
0
SDv6
W
LSG
143
Table 3.6.4. Isolated yields of sortagging ligations from SrtA* reaction
Protein
Isolated Yield (%)
SDvl
61
Svl
41
SDv2
66
Sv3
53
Sv4
76
Sv4-biotin
77
SDv5
14
Sv5
51
Sv5-alkyne
38
SDv6
29
144
Table 3.6.5. EC 50 values of 30-minute protein synthesis inhibition assay. The errors represent
fitting errors from Sigmoidal Boltzmann Fit. (* represents 2 h assay)
Protein
EC 50 (pM)
LFN-DTA
21
SDv1
37+5
SDv2
24
3
SDv5
20
4
Sv5-alkyne
51
15
SDv6
33
6
3
LFN-DTA*
2.0
0.6
SDv4*
3.2
0.7
SDv4-biotin*
7.1
2.5
145
4 ng
Sv-
antiLF
2
LFN
1
*"map 4WOMW *Jjj*
2
3
ONWO
Figure 3.6.1. Immunoblot of media from CHO-Ki cells treated with Svl-3 (Figure 3.2.2c).
Serum-containing F12K with 250 nM wild-type LFN or Svl-3 and 40 nM PA was removed
after
overnight treatment with CHO-Ki cells. The stability of the LFN variants in media was analyzed
by immunostaining with anti-LF.
146
Sv4 (ng)
0.25
0.1
1.0
2.5
3
4
5.0
7.5
10.0
5
6
7
anti-LF
5000000-
*
Sv4
4000000-
3000000-
o 2000000-
1000000-
-
0
0
1
2
8
Lv (ng)
Figure 3.6.2. Linear relationship between Sv4 band intensity and the amount of protein loaded
0.1
(ng): y = 566000x + 107000, R2 = 0.993). The signal intensity of each band was compared to
- 10 ng of pure Sv4 protein loaded on the gel and immunoblotted with anti-LF.
147
a)
b)
0.7
0.6
-
,0.5,
E
-
0.3
-
-
0.2-
400 nM
400 nM
300 nM
200 nM
100 nM
50 nM
25 nM
0.80.69
0.4-
0.1
.
-0
0 0.2-
1
0
100
200
300
400
Time (s)
500
0.0600
700
0
100
200
300
Concentration (nM)
4
400
5
500
Figure 3.6.3. Calibration curve for the interaction between immobilized biotin-' 5-29 p53 and
SUMO-25- 9MDM2 determined using Octet RED96 bilayer interferometry. a) Association and
dissociation curves of various concentrations of SUMO- 2 5-'09MDM2 with biotin-' 5 -29p53
immobilized to super streptavidin sensors. b) A calibration curve was generated using GraphPad
Prism 6 software using non-linear regression analysis (one-site, specific binding fit). The Kd was
found to be 578 170 nM.
148
60
-
A
4
Sv4
1-140
cij
02
L..
0*
0.1
100
10
1
(nM)
Concentration
Inhibitor
1000
Figure 3.6.4. Binding interaction between SUMO-21- '9MDM2 and 4 or Sv4 based on a
competition binding assay at 30 'C. Non-linear regression analysis was used to generate fitted
curves and determine the Kd values based on the equation: Kd = [peptide][MDM2]/[complex].
Non-linear regression analysis was performed using GraphPad Prism 6 software to determine the
Kd value based on the equation: Kd = [peptide][MDM2]/[complex]. We used the following
2
-4(bx); where y is [MDM2] in nM, x
equation to generate fitted curves: y = (b-x-Kd)+ (b+x+Kd)
2
is [inhibitor] in nM, Kd is the dissociation constant, and b is ymax. The Kd values calculated for 4
and Sv4 were 1.0 0.7 nM and 12.3 4.3 nM, respectively.
149
1
4
4
anti-LF
-
+
1
+
-
+
-
+
+
-
5 ng
4
-
PA
PA[F427H] 1 [tM
Sv nutlin
+
a)
+-
4-biotin
4-biotin
-. OWM*
A"WW*OW
anti-MDM2
anti-p53
-ms
anti-p21
A-N-
-si
anti-GAPDH
b)
300250
-
* Sv4 + PA
* Sv4 + PA[F427H]
* Sv4-biotin + PA
a)
a)
C,
200
-
CO,
150-
4-0
a)
50
-
CL
100-
0 I
-
-50
I
MDM2
p53
M9
Sv4-biotin + PA[F427H]
Sv + PA
Sv + PA[F427H]
* Nutlin-3
p21
Figure 3.6.5. Quantification of MDM2, p53, and p21 protein levels for U-87 MG cells treated
with Svl, Sv4, or Sv4-biotin in the presence of PA or PA[F427H] based on western blot data
from Figure 3.2.3d. All levels were normalized to the anti-GAPDH loading control and the PA
only condition.
150
ACys-
45.83
H 6
l-{X*NH
3-44
Thz-
95
-COSF
8
766.4
BCY5-
P-5.
E
ys4-O- f -M+5H],
+M+
M+Hr mar
114 9.1
Bys 33-4 cs-4-61c5H].
M.
+
nvZ
[M+SHr
[+H'
37.3
EM9H
.4
4 H -
H
r
11681
823.2
(M+3Hr
1097.2
800 700 m0 9])0 10001/0
C
860
1100 0
m/z
F
__
___
CYs- Q3.44 -C-4
1
83 7JHCO
ca 7002.1
NH,'0R
1
2
3
4 5
6
7
8
9
10
11
12
13 14
15
16
17
18
19
20
21
1
2
3
4
5
Time (min]
6
7
8
9
12
10 11
Time [nmin]
13
14
15
16
17
18
19
Figure 3.6.6. LC-MS characterization of D-affibody synthesis
151
20 21
A
Th|zf-iOOSA
D
NH
se
.[
-qn
39- CONH
[+8J'
1091.5
9359
Cys- 39-1
ONH
Cys-
29-38
-Cys{
(M8i5H13.
1309.8
JCONH,
7
B
D-CONH 1
Cvs- 29-38 -Cys{
E
(M.7H
[M+7HJ'
NH, 1
+HHr
- Co
fM+qr
1060.9
926.7
1260.6
12968
Lmo
C
R
NH
1
2
3
4
5
6
7
8
9
10
C
11
Time
o
rNH3
12 13 14
[nn]
. bo 90001100 1S
ob.6460.4 10.1
ca. 6480.0
F
15 16 17
16 19 20 21
1
2
3
4
Figure 3.6.7. LC-MS characterization of D-GB 1 synthesis
152
00
5
6
7
8
9
10
11
12
imo [min
13
14
15
18
17
16
19
20
21
30000-I-
*
*
L-affibody
D-affibody
*
20000-0
o 10000ai)
0
U
0-
C>
0
,
-10000-6
*
E
-20000-
U
-z
? -30000210
200
240
230
220
wavelength (nm)
200001500010000-
250
*
0
*
260
L-GB1
D-GB1
p.
5000CD
0)
CD
-0
00.
-5000-
U
-10000-
I
-15000L>
-20000-25000-
0
S
-30000
200
210
240
230
220
wavelength (nm)
250
260
Figure 3.6.8. CD of L- and D-affibody and L- and D-GB 1.
153
A
t=O
L-affibody
t=3h
t=18h
1
2
3
4
5
6
7
Time [min]
8
9
10
11
400
600
WO0
0
1 00
12
400
1600
1800
m/z
B
D-affibody
t=0
t=3h
Lt=18h
L1
154
2
3
4
5
6
7
Time [min]
8
9
10
1
400
600
800
1000
1200
r~z
1400
1600
1800
C
37805.5
t=O
37805.5
t=9h
t=23h
2
1
7
Time [min]
3
8
10
11e
1500
20000
25000
40000
30000
3500
Deconvaluted mass [Dal
45000
37978.0
t=O
37978.0
t=9h
37978.3
t=23h
so
A.o
1
2
3
4
5
71
6
Time [min]
1
15000
000
25000
Ioo
iAI
ohsoo
40000
3A000
3000
Deconvoluted mass [Dal
415000
Figure 3.6.9. Trypsin digestion of L-affibody (A) and D-affibody (B) at t=0, 3, and 18 h and
Sv5-L (C) and Sv5-alkyne (D) at t=O, 9, and 23 h. For A-D, the left panel corresponds to the total
ion current (TIC). For A and B, the right panel corresponds to the mass spectra of the major peak
area and for C and D, the right panel corresponds to the deconvoluted mass of the major peak
area.
155
A
B
Os. 7098.20O
C& 7M008
O.74101
ca. 7713.8
S.M.
S.M
000
1
2
3
4
5
6
7
8
9
10
I1
Trn.
12
ln-m
13
14
;5
;6
17
18
19
&XW
20
000
21
1
2
3
4
5
6
Time
7
8
9
[min
Figure 3.6.10. LC-MS characterization of D-affibody-alkyne (a) and D-affibody-biotin (b)
156
10
800
3.6.1. LC-MS Traces
SDvl
obs.53958.0
0.4
ca. 53957.3
40
PSTG,5Mo
52500
2
1
4
3
7
6
5
8
9
17
15 16
11 12 13 14
Time [min]
10
55000
21
18 19 20
SvO
obs. 32410.7
04
c&. 324 11.4
32200 32400 32600
5
4
3
2
1
11
10
9
8
7
6
Time
[min]
SDv2
obs.53957.9
ca. 53957.3
iI I
PSTGnd -
I I I
0.4
G
52500 55000
2
1
4
3
5
7
6
8
9
10 11 12 13 14 15 16 17 18 19 20 21
time (min)
Sv2
obs.32411 2 t 0 4
ca 324 114
a
@&
ON
|
::E
PSTG,-5
35000
3000
2
1
3
4
5
6
7
8
9
10
11
Time [min}
S15
cb. 32410. 1 .4D 4
caob.
32410.
30000"
1
2
3
4
5
6
7
1
9
10
35000
;1
Time [min]
157
SDv4
obs54290.4
ca. 54291.6
LPSTG, ft
1
2
Sv4
3
SG5
J . k
y
5
4
[III
3 3k
0.4
-H
8
6
7
Time [min)
9
10
11
IIT II~obs.327444 t 0 4
ca. 32744.8
200
1
2
3
7
8
Time [mn]
9
1
1
6400
1
11
SDv4-biotin
obs.55249 1 t04
c, . 55249.Z
1
2
41
3
6
7
9
8
10
11
Time [min]
Sv4-biotin
obs 33702 4 *0
ca 33702 8
c~
W
W
(G2 D),
LPSTG,
'
I
2
Sv50
4
3
1
1
Time
8
7
[minj
0 34000
;1
9
36000
11
SDv5
obs.59430.2 t OA4
ca. 59429.2
C-term
4* (t>TA)LPSTG,
62s5
s5s1"
1
2
3
4
5
6
7
8
9
10
11 12
Time [min)
Sv5
158
13
14
15
16
17
18
19
20
21
4
abs
37582.7
0.4
ca. 37882.4
C-term
LPSTG,"-
35000
2
1
4
3
6
5
7
8
9
10
11
12
Time
(mini
13
15
14
16
17
40000
19
18
20
21
Sv5-alkyne
ca
37977.4
LPSTGJ
40000
35000
4
3
2
1
6
5
9
8
7
10
11
12
13
14
15
16
17
18
19
20 21
Time (min)
Sv5-biotin
38594.5 t 0A
Obs
ca. 38593.2
LPSTG,-H
35000 40
3
2
1
7
6
Tie imin}
5
4
11
10
9
8
SDv6
obW,58907.5t:0.4
ca. 58907 7
LPSTG,
C-term
1
2
3
4
5 6
7
8
9
10
12 13
11
ime [mini
14
15
16
17
IS
19
20
21
159
3.7. References
1.
2.
3.
4.
5.
6.
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8.
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37.
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Simon, M.D. et al. Rapid flow-based peptide synthesis. ChemBioChem 15, 713-720
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Chapter 4: Translocation of Non-Canonical Polypeptides into Cells
Using Protective Antigen
The work presented in this chapter was published in the following manuscript and is open access
from Nature Publishing Group:
Rabideau, A.E.*, Liao, X.,* Akqay, G. & Pentelute, B.L. Translocation of Non-Canonical
Polypeptides into Cells Using Protective Antigen. Scientific Reports 2015, 5:11944.
*: co-first author
163
4.1. Introduction
Nature has evolved several types of translocation systems for delivering toxic proteins
into host cells.' Certain pathogenic bacteria such as Vibrio cholerae, Corynebacterium
diphtheria, and Bacillis anthracis infect host cells with cholera, diphtheria, and anthrax toxin,
respectively. 2 These protein toxins access the cytosol by two main pathways: transport through a
translocase from the host cell endosome or retrograde endocytosis from the endosome to the
Golgi and endoplasmic reticulum. 2 Anthrax toxin is one example that utilizes a translocase to
deliver protein toxins from the endosome into the cell cytosol. 3
Anthrax lethal toxin is comprised of two proteins, protective antigen (PA) and lethal
factor (LF). 3 The pore-forming protein, PA83 binds to an anthrax toxin receptor on the host cell
surface and is then proteolytically activated by furin protease to give PA 6 3 ,4 triggering
oligomerization of PA 63 into a heptameric5 or octameric6 pre-pore. LF binds to the pre-pore
through the 263-residue N-terminal domain (LFN) with nanomolar affinity. 7 Up to three8 or four 6
molecules of LF can bind to the heptameric or optameric pre-pore, respectively. The entire
complex is then endocytosed and the low pH triggers a conformational rearrangement of the prepore to form a pore that spans through the endosomal membrane, 9 initiating translocation of LF
into the cytosol.10
Biophysical and biochemical experiments of LF translocation through the cationselective PA pore have elucidated the translocation mechanism into the cell cytosol."'
12
The
working model points toward a number of key factors that govern translocation through PA and
subsequent escape from the endosome:
164
e
PA pre-pore undergoes conformational rearrangement to form a pore in the endosomal
membrane 5 '
*
1
Charge state-dependent Brownian ratchet drives translocation through the PA pore"
"
* Protein cargo partially unfold in acidic endosome prior to translocation14'
e
2
Cargo passes through the PA conduit of approximately 12 A diameter , 16
Taking advantage of these drivers of translocation, researchers have investigated the delivery of
various protein cargos fused to the C-terminus to LFN through PA pore. Enzymes or enzymatic
domains such as P-lactamase, 18
Pseudomonas exotoxin A, Diphtheriatoxin A chain, 19,20 actin
cross-linking domain from the Vibrio cholerae RTX toxin, 21 Shiga toxin, 2 and Legionella
pneumophila flagellin protein2 3 have been delivered into cells using the PA/LFN system. These
studies showed that the PA pore is efficient in transporting naturally occurring or recombinantly
expressed proteins. However, the question of whether the PA pore can accommodate and
transport molecules beyond natural polypeptides has yet to be investigated. Only one example
has shown the effects on translocation when D-amino acids and cysteic acid were incorporated at
the N-terminus of LFN- 24 Some non-natural molecules, including small molecule drugs and
polypeptides with backbone or side chain modifications, have attracted research interest because
of their therapeutic potential. Investigating the translocation of these non-canonical polypeptides
would not only aid in their cytosolic delivery, but also provide insight into the mechanism and
promiscuity of the PA translocase.
In the present study, we investigated the translocation of various non-canonical
polypeptide moieties through the PA translocase (Figure 4.1.1a). We designed our experiments
to seek answers to three questions: 1, Do non-natural amino acids affect translocation through
165
PA pore? 2, Can PA pore still tolerate cargos containing non-natural peptide backbones? 3, Can
a constrained polypeptide or rigid natural product pass through the 12 A pore? To carry out these
investigations, we leveraged the use of sortase A (SrtA)-mediated ligation. The non-canonical
polypeptide cargos were synthesized with an N-terminal oligoglycine motif and attached to the
C-terminus of LFN-DTA using the LPXTG tag and SrtA2 5 to give LDn constructs (Figure
4.1.1b). DTA (A chain of diphtheria toxin) serves as a reporter of translocation since it inhibits
protein synthesis upon entry into the cytosol. 5 , 22,
26
From our investigations, we found that
peptides containing minor modifications to the backbone or amino acid side chains,
peptidomimetic monomethyl auristatin F (MMAF), and the small molecule drug, doxorubicin
translocate efficiently. In contrast, cyclic peptides and the small molecule drug, docetaxel caused
disruption to the translocation process. Our findings support the hypothesis that the chemical
composition of the cargo does not inhibit the Brownian ratchet provided the cargo is able to
adopt a conformational state in which it can pass through the narrow pore.
166
cytosol
endosome
non-canonical
polypeptide
cargo
LFN
PA pore
bLPSTGG
SrtA
G 5-cargo
cargo
LDn:
LPSTG 5
-
Figure 4.1.1. Delivery of non-canonical polypeptide cargo into the cytosol. a) Non-canonical
polypeptide cargo (green star) ligated to the C-terminus of LFN (pdb: LJ7N) to translocate
through PA pore (pdb: 3J9C) b) Sortase A-mediated ligation of LFN-DTA-LPSTGG and G5
cargo to form constructs are comprised of LFN-DTA and non-canonical polypeptide cargo
(LDn).
167
4.2. Results
4.2.1. Translocation of non-canonical polypeptide cargos with backbone or side chain
modifications
We first investigated the translocation of peptides containing either backbone or side
chain modifications. We chose P-alanine and N-methyl-alanine for backbone modifications, and
propargyiglycine and fluoro-phenylalanine for side chain modifications. These modifications
were incorporated into a model peptide containing all natural amino acids, G 5 -AKFRPDSNVRG
(Table 4.6.1). The peptides were sortagged onto LFN-DTA to give LDn1-5 (Figure 4.2.1a and
Table 4.6.2). We first used a protein synthesis inhibition assay to analyze translocation efficiency
-a
LAcnstrut.
Fru,22,26is assay, ten-fold serial dilutions of each purified conjugate were
added to CHO-Ki in the presence of 20 nM PA and incubated for 30 minutes. Cells were washed
three times with PBS then incubated with medium supplemented with 3H-Leu for 1 h. After
incubation, medium was removed, cells were washed, and 3H-Leu incorporation was measured
by a scintillation counter to determine the translocation efficiency of each cargo. According to
Figure 4.2.1b, each non-canonical cargo (LDn2-5) translocated as efficiently as the controls
(Table 4.6.3), LFN-DTA and LDn1, indicating that these peptides, which contain backbone and
side chain modifications can be appended to the C-terminus of LFN-DTA without major
interference to the translocation process.
We further analyzed the cytosolic presence of the non-canonical peptide conjugates using
digitonin extraction and western blot. CHO-KI cells were treated with 100 nM LDn1-5 in the
presence of 20 nM PA for 12 hours to allow for multiple rounds of endocytosis and
translocation. After trypsin digestion of surface bound materials and washing with PBS, the
168
cytosolic proteins were extracted using a lysis buffer containing 50 pg mL- digitonin, a mild
detergent used to permeabilize only the plasma membrane.2 7 The proteins extracted by digitonin
were analyzed by western blot immunostained with anti-Erkl/2 (a cytosolic marker) and antiRab5 (an early endosome marker) to confirm the presence of only cytosolic proteins. Using
immunostaining by anti-DTA antibody, we observed bands corresponding to LDn2, 4, and 5
with intensities similar to that of LDnl (Figure 4.2.1c). We also analyzed the total cell lysate
using a buffer containing 1% NP-40 and observed comparable band intensities detected by the
anti-DTA antibody (Figure 4.6.3). This data further confirmed the results from the protein
synthesis inhibition assay, indicating that these non-natural modifications did not interfere with
the translocation through PA pore. In addition, we performed control experiments with LDn2 by
incubating the cells at 4'C instead of 37'C, incubating with an inhibitor of endosomal
acidification (Bafilomycin Al), or using a mutant PA (PA[F427H]) that binds LFN but arrests
translocation. In all three cases, we observed no band corresponding to LDn2, indicating the
translocation process followed the same mechanism as LFN-DTA, which requires active
endocytosis, an acidic endosome, and a functional PA (Figure 4.2.1c).
169
a
1 -000000O00-CONH.
F
I.2
b
5
0
F
H
1.4S1.21.0
.8
08
0.61 a-
+LF N-DTA
LDn1
LDn2
+ LDn3
LDn4
8 0-44+
E
'
0.4LLn
+LDn5
-DTA, No PA
+
iZ 0.2
0.0
-14 -13 -12 -11
-10
-9
-8
-
-6
-5
-
-
-
-
-
-
-
+
PA[F427H]
PA 2ng
LDn
1
+
-
+
1
+
2
+
4
+
+
2
+
2
-
M
Log [Protein Concentration
4*C
Ba
5
2
anti-DTA
anti-Erkl/2
anti-Rab5
Figure 4.2.1. Translocation of non-canonical peptides. a) Peptides 1-5 containing noncanonical side-chain modifications were ligated to LFN-DTA using SrtA to form LDnl-5 b)
Protein synthesis inhibition assay in CHO-KI. Ten-fold serial dilutions of LDn1-5 and LFN-DTA
were added to CHO-Ki in the presence of 20 nM PA and incubated for 30 minutes at 37 'C and
5 % CO 2 then washed with PBS. Treated cells were chased with 3H-Leu in Leu-free medium for
1 hour then washed and read using a scintillation counter. c) CHO-Ki cells were treated with
100 nM LDns in the presence of 20 nM PA for 12 hours. The cells were lifted and the cell
surface was trypsin digested for 5 minutes then subsequently washed with PBS. The cell cytosol
was extracted using 50 pg ml' digitonin for 10 min on ice. The cytosol extraction was analyzed
by western blot. The following controls were also analyzed: incubation at 40 C (instead of 37
*C), incubation with Bafilomycin Al, and use of mutant PA, PA[F427H]. Cropped blots are used
in western blot data (c).
170
4.2.2. Translocation of cyclic peptides
We expanded our library of non-canonical peptides under investigation to include cyclic
peptides. In comparison to linear peptides, cyclic peptides have increased structural rigidity and
proteolytic stability, making them attractive therapeutic candidates.28 We cyclized L and D
forms of the model peptide (1) using native chemical ligation (Figure 4.2.2a). As demonstrated
in Figure 4.6.1, we installed a Cys residue on the c-amino group of the Lys residue of the peptide
thioester in both L (7) and D (8) forms. The L model peptide containing an alkylated Cys residue
at the Lys position served as a linear control (6) for the translocation of the cyclic peptides. Each
peptide was sortagged onto LFN-DTA then analyzed using both the protein synthesis inhibition
and western blot assays. We found that there was a substantial reduction in translocation
efficiency for both LDn7 and LDn8, as compared to LFN-DTA and LDn6 (Figure 4.2.2b and
Table 4.6.3). Furthermore, we treated CHO-KI with 100 nM LDn6-8 in the presence of 20 nM
PA overnight then analyzed cytosolic delivery in digitonin extracted materials. The western blot
immunostained with anti-DTA showed no detectable band for LDn7 and LDn8, while the linear
control peptides LDnl and LDn6 showed similar band intensity (Figure 4.2.2c). A small amount
of LDn7 and LDn8 was detected in the total cell lysate (Figure 4.6.3), indicating some cargos
were trapped in endosomes. The western blot result was consistent with the protein synthesis
inhibition assay results, showing that translocation was substantially reduced for the cyclic
peptide conjugates, LDn7 and LDn8, compared to the linear peptide controls.
171
a
6
NH2
0
A K F R
R
P
D
S
N
VOO-CONH2
D
F
7
S
9N
8
R
b
2.0
1.81.6
1.4
--
1.2
-'- LDn8
LF N-DTA
-- LDn6
-A- LDn7
.C 1.0
0.80.6
0.4'
0.2
0.0-0.2-14 -13
-12
-11
-10
-9
-8
-7
-6
-5
C
PA 4 ng
LDn 1
anti-DTA
+
-
+
1
+
6
+
7
+
Log [Protein Concentration (M)]
8
anti-Erkl/2
anti-Rab5
Figure 4.2.2. Translocation of cyclic peptides. a) Linear control peptide and L and D cyclic
peptides to form LDn6-8, respectively b) CHO-Ki were treated with ten-fold serial dilutions of
LDn6-8 and LFN-DTA in the presence of 20 nM PA for 30 minutes. The same experimental
conditions were used as previously described. c) CHO-Ki were treated with 100 nM LD6-8 in
the presence of 20 nM PA for 12 hours then subjected to cytosolic extraction for western blot, as
previously described. Cropped blots are used in western blot data (c).
172
4.2.3. Translocation of complex small molecules
We next investigated the ability of PA to translocate complex, cytotoxic small molecules
that are frequently used in antibody-drug conjugates: doxorubicin, docetaxel, and monomethyl
auristatin F (MMAF). 29 Direct translocation of these small molecules into the cytosol would be
of great interest for the delivery of impermeable chemotherapeutics or other small molecule
-
drugs. These small molecules were conjugated through the Cys residue of the peptide, G5
LRRLRAC, using a maleimide functional group (Figure 4.2.3a and Figure 4.6.2). The peptidesmall molecule products were then sortagged onto LFN-DTA (LDn9- 11, respectively) for
investigation of translocation through PA pore. Again, we used the protein synthesis inhibition
assay and found that LDn9 and LDn1 1 translocated as efficiently as the LFN-DTA control, while
translocation was completely arrested for LDn1O (Figure 4.2.3b and Table 4.6.1). We then
treated the cells with 100 nM LDn9-11 in the presence of 20 nM PA overnight and analyzed
cytosolic delivery by immunostaining with anti-DTA antibody. We observed similar band
intensities for LDn9 and LDnl1 as compared to LFN-DTA, while no band was detectable for
LDn1O (Figure 4.2.3c). We also analyzed the total cell lysate and observed comparable band
intensities with the anti-DTA antibody (Figure 4.6.4). The western blot results were consistent
with protein synthesis inhibition results demonstrating that the PA pore was efficient in
accommodating cytotoxic molecules whose structures were either similar to (i.e. MMAF) or
distinct from (i.e. doxorubicin) polypeptides. We hypothesized that the three-dimensional size of
the molecule was a major factor contributing to translocation efficiency. Therefore, we estimated
the longest linear distance for doxorubicin and docetaxel based on published crystal structures.
Despite different possible conformational arrangements, we found that the longest linear distance
for doxorubicin in the crystal structure to be about 10.3 A,
while that of docetaxel was about
173
13.5 A,3' which is close to the estimated PA pore size (12 A).' 6 Taken together, these data
suggested that PA pore was promiscuous enough to translocate cytotoxic small molecules, but
with a size limitation due to the inherent size of the pore.
174
-Q®®Q®®~ONH
SH
9
10
ON
0
0
0
H
H
0
OH 0
0 N0
Hf4YYH
N
1114N
~0,.
0"
0
b
N
0
1.41.2"
0
0.8
0.6
PA[F427H]
PA
LDn
anti-DTA
-v LDn10
~-"Ln"I
I
-6
14 -13 -12 -11 -10 -9 -9
Log [Protein Concentration (M)]
+
4ng
11 LF,-DTA
4*
9
+
10
11
+
9
BA
+
C
L~9
-
0.01
-15
A
+
u. 0.2
-*-LF N-DTA
+
-0. 4
9
,0-.*
anti-Erki/2
anti-Rab5
Figure 4.2.3. Translocation of small molecules. a) Small molecules doxorubicin, docetaxel,
and monomethyl auristatin F (MMAF) were conjugated at the cysteine residue of G 5-LRRLRAC
then ligated to LFN-DTA to form LDn9- 11. b) CHO-KI cells were treated with ten-fold serial
dilutions of LDn9-11 and LFN-DTA in the presence of 20 nM PA for 30 minutes. The same
experimental conditions were used as previously described. c) CHO-Ki cells were treated with
100 nM LDn9-11 in the presence of 20 nM PA for 12 hours then digitonin extracted and run on
western blot, as previously described. Cropped blots are used in western blot data (c).
175
4.2.4. Translocation of intact cargo
Our studies indicate that a variety of non-canonical polypeptide cargos can translocate
through PA pore into the cell cytosol. To verify that the protein conjugates containing noncanonical polypeptide cargos passed through the pore and into the cytosol without any
truncations, we analyzed the translocation of intact cargos. For this analysis, we installed a biotin
at the C-terminus of each non-canonical polypeptide cargo that translocated efficiently (Figure
4.2.4a and Figure 4.2.4b). The C-terminal biotin provided a small tag for western blot detection
of intact cargos, which were sortagged onto LFN-DTA as previously described (LDnl-bio to
LDn6-bio, LDn9-bio, and LDnll-bio). Translocation of the biotinylated conjugates was
achieved by treating CHO-KI cells with 100 nM LDnI-bio to LDn6-bio, LDn9-bio, and LDn I1bio in the presence of 20 nM PA for 12 hours. The cells were subjected to the same harvest and
digitonin lysis conditions as previously described. Prior to western blot analysis, the SDS-PAGE
gel was run for approximately twice as long as the previous experiments in order to differentiate
the shifts in molecular weights of the conjugates containing intact cargo. The western blot in
Figure 4.2.4c was stained with anti-DTA (subsequently stained with goat anti-mouse IRdye
800CW secondary antibody) and streptavidin IRdye 680LT. The co-staining of anti-DTA and
streptavidin for all the biotinylated conjugates (LDnl -bio to LDn6-bio, LDn9-bio, and LDn 1Ibio) confirmed the presence of the intact protein conjugates in the cytosol of cells. Since
doxorubicin and MMAF were conjugated through non-amide linkages (i.e. maleimide) we relied
on shifts in molecular weight in the western blot to confirm that the conjugates remained intact
after translocation. As demonstrated in Figure 4.2.4c, the shifts in molecular weight for the
biotinylated conjugates corresponded with the 1 ng loading controls (LFN-DTA, LDnl-bio,
176
LDn 1 -bio) as well as the non-biotinylated translocated materials (LDn2, LDn9, and LDn 11)
indicating that the translocated material was indeed intact.
177
aN
NH
G
R
K
ONH
H
0
3-bio 0=
0
-
2-bio
0
HO'-
0
F
NHN0=
=
5-bio
0
NH2
~ ~
C
K,.., O
0
0K
PA
LDn
1ng
LD
1ng
ing
1-bio 11-bio
2
+
+
9
11
+
+
+
+
+
+
+
+
C
0
H
0
+
6-bio
F
+
4-blo
F
1-blo 2-bio 3-bNo 4-blo 5-bio 6-bio 9-bio 11 -bio
anti-DTA
streptavidin-680
anti-Erk1 /2
"'
""*
" ""w
"
"'
O
anti-Rab5
Figure 4.2.4. Translocation of C-terminally biotinylated cargo. a) Non-canonical peptides 1-6
were modified to contain biotin on a C-terminal lysine residue (1 -bio to 6-bio) then ligated to
LFN-DTA to form LDnl-bio to 6-bio b) Peptides containing the small molecules, doxorubicin
and monomethyl auristatin F (MMAF) were modified to contain biotin on a C-terminal lysine
residue (9-bio and 11 -bio, respectively) then ligated to LFN-DTA to form LDn9-bio and LDn 11bio c) CHO-KI cells were treated with 100 nM LDn2, LDn9, LDnl 1, LDnl-bio to LDn6-bio,
LDn9-bio, and LDn 11 -bio in the presence of 20 nM PA for 12 hours then digitonin extracted and
run on western blot, as previously described. Cropped blots are used in western blot data (c).
178
4.3. Discussion
In this paper, we investigated whether PA pore could efficiently transport non-canonical
polypeptides into the cell cytosol. We used two separate assays to evaluate translocation
efficiency. The protein synthesis inhibition assay based on DTA activity is widely used in the
anthrax toxin field to probe translocation, enabling direct comparison to the previous reports. We
also used cytosolic extraction by digitonin and western blot analysis to further investigate
delivery of the cargos in the cytosol.
Based on these two assays, we found that several variants did not perturb translocation
through PA pore. The peptide cargos contain backbone modifications, or side chain
modifications that can be used to increase proteolytic stability (e.g. P-alanine, N-methyl-alanine),
binding affinity (e.g. fluoro-phenylalanine), or serve as chemical handles (e.g. alkyne or biotin
groups) for follow-up analysis. We also demonstrated the translocation of small molecule drugs
such as doxorubicin and MMAF through PA pore. All together these examples indicate that the
PA pore is relatively promiscuous in terms of the substrates that can be translocated. Once
unfolding and translocation is initiated by the interaction of LFN with the pore in the acidic
endosome, the pore is able to accommodate the trailing segment, with high tolerance for
chemical modifications.
The digitonin extraction and western blot analysis not only confirmed the cytosolic
delivery of these materials through PA pore, but also supported the reported translocation
mechanism. The requirement of endocytosis, endosome acidification, and active PA pore was
indicated by the control experiments where no bands were detected using 4 'C, Bafilomycin Al,
or PA[F427H].
179
Model cyclic peptides and complex small molecules such as docetaxel perturbed
translocation through PA pore, as indicated by the substantial reduction or abolishment of protein
synthesis inhibition. We hypothesize that the three-dimensional size of the molecules was
responsible for arresting translocation. For the cyclic peptides, although the structures are
unknown, the potential rigidity and large size could perturb translocation. Furthermore,
translocation efficiency is independent of stereochemistry, as evidenced by the efficient
translocation of linear mirror peptides3 2 and inhibited translocation of the D cyclic peptide cargo.
Although there was some detectable protein synthesis inhibition activity with the cyclic peptides,
we observed no material by western blot of the cytosolic fractions. We note that the protein
synthesis inhibition assay is a more sensitive assay since its readout is correlated with the activity
of the DTA enzyme, which turns over at very low concentrations, while the western blot
approach does not allow us to detect very low levels of protein. Nevertheless, both detection
methods demonstrated that these large and constrained cargos had difficulty passing through PA
pore. These results provide additional evidence to support the translocation mechanism, where
cargos need to be in an extended confirmation to pass through the pore; large and rigid molecules
cannot efficiently pass through the pore. This result also provides design principles for different
cargos of interest.
Moreover, we analyzed the translocation of intact polypeptide cargo. The non-canonical
polypeptide cargos that were found to translocate efficiently were biotinylated on the C-terminus
then translocated into CHO-Kl cells. After translocation and digitonin extraction, the western
blot was analyzed for the presence of DTA as well as biotin using streptavidin labeled with an
JRdye. Co-staining of DTA and streptavidin for each conjugate as well as band shifts
corresponding to the correct molecular weights indicated that the protein conjugates containing
180
non-canonical polypeptide cargos remained intact after translocation through PA pore into the
cytosol.
Nature evolved to utilize 20 amino acids to make up proteins. Recently, there has been
great interest to incorporate non-canonical amino acids into polypeptides in order to enhance
biological properties or to study biological processes. For our study, we asked the basic question
of whether these non-natural amino acids could be efficiently translocated into the cell cytosol
through the anthrax toxin PA pore. Indeed, we found the PA translocase still functions despite
the presence of non-canonical cargo.
4.4. Experimental
4.4.1. Materials
All chemicals were reagent grade and used as supplied except where noted. 2-(1HBenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU), N-Fluorenyl-9-
methoxycarbonyl (Fmoc) and di-tert-butyl-dicarbonate (Boc) protected amino acids were
purchased from CreoSalus or Chem-Impex International. 4-Methylbenzhydrylamine (MBHA)
resin was obtained from Anaspec, CA. NN-dimethylformamide (DMF), dichloromethane
(DCM), methanol (MeOH), diethyl ether, HPLC-grade acetonitrile (MeCN) and guanidine
hydrochloride (guanidine-HCl) were from VWR. Trifluoroacetic acid (TFA) was purchased from
NuGenTec and Sigma-Aldrich. 3-maleimidopropionic acid was purchased from Toronto
Research Chemicals, Toronto, Ontario. Doxorubicin hydrochloride was purchased from
AvaChem Scientific, docetaxel was purchased from A ChemTek Inc., and maleimidomonomethyl auristatin F (MMAF) was purchased from Concortis. All other reagents were
purchased from Sigma-Aldrich and Life Technologies.
181
All moisture sensitive reactions were performed under argon atmosphere in oven-dried
glassware and in anhydrous solvents. Reactions were monitored by thin-layer chromatography
(TLC) carried on silica gel 60F 25 4 plates (EMD Chemical Inc.) and were visualized under an UV
lamp or by
charring with phosphomolybdic acid or ninhydrin
stain. Flash column
chromatography was performed on 60A silica gel (230-400 mesh) purchased from Whatman Inc.
The following primary and secondary antibodies were used goat anti-LF (bD- 17, Santa
Cruz Biotechnology), rabbit anti-DTA (ab8308 abcam), anti-Erkl/2 (137F5 cell signaling), antiRab5 (C8B1 cell signaling), goat anti-mouse IRdye 680RD (LI-COR Biosciences), goat antimouse IRdye 800CW (LI-COR Biosciences), goat anti-rabbit IRdye 800CW (LI-COR
Bis
e)
dny
-g
IDy
Lc
T IC1R1
Biosciences),
and streptavidin iRdye
680LT (LI-COR Biosciences).
4.4.2. 'H Nuclear magnetic resonance ('H NMR)
H NMR spectra were obtained with a Bruker Advance 1II 400 MHz instrument and
referenced to tetramethylsilane (TMS) at 0.00 ppm. Chemical shifts are recorded as parts per
million (ppm) in 8 scale and coupling constants J, are in hertz (Hz). Multiplicities are indicated
as "s" (singlet), "bs" (broad singlet), "d" (doublet), "t" (triplet), "dd" (doublet of doublets), "ddd"
(doublet of doublet of doublets), or "m" (multiplet).
4.4.3. Synthesis of docetaxel-maleimide
Docetaxel (100 mg, 0.124 mmol) and maleimidopropionic acid (25 mg, 0.149 mmol)
were taken in anhydrous DCM (1.5 mL), followed by addition of Mukaiyama's reagent; 2chloro-l-methylpyridinium iodide (57 mg, 0.225 mmol) and excess triethylamine (0.2 mL) at
182
00 C. The reaction mixture was slowly warmed up to room temperature and stirred 16 hours, at
which TLC analysis (5% v/v methanol in dichloromethane) indicated consumption of starting
materials and formation of a major product. The reaction was quenched by addition of ethanol
and additional stirring for 10 min, followed by concentration to dryness. The crude material was
subjected to silica flash chromatography to give the thiol reactive docetaxel derivative,
docetaxel-maleimide, 62 mg (52.2 % yield). The identity of the product was confirmed by high
resolution LC-MS and 'H-NMR.
m/z.'HMR (500 MHz, CDCl 3):
8
Calculated mass: 958.4 Da; observed [M+H]+: 959.4
8.16 (d, J=7.55 Hz, C25, C29-H, 2H), 8.02 (s, 2H), 7.60 (t,
J=7.30, 111), 7.50 (t, J=7.6, 2H), 7.48-7.30 (m, 8H+CDCl 3), 6.70 (s, maleimide, CH, 2H) 6.58 (d,
J=9.15 Hz, 2H) 6.15 (m, 2H), 5.75 (d, J= 9.80 Hz, lH), 5.70 (d, J=6.95 Hz, C2-CH, lH), 5.50
(bs, C3'-CH, 1H), 5.32 (bs, 1H), 5.26 (s, C1O-CH, lH), 4.88 (dd, J=8.20 Hz, C5-CH, IH) 4.32
(d, J= 8.55 Hz, C20-CHb, 1H), 4.27 (in, C7-CH, 1H), 4.2 (d, J=4.75 Hz, C20-CHa 1H), 3.95 (d,
J=6.30 Hz, 1H), 3.85 (m, 1H), 3.70 (m, lH), 3.50 (m, 7H), 2.55 (m, 2H), 2.48 (s, C22-CH 3, 3H),
1.98 (s, 2H), 1.90 (d, J=11.95 C14-CH2 ,2H) 1.80 (s, C18-CH 3,3H), 1.72 (s, C19-CH 3,3H), 1.35
(9Hs of -tBu), 1.22 (s, C16-CH3 , 3H), 1.10 (s, C17-CH 3 , 3H)
4.4.4. Synthesis of doxorubicin-maleimide
Doxorubicin (50 mg, 0.086 mmol) and N-succinimidyl ester of maleimidopropionic acid
(45.78 mg, 0.172 mmol) were taken in DMF (1.6 mL) and reacted for 1 hour in the presence of
N,N-diisopropylethylamine DIEA (50
iL), at which TLC analysis (20% v/v methanol in
dichloromethane) indicated completion of the reaction and formation of a major product. The
reaction mixture was quenched by diluting with DCM, followed by repetitive aqueous
extractions to remove DMF and unreacted doxorubicin. The combined organic phase was dried
over magnesium sulfate (MgSO 4 ), inorganic salts were filtered off and concentrated in vacuo to
183
dryness. The crude material was purified by silica flash chromatography (20% v/v methanol in
dichloromethane) to give the thiol reactive doxorubicin derivative, doxorubicin-maleimide, 47.7
mg (80% yield). The identity of the product was confirmed by MALDI and 'H-NMR. Calculated
mass: 694.2; Observed [M+Na]+ 717.0 m/z and [M+K]+ 732.9 m/z. 'HMR (500 MHz, CH 30D):
8 8.25 (d, J=7.65 Hz, IH), 8.00 (s, 1H), 7.85 (t, J=8.05 Hz, IH), 7.61 (d, J=8.65 Hz, I H), 6.78 (s,
maleimide, CH, 2H), 5.42 (d, J=3.65 Hz, 1H), 5.21 (bs, lH), 4.73 (d, J=3.5 Hz, 2H), 4.56 (s, COCH 2 -OH, 2H), 4.25 (dd, J=6.75, 10.15 Hz, 2H), 4.08 (m, 2H), 4.05 (s, 3H, OCH3 ), 3.72 (t, J=
6.95 Hz, 2H)) 3.60 (s, lH), 3.44 (s, 2H), 3.20 (s, 2H), 3.12 (d, 2H), 2.90 (s, 2H), 2.45 (t, J=6.90
Hz, 2H) 2.40 (d, J=14.80 Hz, 2H) 2.20 (dd, J=4.80, 9.57 Hz, 2H), 1.90 (ddd, 1H), 1.68 (dd,
)
J=4.35 and 12.95 Hz, 2H), 1.30 (s, 4H), 1.28 (d, J=6.60 Hz, 3H, CH3
4.4.5. Solid phase peptide synthesis (Boc)
Select peptides were synthesized using in situ neutralization boc chemistry. Peptides were
synthesized on 0.2 mmol scale on MBHA resin and the following side chain protection was used
for L- and D-amino acids: Arg(Tos), Asn(Xan), Asp(OcHex), Lys(2-ClZ), Lys(Alloc), and
Ser(Bzl). For the cyclic peptides, peptide thioesters were prepared using the S-trityl
mercaptopropionic acid (MPA) strategy. After peptide synthesis, the peptides were cleaved from
the resin and side chains were deprotected using 10% (v/v) p-thiocresol and 10% (v/v) p-cresol
in anhydrous HF for 1 h at 0 'C. Peptides were then triturated with cold diethyl ether, dissolved
in 50:50 A:B (A: water + 0.1% TFA and B: acetonitrile + 0.1% TFA), and then lyophilized.
4.4.6. Solid phase peptide synthesis (Fmoc)
Select peptides were synthesized using fast flow Fmoc synthesis on a 0.1 mmol scale on
aminomethyl resin with the Rink amide linker.3 3 Side-chain protection for the amino acids
184
included: Arg(Pbf), Asn(Trt), Glu(OtBu), Lys(Boc), Lys(Alloc), Ser(tBu), Thr(tBu), Trp(Boc),
and Tyr(tBu). After synthesis, peptides were cleaved from the resin with 94% TFA containing
2.5% EDT, 2.5% H 20, and 1% TIPS (v/v) for 7 min at 60 'C. After cleavage, TFA was dried
under N 2 (g), triturated with cold diethyl ether, dissolved in 50:50 A:B, and then lyophilized.
The allyloxycarbonyl (Alloc) protecting group was removed using 4.85 mmol
phenylsilane and 39.5 pimol tetrakis(triphenylphosphine)palladium(0) in DCM for 20 min at RT.
The resin was washed with DCM then DMF.
4.4.7. Protein expression and purification
His6 -SUMO-LFN-DTA(C 1 86S)-LPSTGG-His 5 , His 6 -SUMO-LFN-DTA(C186S),
SrtA*-
His 6 , wild-type protective antigen (PA), and PA[F427H] were expressed in E coli BL21 (DE3)
cells at New England Regional Center of Excellence/Biodefense and Emerging Infectious
Diseases (NERCE). Each His6-tagged protein was purified using Ni-NTA columns. Each cell
pellet (approximately 40 g) was resuspended in 100 ml of 50 mM Tris-HCI, 150 mM NaCl, pH
7.5 buffer containing 200 mg lysozyme, 4 mg Roche DNAase I, and 2 tablets of Roche protease
inhibitor cocktail. The suspension was sonicated on ice three times for 20 seconds. After
sonication, the suspension was centrifuged at 17,000 rpm for 40 minutes. The lysate was loaded
onto three 5 ml GE HisTrap FF crude Ni-NTA column pre-equilibrated with binding buffer (20
mM Tris-HCl pH 8.5, 150 mM NaCl, at pH 8.5). After loading the lysate, the columns were
washed with 100 mL binding buffer then 100 mL 40 mM imidazole in 20 mM Tris-HCl pH 8.5,
500 mM NaCl. The protein was eluted using 500 mM imidazole in 20 mM Tris-HCl pH 8.5, 500
mM NaCl. The eluted protein was buffer exchanged to remove the imidazole using a HiPrep
26/10
Desalting column (GE Healthcare,
UK). Wild-type PA and PA[F427H]
were
185
overexpressed in the periplasm of E. coli BL21 (DE3) cells and purified by anion exchange
chromatography.
4.4.8. Sortase-mediated ligation
Sortase A was used to ligate peptides containing the N-terminal oligoglycine motif to
LFN-DTA-LPSTGG (LDn). Staphylococcus aureus 59-21ISrtA (P94S/D160N/K196T; SrtA*)
evolved by Chen, et al. was used for our SrtA-mediated ligations. In order to obtain a native Nterminus, the small ubiquitin-like modified (SUMO) was cleaved off LFN-DTA-LPSTGG.
SUMO cleavage was achieved using I pg SUMO protease per mg of protein substrate at RT for
1 hour followed by gel or LC-MS analysis to confirm complete cleavage. We performed the
SrtA-mediated ligations in the presence of Ni-NTA beads in order to bind all His 6-tagged
reagents and release the His6 -free product in the supernatant. Ni-NTA beads were equilibrated
with SrtA buffer (10 mM CaCl 2 , 50 mM Tris-HCl, 150 mM NaCl, pH 7.5). In one pot, 50 [LM
LFN-DTA-LPSTGG-Hiss, 5 pM SrtA*, and 300 pM Gs-peptide were incubated with Ni-NTA
beads in SrtA buffer for 30 min at RT while rotating. After incubation, the beads were spun
down at 4 'C and the supernatant was collected. The beads were washed twice with SrtA buffer
and twice with 10 mM imidazole in 20 mM Tris-HCl pH 7.5, 150 mM NaCI (to remove any nonspecifically bound LFN). The supernatant and all washes were combined, concentrated, and
buffer exchanged three times into 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 to remove the excess
G 5-peptide. The purity of the ligated product (LDn) was analyzed by LC-MS. Concentrations of
the ligated products containing non-natural functionalities were determined using Bradford
assay.
4.4.9. LC-MS analysis
186
The purity of all peptides and proteins were analyzed by high resolution LC-MS (Agilent
6520 Accurate-Mass quadrupole time-of-flight liquid chromatography mass spectrometry
system). The samples were analyzed on an Agilent Zorbax 300SB C 3 column (2.1 x 150 mm, 5
+
pm) using 0.8 mL min~' and a linear gradient of 1-61% or 5-65% B' over 9 min (A': water
0.1% formic acid; B': acetonitrile + 0.1% formic acid). The observed mass was reported by
averaging the major peak in the total ion current (TIC). For each protein, the charge state series
was deconvoluted (maximum entropy setting) using Agilent MassHunter Bioconfirm.
4.4.10. Preparative, semi-preparative, and analytical RP-HPLC
Crude peptides were purified by reverse phase-HPLC. The peptides were dissolved in
99:1 or 95:5 A:B and 6 M guanidine hydrochloride was added depending on the solubility of the
peptide. For preparative RP-HPLC purification, we used an Agilent Zorbax SB C8 column (21.2
x 250 mm, 7 tm) at a flow rate of 10 mL min' at 1-41% or 5-45% B over 80 min. For semipreparative RP-HPLC purification, we used an Agilent Zorbax SB C 18 column (9.4 x 250 mm, 5
ttm) at a flow rate of 5 mL/min over the same gradient. UV absorbance was monitored at 214 nm.
Purity of the fractions was analyzed by MALDI or LC-MS. HPLC fractions from preparative or
semi-preparative HPLC were spotted with MALDI matrix alpha-cyano-4-hydroxycinnamic acid
(CHCA) in 50% A: 50% B and checked for the correct molecular masses. The analytical RPHPLC Agilent C 18 Zorbax SB column (2.1 x 150 mm, 5 pim) was used to confirm the purity of
fractions at a flow rate of 0.5 mL/min over a linear gradient of 1-51% B over 12 min. Analytical
HPLC UV absorbance traces were measured at 214 nm.
4.4.11. Conjugation of docetaxel, doxorubicin, and MMAF to G 5-LRRLRAC
187
Doxorubicin-maleimide (16 mg) or docetaxel-maleimide (30 mg) was added to a 16.5
mM solution of peptide G 5-LRRLRAC in DMF to a final concentration of 16.5 mM (1:1). The
reaction mixtures were allowed to stir at 36'C for 5 hours, followed by additional 10 hours at
room temperature, at which time LC-MS analysis indicated consumption of the starting peptide
and formation of G5-LRRLRAC(doxorubicin) and G5-LRRLRAC(docetaxel) peptide conjugates.
-
The products were purified by semi-preparative RP-HPLC to give 35 mg (82% yield) of G5
LRRLRAC(doxorubicin) and 50 mg (75% yield) of G5-LRRLRAC(docetaxel).
MMAF was also ligated to G 5-LRRLRAC using 2 mg maleimide-MMAF (20 mM) in
DMSO and 20 mM G 5-LRRLRAC (2.5 mg). Reaction was incubated at room temperature for 2
-
h. LC-MS indicated consumption of the peptide starting material and formation of G5
LRRLRAC(MMAF) product. The product was purified by semi-preparative RP-HPLC to give
4.8 mg (86% yield).
strategies
LRRLRAC(doxorubicin)K(biotin)
and
were
utilized
for
the
synthesis
G5-LRRLRAC(MMAF)K(biotin)
in
of
G5
which
G5
-
coupling
-
Similar
LRRLRACK(biotin) was used as a starting material.
4.4.12. Cyclization of linear peptide using native chemical ligation
Cyclization of the L-linear precursor peptide was performed by conversion of the
thiazolidine residue into a Cys residue and subsequent native chemical ligation (NCL), according
to Figure 4.6.1. To convert the thiazolidine residue into a Cys residue, 30 mg peptide (2 mM)
was dissolved in 0.2 M sodium phosphate buffer containing 6 M guanidine-HCl, 20 mM
TCEP-HC, and 0.2 M methoxyamine hydrochloride (MeONH 2 HCl). The reaction was
incubated at pH 4.0 at RT for 6 h. In the same pot, 4 NCL was performed to cyclize the peptide
188
by adding 10 mM 4-mercaptophenylacetic acid (MPAA) and incubating at pH 7.0 for I h at
RT.35 The cyclized product was purified by semi-preparative RP-HPLC. Pure fractions that were
confirmed by MALDI and analytical RP-HPLC were combined and lyophilized to give 7 mg, 4.3
[tmol (26.1% yield). The Cys residue in the cyclized peptide was alkylated using 50 mM
bromoacetamide and 20 mM TCEP-HCl in 0.2 M sodium phosphate buffer at pH 7.0 for 15 min
at RT. Once complete, the reactions were quenched with 100 mM 2-mercaptoethanesulfonate
(MESNa) and purified by semi-preparative RP-HPLC. Fractions containing the pure, alkylated
L-cyclic peptide were combined and lyophilized to give 2.3 mg, 1.4 pimol (31.7% yield).
Cyclization of the D peptide was performed using the same method as described for the L cyclic
peptide. The pure D cyclic peptide yield was 6.0 mg, 3.7 jimol (19.1% yield) and the alkylated D
cyclic peptide yield was 2.2 mg, 1.3 pimol (35.4% yield).
4.4.13. Protein synthesis inhibition assay
CHO-K1 cells were maintained in F-12K medium containing 10% (v/v) fetal bovine
serum, 100 U mL' penicillin and 100 ptg mUL streptomycin at 37 'C and 5% CO 2 . CHO-KI (3.0
X 104
per well) were seeded into a 96-well plate and incubated overnight. The following day, the
cells were treated with ten-fold serial dilutions of each construct (LDn 1-11) in the presence of 20
nM PA for 30 minutes at 37 'C and 5% CO2 . After treatment, the cells were washed three times
with PBS and then treated with 100 jiL leucine free medium containing I pCi ml]' 3H-Leucine
(PerkinElmer) for 1 hour at 37 'C and 5% CO2 . After treatment with 3H-Leu, the cells were
washed three times with PBS and then suspended in 150 gL of scintillation fluid. 3H-Leu
incorporation was measured using a scintillation counter to determine the inhibition of protein
synthesis by LFN-DTA in the cytosol. Cells treated with PA only were used for normalization.
189
Each experiment was done in triplicate. The data were plotted using Origin8 using Sigmoidal
Boltzmann Fit using equation y = A2 +
1+e
0 and
xo represents the log(EC5 o) values.
4.4.14. Translocation of LDn1-11 and cytosolic and total cell lysate extraction
CHO-KI (2.0 x 105 per well) were seeded in a 12-well plate and incubated overnight.
The cells were treated with 100 nM LDn1-11, LDnI-bio to LDn6-bio, LDn9-bio, and LDnl l-bio
in the presence of 20 nM PA for 12 hours at 37 'C and 5% C02. After treatment, the cells were
lifted and the cell surface was digested with 0.25% trypsin-EDTA for 5 minutes. The cells were
then washed twice with PBS. For cytosolic protein extraction, 0.5 x 105 cells were resuspended
in 50 pL of 50 pg mL-1 digitonin in 75 mM NaCl, 1 mM NaH 2PO 4, 8 mM Na2 HPO 4. 250 mM
sucrose supplemented with Roche protease inhibitor cocktail for 10 min on ice, and centrifuged
for 5 minutes at 13,000 rpm. For total protein extraction, cells were lysed in total cell lysis buffer
(25 mM Tris, 150 mM NaCl, I% v/v NP-40, pH 7.5) supplemented with Roche protease
inhibitor cocktail on ice for 30 minutes and centrifuged for 10 minutes at 13,000 rpm. Both
supernatants were collected for western blot.
4.4.15. Western blot of extracted material
Extracted material was run on an SDS-PAGE gel (Life Technologies) for 35 minutes at
165 V and then transferred onto a nitrocellulose membrane soaked in 48 mM Tris, 39 mM
glycine, 0.0375% SDS, 20% methanol and using a TE 70 Semi-Dry Transfer Unit (GE) for 1
hour at 17 V. The membrane was blocked at room temperature for 2 hours using LI-COR
Odyssey blocking buffer (PBS). The membrane was immunostained overnight with anti-DTA,
anti-LF, anti-Erkl/2, or anti-Rab5 in TBST at 4 'C. After incubation, the membrane was washed
190
with TBST and incubated with the appropriate secondary antibody in TBST for 1 hour at room
temperature then washed with TBST. The membrane was imaged by LI-COR Odyssey infrared
imaging system.
4.5. Acknowledgements
This work was funded by MIT start-up funds, MIT Reed Fund, Damon Runyon Cancer
Research Foundation Innovation Award, National Science Foundation (NSF) CAREER Award
(CHE-1351807) for B.L.P, and NSF Graduate Research Fellowship for A.E.R. We thank Prof. R.
J. Collier (Harvard) for his continued support, the NERCE facility (grant: U54 A1057159) for
expression of toxin proteins, and Prof. D. Lauffenburger for access to the LI-COR Odyssey
Infrared Imager. We also thank Jingjing Ling and Mike Lu their support and comments.
191
4.6. Appendix
Table 4.6.1. Peptides used in this investigation (*: alkylation)
Sequence
Observed (Da)
Calculated
(Da; mono.)
I G 5-AKFRPDSNVRG-CONH 2
1529.8
0.1
1529.8
2 G 5-(P-Ala)KFRPDSNVRG-CONH2
1529.8
0.1
1529.8
3 G 5-(N-Me-Ala)KFRPDSNVRG-CONH 2
1543.8
0.1
1543.8
4 G 5-(propargyl-Gly)KFRPDSNVRG-CONH 2
1553.8
0.1
1553.8
5 G 5-AK(PheF 3)RPDSNVRG-CONH 2
1583.8
0.1
1583.7
6 G 5-AK(C*)FRPDSNVRG-CONH
1689.8
0.1
1689.8
7 G 5-AK(C*)FRPDSNVRG (cyclic)
1672.8 +
0.1
1672.8
8 (D)-G 5-AK(C*)FRPDSNVRG (cyclic)
1672.8
0.1
1672.8
9 G 5-LRRLRAC(doxorubicin)-CONH
1864.9
0.1
1864.9
10 G 5-LRRLRAC(docetaxel)-CON H 2
2129.0
0.1
2129.0
1I G 5-LRRLRAC(MMAF)-CONH
2095.2
0.1
2095.2
1-bio G 5-AKFRPDSNVRGK(biotin)-CONH-,
1884.0
0.1
1884.2
2-bio G 5-(p-Ala)KFRPDSNVRGK(biotin)-CONH 2
1884.0
0.1
1884.2
3-bio G 5-(N-Me-Ala)KFRPDSNVRGK(biotin)-CON H 2
1898.0
0.1
1898.0
1908.0
0.1
1908.0
5-bio G 5-AK(PheF 3)RPDSNVRGK(biotin)-CON H2
1938.0
0.1
1938.0
6-bio G 5-AK(C*)FRPDSNVRGK(biotin)-CONH
1987.0
0.1
1987.0
2219.1
0.1
2219.0
2449.4 + 0.1
2449.4
2
2
2
4-bio G 5-(propargyl-Gly)KFRPDSNVRGK(biotin)-CONH
2
9-bio G 5-LRRLRAC(doxorubicin)K(biotin)-CONH
11-bio G 5-LRRLRAC(MMAF)K(biotin)-CONH,
192
2
2
Table 4.6.2. List of variants
Variant
LDnI
Sequence
PSTG(
Jn(O
JD
LDn2
LDn3
LDn4
Ok:PSG.'
Go
LDn5
LDn6NHa
LDn7
R
P
SG
LDn8
d
r
UA PSTG,
faaI.
F.I
LDn9
LDnIO
>(o
o
L-InIIA
gn !A
TAPSTGXK13
LDnI Io
@WC:T::)4PSTG--
0,~XJ
o
LDn3-bio
PffGL-AySXCOXIYSTG,
193
LDn4-bio
X
PSTG
..
LDn5-bio
TAPSTG
aJ
LDn6-bio
LDn9-bio
CL& -C
T A)-PSTGJX~1Df
,2
LDnlI1-bio
)-PSTGJ&kyZ)Do(
D
194
r
R
Table 4.6.3. EC 50 values of 30-minute protein synthesis inhibition assay. The errors represent
fitting errors from Sigmoidal Boltzmann Fit. #represents data from a separate assay. n/a indicates
that the data could not be properly fit with the Sigmoidal Boltmann Fit.
Protein
EC50 (pM)
Protein
EC5o (pM)
LFN-DTA
21
3
LFN-DTA#
74+ 12
LDnI
37
5
LDn9"
128
LDn2
31
10
LDn10'
LDn3
31
5
LDnI1'
LDn4
48
15
LDn5
24
3
LDn6
36
5
LDn7
n/a
LDn8
n/a
18
n/a
428
90
195
0
0H
-
HN
S
G, A
NH 2
SH
K- F~7f RXPYDs8 NYVYR G A
6 MGnd-HC
20 mM TCEP-HC
0.2 M MeONH 2-HCI
0.2 M NaHPO4
pH 4.0, RT 6 h
R
10 mM MPAA
pH 7.0, RT, 1h
D
R
S
s,
F
F
GGGGGAN
Alkylation
0
G-
V
RG0.2
>
G
H2 N
NCL
1. 20 mM TCEP HCI
50 mM bromoacetamide
M NaHPO,
pH 7.0, RT, 15 min
2. 100 mM MESNa
GN
0
Figure 4.6.1. Cyclization of L-linear peptide using native chemical ligation
196
D
F
N
V
G
(H
R
HMkyaasONH
0- *.H
a)
OH
0
0
0
OH
0
OH 0
0
OHOH
+
O
0
OH
0
doxorubicin
0
0
D
0
0
F
/0
lhr, RT, 80%
doxorubicin-maleimide
NH 2
OH
KrNHOH
1tNH
b)
)
0
0HO
O. ,NH
,
OH
k~k
0
H .. Ho
COHi
0
docetaxel
0
0
OH
0
O
+ H
OH
OH
HO
0
Mukaiyama's reagent
H
OHO
.400
E
Anhydrous DCM
16hr, O*C, 72%
0
0
N
0
docetaxel-maleimide
Figure 4.6.2. Synthesis of doxorubicin-maleimide (a) and docetaxel-maleimide (b).
197
2 ng
1
+
-
LFN-DTA
+
+
+
+
1
2
3
5
+
6
+
7
+
PA
LDn
+
"Ic
8
anti-DTA
anti-Erki /2
anti-Rab5
Figure 4.6.3. Western blot of total extraction of LDn 1-8. Cropped blots are used in western blot
data.
198
+
LF..-DTA
9
+
10
+
4ng
9
+
PA
LDn
11
anti-DTA
anti-Erk1/2
anti-Rab5
Figure 4.6.4. Western blot of total extraction of LDn9-l 1. Cropped blots are used in western blot
data.
199
obs.53958.O
0
4
ca. 53957.3
LPSTG, -( ) C., C, ) -I-op
)(-!J
D,(-R
G),,,xi,
55000
52500
1
2
3
4
5
6
7
8
9
10
11
12 13
Time [min]
14
15
16
18
17
19
20
21
LDn2
obs.53957.8 t 0.4
ca. 53957.3
PSTG 5~ -
F
52500 55000
I I I I I
1
2
3
4
5
I I I
7
6
8
9
10
11
12 13
Time [min]
14
15
16
17
18
19
20
21
LDn3
ca. 53971 3
JM-DCA:LPSTG>
OO®OO()CDD@-
&cNI
52500 55000
1
2
3
4
5
6
7
8
9
10
11
12 13
Time [min]
14
15
16
18
17
19
20
21
LDn4
ca. 53981.3
LPSTG,
,
III
4.6.1. LC-MS Traces
LDnl
52500 55000
1
2
3
4
5
6
7
8
9
10
11
12
13
I
14
15
16
17
18
19
20
21
Time [min]
LDn5
ca. 54011.3
-cN
LPSTG
I
.
1
200
2
52500 55000
I
3
I
4
I
I
I
5
6
7
I
8
I
9
I
I
I
I
10
11 12 13
Time [min]
I
I
I
I
I
I
I
I
14
15
16
17
18
19
20
21
LDn6
PSTG
1
@VIs
-1
obs.54118.3
Ca 54118.4
0
NH2
5
O S-
1
1
0.4
N ,oNH
(D-
@@
l----lGO
52500 55000
04
401.
IL
I I I I I I I I I I I I I I I I I I 19I
11 12 13
Time [min]
10
9
8
7
6
5
4
3
2
1
14
15
16
21
20
18
17
LDn7
R
obs.54101.2 * 0.4
D
F
ca. 54100.6
s
N
aw(fLPSTG,
NHV
R
G
S2500 55000
I
I
IIIII
IIIIIII II
1
2
4
3
6
5
8
7
14
11 12 13
Time [min]
10
9
15
17
16
19
18
21
20
.Dn8
LEd
obs.54101.0 *
ca. 54100.6
LPSTGF
50000
2
1
4
3
6
5
8
7
14
12 13
11
Time [min]
10
9
15
17
16
0.4
55000
19
18
21
20
LDn9
obs. 54293.7 0.4
"ooOH o o
0
ca. 54291 5
NH
005000
5
oNH,
LPSTG
~Th
1
2
4
3
5
I
I
6
7
Time [min]
8
9
11
10
LDn1O
0
NH
obs.54558.3
ca. 54555.6
0
0.4
OH0
C0
N
5600055000
H
P
6
Time [min]
9
10
11
201
LDn11
obs.54524.0 * 0.4
ca. 54521.8
LPSTG,-D&
G
(D
50000 55000
I
1
2
3
IL
4
5
6
7
Time [min]
8
I
9I
10
1
11
1
LDnl-bio
obs.54310.8
ca. 54312.4
LPSTG, - A (i ( Qi--(-))( -0(C
0 WN
-) -
1
2
3
4
5
('a
,
V )( Xi
6
Time [min]
ONH
&4-0 00 5-60 0
K
7
9
8
10
11
LDn2-bio
ca1
54000
1
2
3
4
5
6
7
Time [min]
8
9
0.4
54312.4
56000
10
11
LDn3-bio
*
obs.54328.2
ca. 54325.8
0.4
PSTG>)~
52500 55000
1
2
1
2
3
7
'6
3
Time [min]
8
9
10
11
LDn4-bio
obs.54338.7
0.4
ca. 54335.8
52500 55000
1
202
2
3
4
5
6
7
Time [min]
8
9
10
11
0.4
LDn5-bio
ca. 54365.7
DA PSTG5-
o
()C.Q's
(-)-
52500
5
4
3
2
1
6
-ime [mb]
8
7
9
55000
10
11
LDn6-bio
obs54475.5
0.4
ca. 54471.9
1H2
PSTGOA
(
NH
ON
N
52500 55000
1
2
3
4
5
T6 [min
mn 7
9ime
8
10
9
11
LDn9-bio
Wi)5
1
2
3
obs.54650.3 * 0.4
ca. 54647.1
LPTG
4
5
6
Time [min]
7
8
10
9
11
LDn1I-bio
*
obs.54880.5
0.4
ca. 54878.6
LPSTG5 J~K~XCX.~
520
1
2
3
4
5
6
Time [min[
7
8
9
5057500
10
11
203
4.7. References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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(2006).
Krantz, B.A. et al. A phenylalanine clamp catalyzes protein translocation through the
anthrax toxin pore. Science 309, 777-781 (2005).
Nassi, S., Collier, R.J. & Finkelstein, A. PA(63) channel of anthrax toxin: An extended
beta-barrel.Biochemistry 41, 1445-1450 (2002).
Thoren, K.L., Worden, E.J., Yassif, J.M. & Krantz, B.A. Lethal factor unfolding is the
most force-dependent step of anthrax toxin translocation. Proceedings of the National
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tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer
membranes. J. Gen. Physiol. 96, 943-957 (1990).
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Hobson, J.P., Liu, S.H., Rono, B., Leppla, S.H. & Bugge, T.H. Imaging specific cellsurface proteolytic activity in single living cells. Nat. Methods 3, 259-261 (2006).
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21.
22.
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24.
25.
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27.
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32.
33.
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35.
Arora, N., Klimpel, K.R., Singh, Y. & Leppla, S.H. Fusions of anthrax toxin lethal factor
to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which
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diphtheria toxin enzymatic domains are toxic to mammalian cells. Infect. Immun. 62,
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through the anthrax toxin pore. Angew. Chem. Int. Ed. Engl. 50, 2294-2296 (2011).
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versatile method for protein labeling. Nat. Chem. Biol. 3, 707-708 (2007).
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Adam, S.A., Marr, R.S. & Gerace, L. Nuclear-protein import in permeabilized
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Nogales, E., Wolf, S.G. & Downing, K.H. Structure of the alpha beta tubulin dimer by
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Simon, M.D. et al. Rapid flow-based peptide synthesis. ChemBioChem 15, 713-720
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Johnson, E.C.B. & Kent, S.B.H. Insights into the mechanism and catalysis of the native
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205
Chapter 5: A D-Amino Acid at the N-terminus of a Protein
Abrogates its Degradation by the N-End Rule Pathway
Portions of the work presented in this chapter have been submitted for publication:
Rabideau, A.E. & Pentelute, B.L. "A D-Amino Acid at the N-terminus of a Protein Abrogates its
Degradation by the N-End Rule Pathway."
206
5.1. Introduction
The chirality of biomolecules in nature is critical for substrate recognition, protein
binding, and product formation. While E3 ubiquitin (Ub) ligases of the Ub/proteasome system
have promiscuous substrate binding sites, the chirality of protein substrates has never been
investigated. 1-3 Since the homeostasis of a cell's protein and amino acid concentrations is
regulated by the proteasome, perturbation of proteasomal activity through substrate modification
can drastically affect the intracellular equilibrium. Here, we investigated the effect on
proteasomal degradation after the incorporation of one mirror image (D)-amino acid on the Nterminus of otherwise all L-proteins.
Varshavsky and co-workers have characterized the N-end rule as it relates to the
intracellular stability of proteins.4-6 According to the N-end rule, the identity of the N-terminal
amino acid mediates the selective degradation of specific proteins through the Ub/proteasome
system. The N-terminal degradation signals are termed N-degrons, which can range from
stabilizing to destabilizing residues. The primary destabilizing residues are R, K, H, L, F, Y, W,
and I, while D, E, N,
Q,
and C are also destabilizing after modifications. N-degrons are
recognized by N-recognins, or E3 Ub ligases, which interact with E2 Ub ligases to
polyubiquitinate proteins for proteasomal degradation. 6 To date, the N-end rule has only been
defined for L-amino acids.7- 9 The main challenge with probing the N-terminus with non-natural
functionalities is cytosolic delivery of the proteins. To overcome this, we utilized a platform
derived from nature that enables the delivery of different types of proteins into the cytosol of
cells.
207
Anthrax lethal toxin from Bacillus anthracis utilizes the protective antigen (PA) pore to
deliver lethal factor (LF) into the cytosol.' 0 Protein translocation by anthrax lethal toxin has been
extensively characterized. In short, to obtain entry into the cell, PA binds to an anthrax receptor
on the cell surface and forms the PA pre-pore.' 1-16 LF binds to the PA pre-pore then the entire
complex is endocytosed and endosomal acidification triggers a conformational rearrangement of
the PA pre-pore to form a pore.1 7-
18
LF translocates through the pore via a charge state-
dependent Brownian ratchet into the cytosol.12, 19 The N-terminal domain of LF (LFN) is
sufficient to bind to the PA pre-pore, but does not cause any intracellular toxicity. 2' The PA/LFN
delivery system has been engineered to deliver various peptide, protein, and small molecule
cargos into the cell cytosol.2 1 2 3
The majority of eukaryotic proteins from ribosomal translation are composed of L-amino
acids and achiral glycine. In order to study the intracellular stability of proteins containing Damino acids, we used Sortase A (SrtA) from Staphylococcus aureus to ligate one D-amino acid
onto the N-terminus of natural proteins for the delivery though PA pore. 24 Furthermore, we
incorporated a cleavable linker that releases the cargo protein from LFN after translocation into
the cytosol further allowing us to characterize N-terminal D-amino acids on proteins other than
LFN,
including A-chain of diphtheria toxin (DTA),2 5
(DARPin), 27,
28
2
26
designed ankryin repeats protein
and a Ras/Rap 1-specific endopeptidase (RRSPc 2 ).
29, 30
2
We opted to use a
hindered disulfide cleavable linker that was small in structure such that it could translocate
'
through the PA pore efficiently and readily cleaved in the reducing environment of the cytosol. 3
Furthermore, we applied this methodology to the targeted delivery of stabilized RRSPc 2 into
pancreatic cancer cells (AsPC-1) using the epidermal growth factor receptor (EGFR) to interrupt
the mitogen activated protein kinase (MAPK) pathway.
208
5.2. Results
5.2.1. Sortase A Attaches One D-Amino Acid onto the N-Terminus of LFN-DTA
The X-LFN-DTAmut constructs were produced through enzyme-mediated ligation of
XALPSTGG onto the N-terminus of the LFN-DTAmut. The N-terminal amino acid (X) represents
a natural L-amino acid (LX) or its mirror image D-amino acid (DX), while the remaining residues
had all L conformation (Figure 5.2.la). Each XALPSTGG peptide was ligated to G5 -LFNDTAmut using Staphylococcus aureus sortase A to yield XALPSTG-LFN-DTAmut constructs (XLFN-DTAmut; Figure 5.2. 1b). A one-pot ligation scheme was used for each reaction.
5.2.2. One N-Terminal D-Amino Acid Stabilizes LFN-DTA to Proteasomal Degradation
We used the A chain of diphtheria toxin (DTA) as a first measure of proteasomal
degradation inside cells. We specifically used a mutant form of DTA (E148S; DTAmut) that is
reported to be 300-fold less active than wild-type DTA,
allowing us to capture the translocation
efficiency of each X-LFN-DTAmut construct over a wider dynamic range than wild-type DTA.
DTA inhibits protein synthesis by ADP ribosylating elongation factor-2.2 5 ,
26
To a first
approximation, less protein synthesis inhibition (i.e. fewer DTA molecules) indicates decreased
protein stability or hampered translocation. In order to analyze protein stability, we used two
assays: 1, protein synthesis inhibition due to DTA; and 2, western blot analysis of the cytosolic
fraction. Utilizing these two established assays we probed the intracellular stability of constructs
containing N-terminal D-amino acids after translocation through PA pore (Figure 5.2.1c).
209
a.
0
4
.H2 N
H2N
R
R
L-amino acid
D-amino acid
LX
DX
b.
SrtA
X-ALPSTGG+G
X-ALPSTG
PA
receptor
proteasome
endosome
?
cytosol
Figure 5.2.1. Intracellular stability was monitored for X-LFN-DTA constructs delivered
through protective antigen pore. a. LX- (left) and DX- (right) amino acids ligated to the Nterminus of LFN-DTA (LFN: green; DTA: orange). b. XALPSTGG peptides, where X represents
either L or D amino acids, are ligated onto G5 -LFN-DTA using Sortase A (SrtA) to
form X-LFNDTA constructs. c. Translocation of X-LFN-DTA constructs is achieved using protective antigen
(PA) of anthrax toxin.
210
For the protein synthesis inhibition assay, Chinese hamster ovary (CHO-Ki) cells were
treated with ten-fold serial dilutions of each construct for 6 hours followed by incubation with
3
H-Leu in leucine-free medium for 1 hour. The fraction of protein synthesis was counted by a
scintillation counter (Figure 5.6.1 and Table 5.6.1). According to Figure 5.2.2a, regardless of
chirality, stabilizing amino acids such as A and V on the N-terminus of LFN-DTAmut had
relatively similar EC5o values as the wild-type control (LFN-DTAmIt), which contains LA at the
N-terminus. Furthermore, destabilizing residues like LW on the N-terminus of LFN-DTAm,,t had
significantly higher EC 5o values (i.e. less DTA activity) than the wild-type control while the
DW-
LFN-DTAmut construct had similar activity as the control, which suggests that D-amino acids act
as stabilizing residues.
As a second measure of protein stability endowed by N-terminal D-amino acids, the XLFN-DTAmut constructs were delivered into CHO-KI cells, lysed using digitonin lysis buffer, and
analyzed by western blot. CHO-Kl cells were treated with X-LFN-DTAmut constructs in the
presence of PA for 6 hours. As a control, select conjugates (LV-,
DV_.
LA-, DA-,
LW, and DW-
LFN-DTAm
1 Ut) were incubated in the presence of lactacystin, a proteasome inhibitor. After 6
hours, the cells were lysed using a digitonin lysis buffer then analyzed by western blot. The
western blot was immunostained with LF, Erkl/2, and Rab5 antibodies. Based on the findings in
Figure 5.2.2b, the western blot results corroborated the protein synthesis inhibition data. The
samples treated with lactacystin all showed strong anti-LF bands, indicating that the proteasome
played a key role in degrading the LX-LFN-DTAmut constructs but had no observable effect on the
DX-LFN-DTAmut
constructs. Furthermore, these data indicated that each construct translocated
efficiently into the cells, regardless of N-terminal amino acid. A similar analysis was made for
211
the remaining constructs. These data support the N-end rule for N-terminal L-amino acids, while
all N-terminal D-amino acids stabilized X-LFN-DTAmt to degradation.
To verify the mechanism of translocation and endosome escape, we incubated the X-LFNDTAmut constructs with a mutant PA (PA[F427H]),
4
a vacuolar H+-ATPase inhibitor
(Bafilomycin Al), or at 4 'C with CHO-Ki for 6 hours. In all cases, no material was found to
translocate into the cytosol by western blot (Figure 5.6.2). These controls indicated that delivery
of the X-LFN-DTAmut constructs into the cytosol is dependent on functional endocytic machinery
and PA. Moreover, we found that our observations were not cell-specific. After translocation, we
observed protein stabilization in human embryonic kidney cells (HEK-293T) and human cervical
cancer cells (HeLa), similar to the stabilization obscrvcd in CO-KI
cells (Figure 5.6.3).
5.2.3. Proteasomal Stabilization is Not an Artifact of the Sortag
SrtA ligation adds a short linkage (i.e. LPSTG5 ) between the N-terminal amino acid (X)
and the start of the protein. We used native chemical ligation (NCL)35 to prepare constructs with
native sequences for comparison with sortagged proteins. For our analysis. native LF1-T)TA.a
was synthesized containing L-alanine at the N-terminus and compared to NCL synthesized
constructs containing N-terminal
DA,
'W, or
DW.3 6
Each native construct was translocated in
CHO-Ki cells and their protein stability was compared to the sortagged conjugates using
western blot. Based on western blot of the translocated material in Figure 5.6.4, both native and
sortagged constructs containing LA,
DA,
and
DW
had similar protein stability, while LW in both
cases was highly degraded. These observations indicated that stabilization of LFN-DTAmnt
through the incorporation of one N-terminal D amino acid is not an artifact of the sortag.
212
a.
10.0-
-r
DX-LF N-DTAt
E LX-LFN-DTA,
1.0--
C
0.
E LFN-DTAt
-
'
.
E
DYDWDMD
FDHj Dp LP DKDL DRDE DTDV LV DALM LI
DD
LDNLT DS LA LL SLD LN LR LE LH LF LK LYLW
X-LF N-DTAmut
b.
1 ng
X-LF N-DT \
LDO
20 [M lactacystin
LV
DV
LA
(A
LW
DW
LD
DD
LE
DE
LF
DF
LM
DM
LN
DN
LP
Dp
LV
DV
LA
DA
LW
DW
LDm
LH
DH
LI
DI
LK
DK
LL
DL
LR
DR
LS
DS
LT
DT
LD,
anti-LF
anti-Erk1/2
anti-Rab5
1 ng
X-LFN-DTAm
LD
anti-LF
anti-Erk1/2
anti-Rab5
1 ng
X-LFN-DTAmt
LDm
LY
DY
anti-LF
anti-Erk1/2
anti-Rab5
Figure 5.2.2. One N-terminal D-amino acid on LFN-DTA enhances protein stability. a.
Translocation X-LFN-DTA constructs was analyzed by protein synthesis inhibition assay in
CHO-Ki cells after 6 hours (n = 3). EC5o values from the protein synthesis inhibition assay were
graphed for all LX-LFN-DTA or DX-LFN-DTA constructs. EC50 values (and error bars) were
determined using a Boltzmann distribution fit. b. LV, DV-, L A-, DA-, LW, and DW-LFN-DTAmut
were translocated into CHO-Ki cells in the presence of 20 nM PA for 6 hours then extracted
using digitonin lysis buffer and analyzed by western blot. As a groteasomal inhibitor, 20 1 M
lactacystin was used. Translocation of all LX-LFN-DTA or X-LFN-DTA constructs was
analyzed by western blot.
213
5.2.4. LFN-DTA with One N-terminal D-Amino Acid is Stable In Vitro
Protein translocation through the PA pore requires docking of LFN, specifically the Nterminal portion on the surface of the PA pre-pore." To decouple protein translocation and
protein degradation, we analyzed the in vitro rates of degradation of the X-LFN-DTAmut
constructs in rabbit reticulocyte lysate (RRL). Pure X-LFN-DTAmut proteins were incubated in
the presence of 70% RRL at 37 'C. Samples at various time points were pulled and analyzed by
western blot using LFN and P-actin antibodies (Figure 5.2.3a). As indicated in Figure 5.2.3b, only
LW-LFN-DTAmut
experienced significant protein degradation after 120 minutes. These data
further support the in vivo protein synthesis inhibition and western blot analyses, which
collectively suggest that N-terminal D-amino acids stabilize LFN-DTAmUt to protein degradation.
5.2.5. LFN-DTA with One N-terminal D-Amino Acid is Not Ubiquitinated
Polyubiquitination of proteins by El, E2, and E3 Ub ligases is a critical step before
proteasomal degradation.3 7 In order to identify the mode in which proteins with N-terminal Damino acids are stabilized, we used a pull-down assay. Protein constructs containing biotin (XK(bio)-LFN-DTAmut, where K(bio) represents biotin and X represents
LV, LW, DW, LR,
or
DR)
were synthesized (Figure 5.6.5 and Figure 5.6.6). Each construct was incubated with 70% RRL
for 10 minutes to allow for polyubiquitination followed by pull-down using streptavidin beads
and western blot analysis (Figure 5.2.3c). Streptavidin and anti-ubiquitin staining indicated that
only the constructs containing N-terminal LW and 'R were ubiquitinated, while the negative
control LV- as well as both
DW-
and DR-K(bo)-LFN-DTAmut constructs contained no detectable
ubiquitination. These results indicate that destabilizing amino acids like LW and
LR
recognized by the Ub/proteasome system and are readily degraded, while
are not
ubiquitinated nor degraded by the proteasome. (Figure 5.2.3d).
214
DW
and
DR
are
a. X-LF-DTA,
anti-LF
60
%pW W
DV
X-LF N-DTAMA
time (min) 0
60
10
anti-LF *-W"
%
-
#
0
60
10
-W
120
0
10
60
10
DA
120
0
10
DW
60
120
0
$woo
W
.-
120
oW
WO
M o
%=0
anti-p-actin %
anti-f-actin
120
:: : : I! :: t- .
b . 1004 A : --
LW
LA
LV
10
0
%
time (min)
OW
10
u-
60
120
tow
%WW W W
1
--. LV-LF N-DTA
-+-'A-LFN-DTA,.
,k'W-LFN-DTAm~
- ,- DV-LF i-DTAm
N
mul
--- DA-LFk-DTA,
+-DW-LF -DTA
0
20
40 60 80 100
Time (minues)
120
Pull down: Streptavidin
ladder RRL RRL
X-K(bio)-LFN-DTAmuT (kDa) only only LV LW OW LR DR
C.
188
Streptavidin
49
98
anti-Ubiquitin
62
Figure 5.2.3. One N-terminal D-amino acid prevents ubiquitination of LFN-DTA. a. The
stability of LV-, DV-, LA- , DA-, LW-, and DW-LFN-DTAmut (4 ng) was monitored in 70% RRL
over time at 37 'C and then analyzed by western blot. b. The concentration of X-LFN-DTAmut
(%) was plotted against time, based on the western blot in Figure 5.2.3a. c. X-K(bio)-LFNDTAmut constructs (1 [tM; X represents 'V, LW, DW, LR, and DR) were incubated in 70% RRL
for 10 minutes at 37 'C then pulled down using streptavidin beads for 1 hour. Elution samples
were analyzed by western blot (streptavidin and anti-ubiquitin staining).
215
5.2.6. N-terminal Stabilization is Not Protein-Specific
In order to study the stabilization of proteins other than LFN, we incorporated a cleavable
linker to separate the attached cargo from LFN once the entire construct translocated into the
cytosol but was also stable outside of the cell. The cleavable linker allowed for the intracellular
stabilization of different types of cargo to be explored using the PA/LFN delivery platform. We
used a hindered disulfide cleavable linker (Figure 5.6.7), which contains steric hindrance around
a disulfide bond thus increasing its stability toward reduction.3 1 While hindered disulfide bonds
have a wide range of rates of reduction, the penicillamine-cysteine bond was chosen since it is
more stable than unhindered disulfide bonds, but can be readily reduced at physiological
conditions over the time scale of our experiments (Figure 5.6.8).
Enabled by the hindered disulfide cleavable linker, we analyzed the stability of X-DTAm,,t
and X-DARPin protein cargo after translocation into the cell cytosol. For our analyses, 1-X
(Figure 5.2.4a) and 2-X (Figure 5.6.9a) conjugates were synthesized containing X-DTAmut and
X-DARPin linked to LFN through a hindered disulfide, respectively, where X represents LV,
DV, LA, DA, LW, and DW. The protein stability of X-DTAmut was analyzed using the protein
synthesis inhibition assay after translocation in CHO-KI cells for 6 hours. According to the
results in Figure 5.2.4b, the activity of X-DTAmut was stabilized through the addition of one Nterminal D-amino acid. These data were confirmed through the use of western blot analysis.
CHO-Ki cells were treated and lysed using the same conditions previously described. According
to the anti-LF immunostaining in Figure 5.2.4c, there was no detectable full-length material after
the 6 hour incubation. These results indicate sufficient cleavage of the hindered disulfide
material in the reducing environment of the cytosol after translocation. The bands corresponding
to cleaved LFN further demonstrate the reduction. The presence of bands corresponding to DTA
216
in Figure 5.2.4c corroborated the protein synthesis inhibition assay data; the cleaved X-DTAmut
proteins in which X represented a D-amino acid were stabilized to degradation upon cleavage
from LFN. A similar analysis was made for the X-DARPin protein cargo, in which we
demonstrated the stabilization of biotinylated DARPin using one N-terminal D-amino acid
(Figure 5.6.9b). Regardless of the side-chain functionality, both DTAmut and DARPin proteins
were stabilized using one N-terminal D-amino acid.
217
a.
LA
S0
LPSTGLRLA-N
X-ALPSTG
C.
)kNH2
media
1 ng
LFNDTA
1-X
- LD G5
VL
'V OV LA DA LWDW LV
anti-LF
OH
H0
anti-DTA
1-X
anti-Erk1/2
W40U5RW9
anti-Rab5
b.
'A AIWLPSTGLRLkN.ANH2
biotin
X-AGAKLPSTG
.
2-X
NOH
d.
X-K(bio)-DARPin
2-X 'V
1 ng
LFN
D
V
media
LV DV
-
LV DV LA
DA
LW DW LV DV
Q*su"usmma&#6 4
anti-LF
Streptavidin
anti-Erki /2
-
~.
-
_-
-~
anti-Rab5
Figure 5.2.4. N-terminal D-amino acid stabilization is not limited to LFN. a. Molecular
composition of X-DTAmut conjugated to LFN through a hindered disulfide (1-X), where X
represents G5 , LV DV LA, DA, LW, or DW. b. Molecular composition of X-DARPin conjugated
to LFN through a hindered disulfide (2-X), where X represents LV, DV, LA,
DA, LW,
or
DW.
c.
CHO-Ki cells were treated with 100 nM 1-X conjugates in the presence of 20 nM PA for 6
hours then extracted using digitonin lysis buffer and analyzed by western blot. The absence of
full-length material suggests that each construct was appropriately reduced in the cytosol.
Furthermore, LFN (LA as the native N-terminus) and X-DTAmut bands indicated cleavage and
stabilization of the X-DTAmut cargo with one N-terminal D-amino acid. The post-incubation
media was analyzed by Western blot to indicate the stability of the hindered disulfide over the
time of the experiment. d. CHO-KI cells were treated with 100 nM 2-X conjugates in the
presence of 20 nM PA for 6 hours then extracted using digitonin lysis buffer and analyzed by
western blot using anti-LF and streptavidin staining. LFN and X-DARPin bands indicated
cleavage and stabilization of the X-DARPin cargo with one N-terminal D-amino acid.
218
5.2.7. RRSPC 2 is a Ras/Rapl-Specific Endoprotease
Ras is a GTPase responsible for activating signal transduction pathways. Interrupting the
Ras GTPase activity interferes with the signal transduction of the PI3K/Akt and MAPK/Erk
pathways, which deregulates cell growth and survival processes.38 Recently, a Ras/Rap 1-specific
endopeptidase (RRSP) has been characterized from the Vibrio vulnificus MARTX toxin.2 9'
30
Since RRSPC 2 can recognize mutant forms of Ras, it is an attractive candidate for treating cancer
cells expressing mutant Ras proteins. 39 The N-terminus of RRSPC 2 is a destabilizing residue (i.e.
Phe3669); therefore, delivery would lead to rapid degradation in the cytosol. Thus, we applied
our methodology of protein stabilization to stabilize the catalytic domain of RRSP (RRSPc 2) for
its targeted delivery into pancreatic cancer cells for interruption of the MAPK pathway.
LFN conjugates containing X-RRSPc 2 were synthesized (3-X), where X represents LF or
DF
(Figure 5.2.5a). The Ras protease activity of each conjugate was analyzed in vitro (Figure
,
5.6.10 and Figure 5.6.11) using 25-fold excess KRas substrate. In all cases containing RRSPC 2
the KRas substrate was completely cleaved after 10 minutes. These results indicated that Nterminal modifications on RRSPC 2 did not affect the Ras protease activity. Furthermore, the
translocation efficiency of RRSPC 2 through PA pore was analyzed by the protein synthesis
inhibition assay. According to the results, RRSPC 2 translocated as efficiently as the wild-type
LFN-DTA control (Figure 5.6.12).
5.2.8. EGFR-Targeted Delivery of RRSPc 2 Interrupts the MAPK Pathway
Modified PA proteins have recently been developed to retarget PA for the recognition of
non-native receptors.
40-42
Delivery of LFN can be targeted to specific cells through a mutant PA
(N682A/D683A; mPA) and an added targeting domain, such as EGF to target EGFR. Since
219
EGFR is overexpressed in certain types of cancer cells, including pancreatic cancer, it is an ideal
target for specific delivery of bioactive cargos.
We investigated the delivery of stabilized RRSPC 2 (3-DF) using EGFR-targeted PA
(mPA-EGF), which recognizes EGFR.
We analyzed the effect of RRSPC 2 on the MAPK
pathway in the AsPC-1 pancreatic cancer cell line, which overexpresses EGFR (Figure 5.6.13)
and contains mutant KRas (G12D). AsPC-l cells were treated with the 3-X conjugates
containing X-RRSPc 2 in the presence of mPA-EGF, a mutant mPA-EGF (mPA[F427H]-EGF)
that inhibits translocation, or I pM EGF to outcompete mPA-EGF for EGFR. In addition,
constructs that lack the cleavable linker ('A-LFN-RRSPc 2 and LF-LFN-RRSPC2) were used as
controls. After treatment, the cells were lysed with digitonin lysis buffer or
RIPA
buffer to
analyze translocation or bioactivity, respectively. According to Figure 5.2.5b, after cleavage,
RRSPC 2 was significantly more stable than 'F-RRSPC
2
DF-
in the cytosol. Furthermore, 3- F-treated
cells contained cleaved KRas and had approximately 6-fold reduction in pErkl/2 protein levels
in the total cell lysate, similar to the positive control, LA-LFN-RRSPC 2 (no disulfide linker), with
respect to the mPA-EGF only control (Figure 5.6.14). The 3-LF-treated cells showed no obvious
Ras protease activity, similar to the negative control LF-LFN-RRSPC 2 (no disulfide linker). These
results indicated that one N-terminal
DF
stabilized RRSPC 2 to degradation and perturbed the
MAPK signaling pathway in AsPC-1 pancreatic cancer cells.
220
a.
LRLA-N
LPSTG
5
'A -
NH,
biotin
X-AG 5 KLPSTG5 RRSP
NOH
3-X
1 [tM EGF
-
-
-
-
-
-
-
LF DF LF DF LF DF
+
+
-
-
+
+
-
-
-
-
+
+
+
-
-
mPA[F427H]-EGF
-
-
+
+
mPA-EGF
-
LFN
DF
LA
-
-
LF
LA
+
3-X
-
-
2
.5ng
---
-
X-LFN-RRSPC
-
DF-K(bio)-RRSPC 2
-
b.
Streptavidin
2>
L
CO
0
anti-Erki /2
anti-Rab5
anti-KRas
C
KRas
KRasc
U-
Aii-
a
ftw
a
0
anti-pErki /2
0
low" 00 of Is to 01 Nfa,&-" is us
-
anti-Erk1 /2
00000
Figure 5.2.5. Precision delivery of stabilized RRSPC 2 through EGFR into pancreatic cancer
cells interrupts the MAPK pathway. a. Molecular composition of X-RRSPC 2 conjugated to
LFN through a hindered disulfide (3-X), where X represents 'F or DF. b. AsPC-1 cells were
treated with 100 nM 3-DF or 3-LF in the presence of 10 nM mPA-EGF for 9 hours. Controls
included mPA-EGF only, -DF only, X-LFN-RRSPC2 (X represents 'A or LF) plus mPA-EGF,
and wild-type LFN plus mPA-EGF. In addition, incubation with 1 tM EGF was used to
outcompete mPA-EGF or 10 nM mPA[F427H]-EGF to arrest translocation from the endosome.
The cytosolic fraction was extracted using digitonin lysis buffer and total cell lysate was
C-terminal
extracted using RIPA buffer. Both lysates were analyzed by western blot (KRasc:
fragment of KRas).
221
5.3. Discussion
For the first time, we have demonstrated the stabilization of proteins to proteasomal
degradation using one D-amino acid at the N-terminus. This phenomenon was evident for LFNDTAmut, DTAmut, DARPin, and RRSPC 2 proteins delivered into the cytosol using the PA/LFN
delivery system. Our findings suggest that stabilization using D-amino acids at the N-terminus of
proteins is not protein-specific. Taken together, we updated the N-end rule to include D-amino
acids as stabilizing residues.
In the ubiquitin/proteasome system, an E3 ubiquitin ligase forms a complex with an E2
ubiquitin ligase, in order to conjugate ubiquitin onto the fated protein. 43 ' 44 In particular, the UBR
box domain within E3 ubiquitin ligases is responsible for recognition of the N-terminus of
protein substrates. Analysis of crystal structures of the UBR box domains from the Ubrl and
Ubr2 E3 ubiquitin ligases revealed critical hydrogen bonds between the first two residues of the
substrates. 45' 46 We hypothesized that inverted stereochemistry at the alpha carbon of N-terminal
residue would interrupt the hydrogen bonding network, prevent substrate binding in the UBR
box, and inhibit ubiquitination. Through a pull-down assay, we demonstrated that LFN-DTAmUt
constructs containing one N-terminal D-amino acid were not ubiquitinated, while the constructs
containing an N-terminal L-amino acid were highly ubiquitinated. Thus, proteins with Nterminal D-amino acids cannot be ubiquitinated for proteasomal degradation.
The PA/LFN delivery system has been used to deliver a variety of cargos into the cytosol
of cells. Many examples of delivery incorporate the protein, peptide, or small molecule cargo on
the C-terminus of LFN, leaving cargo attachment to the N-terminus of LFN relatively
unexplored. 2 1 , 22,
222
47
Through these studies, we have found that the PA pore can tolerate short
peptide modifications at the N-terminus of LFN. Furthermore, we analyzed the intracellular
stability of three different proteins, DTAmut, DARPin, and RRSPC 2 , by installing a hindered
disulfide cleavable linker between LFN and the protein cargo. The hindered disulfide was shown
to be stable outside of the cell, yet readily reduced in the cell cytosol, freeing the cargo for
interactions with intracellular substrates. Previous explorations of translocation have involved
the delivery of proteins from N- to C-termini. For the first time, we demonstrated that the PA
pore is capable of translocating protein cargo from C- to N-termini, providing further support to
the payload promiscuity of PA.
Using the PA/LFN delivery platform, we aimed to target stable, bioactive protein cargo to
specific cancer cells. Since several pancreatic cancer cell types overexpress EGFR and more than
80% have mutant KRas, the AsPC-l pancreatic cancer cell line was an excellent model for
applying our recent findings. We targeted the delivery of the stabilized Ras/RapI endopeptidase,
RRSPc 2 to AsPC-l cells through EGFR using a mPA-EGF fusion protein. The EGFR-directed
delivery of stabilized
DF-RRSPC 2
caused cleavage of KRas, which resulted in downstream
interruption of the MAPK pathway. We believe this methodology can be expanded to stabilize
disordered proteins prone to degradation as well as to drastically extend the intracellular halflives of therapeutic proteins.
D-amino acid incorporation into polypeptides occurs in nature, albeit infrequently when
compared to L-amino acid incorporation. Select organisms including bacteria and some
eukaryotes utilize racemases to convert L- to D-amino acids or nonribosomal protein synthetases
to site specifically insert a D-amino acid within a growing peptide chain 48, . The precise roles
of naturally occurring D-amino acid containing polypeptides remains an active area of
223
investigation; however, early studies have shown that polypeptides containing D-amino acids
generally occur in higher abundance and are often more active compared to their L-counterparts
48.
Furthermore, naturally occurring cyclic peptides which would also evade the N-end
rule have
been demonstrated to have enhanced stability in cells
ways to circumvent the N-end rule.
224
50.
Perhaps nature has evolved unexpected
5.4. Experimental
5.4.1. Materials
Peptides were synthesized using Fmoc-protected L- and D-amino acids, N,NN',N'Tetramethyl-O-(1 H-benzotriazol- 1 -yl)uronium
(HBTU),
hexafluorophosphate
and
1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU)
purchased
from Creosalus and
Chemlmpex.
Dimethylformamide,
piperidine,
diisopropylethylamine, trifluoroacetic acid, and triisopropylsilane were purchased from VWR or
Sigma Aldrich. All cloning was accomplished using the QuikChange Lightning kit (Agilent) or
HiFi
DNA
Taq
Polymerase
(LifeTechnologies)
and
pET
SUMO
Champion
kit
(LifeTechnologies). All proteins were expressed in BL21(DE3) from LifeTechnologies. All
media for tissue culture was from LifeTechnologies and fetal bovine serum was from Sigma
Aldrich. For western blots, nitrocellulose membranes (GE), filters (BioRad), and PBS blocking
buffer (LI-COR) were used. We used the following primary and secondary antibodies: LF (Santa
Cruz), DTA (abcam), Erkl/2 (Cell Signaling), Rab5 (Cell Signaling), p-actin (Sigma Aldrich),
ubiquitin (Santa Cruz), KRas (Cell Signaling), pErk1/2 (Cell Signaling), donkey anti-goat
IRdye800 (LI-COR), donkey anti-goat IRdye680 (LI-COR), goat anti-mouse IRdye800 (LICOR), goat anti-mouse IRdye680 (LI-COR), goat anti-rabbit IRdye680, goat anti-rabbit
IRdye800 (LI-COR), and streptavidin IRdye680 (LI-COR). Unless specified otherwise, all other
reagents were purchased from VWR, Sigma Aldrich, or LifeTechnologies.
5.4.2. Protein Expression and Purification
G 5-LFN-DTAmUt,
LFN-LPSTGG-H 6 ,
G 5-DTAnut-C,
17C-LFN-DTAmut,
LPSTGG-H5 , G 5-RRSPc 2, and Gs-RRSPc 2 -C were expressed in BL21(DE3)
LFN-DTA-
. coli cells. The
cells were grown at 37 'C to an OD600 0.6-1.0. The proteins were induced with 0.4 mM IPTG
225
overnight at 30 'C. After induction, the cells were pelleted and resuspended in 20 mM Tris pH
7.5 and 150 mM NaCl with protease inhibitor cocktail (Roche), DNaseI (Roche), and lysozyme.
The cells were sonicated three times for 20 seconds on ice then spun down for 30 minutes at
35,000 g and 4 IC. The lysate was purified over a HisTrap FF NiNTA column (GE Healthcare)
pre-equilibrated with 20 mM Tris pH 8.5 and 150 mM NaCl. Each protein was loaded onto the
column then washed with the equilibration buffer and then with 20 mM Tris pH 8.5, 500 mM
NaCl, and 40 mM imidazole. Finally, each protein was eluted in buffer containing 20 mM Tris
pH 8.5, 500 mM NaCl, and 500 mM imidazole. The protein elutions were desalted using a
HiLoad 26/10 desalt column (GE Healthcare) into 20 mM Tris pH 7.5 and 150 mM NaCl. After
desalting the proteins, the native N-termini were obtained by cleaving the SUMO protein fusion.
For SUMO cleavage, I pg SUMO protease was added per mg protein for 1 hour at room
temperature. The cleaved sample was run over a second NiNTA column in order to obtain the
pure protein in the flow through and buffer wash.
Wild-type PA, PA[F427H], PA-EGF, and PA[F427H]-EGF were expressed in the
periplasm of E. coli BL21 (DE3) cells and purified by anion exchange chromatography followed
by size exclusion chromatography.
5.4.3. Fmoc Solid Phase Peptide Synthesis
All peptides were synthesized using Fmoc fast flow solid phase peptide synthesis (SPPS).
All peptides were synthesized on a 0.1 mmol scale on aminomethyl resin with the Rink amide
linker. Side-chain protection for the amino acids included: Arg(Pbf), Asn(Trt), Glu(OtBu),
Lys(Boc), Lys(Alloc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). For each coupling, 1 mmol
(10 eq) amino acid was dissolved in 0.4 M HBTU or HATU in DMF and diisopropylethylamine
226
was used as the activating reagent. Peptides were synthesized according to the protocol in Simon,
et al.5 using 3 minute cycles at 60 'C of 40 sec amino acid coupling then 1 min DMF wash then
20 sec 20% piperidine in DMF (v/v) deprotection and finally 1 min DMF wash. After synthesis,
peptides were cleaved from the resin with 94% TFA containing 2.5% EDT, 2.5% H 20, and 1%
TIPS (v/v) for 7 min at 60 'C. After cleavage, TFA was dried under N2(g>, triturated three times
with cold diethyl ether, dissolved in 50:50 A:B, and then lyophilized.
For biotinylated peptides, the allyloxycarbonyl (Alloc) protecting group was removed
using 4.85 mmol phenylsilane and 39.5 pimol tetrakis(triphenylphosphine)palladium(0) in DCM
for 20 min at RT. The resin was washed with DCM then DMF. Biotin (1 mmol) was coupled
using 0.2 M HBTU in DMF for 20 min at RT.
5.4.4. Sortase A-Mediated Ligation of X-LFN-DTAmut Constructs
The X-LFN-DTAmut constructs were synthesized using the one-pot SrtA-mediated ligation
strategy with an evolved SrtA (SrtA*), as reported in Liao, et al. 2 1, 24 GS-LFN-DTAmut was ligated
onto each XALPSTGG peptide using the following conditions: 50 jtM GS-LFN-DTAmut, 1 mM
XALPSTGG, and 5 ptM SrtA*. These conditions were optimized to maximize the amount of
product formed with respect to G5-LFN-DTAmut. The sortagging reactions were incubated at
room temperature for 25 minutes then NiNTA was added to each reaction mixture and rotated
for an additional 5 minutes to remove the SrtA* from the reaction. At the completion of the
reaction, the samples were spin filtered at 4 'C and then buffer exchanged three times into 20
mM Tris pH 7.5 and 150 mM NaCl to remove the excess peptide. LC-MS was used to analyze
the purity of each X-LFN-DTAut construct.
227
5.4.5. Protein Synthesis Inhibition Assay with X-LFN-DTAmut Constructs
Chinese hamster ovary (CHO-Ki) cells (ATCC) were grown in F-12K medium
.
containing 10% (v/v) fetal bovine serum and IX penicillin-streptomycin at 37 'C and 5% CO2
For the protein synthesis inhibition assay, 20,000 CHO-KI cells were plated per well in 96-well
plates 16 hours before the assay. Each X-LFN-DTAmut construct was diluted ten-fold and in
triplicate then PA was added to each well for a final concentration of 20 nM. The plates were
incubated for 6 hours at 37 'C and 5% CO 2 . After incubation, the medium was removed and the
cells were washed three times with PBS. Leucine-free F-12K containing 3H-leucine (1 ptCi mL-,
PerkinElmer) was added to each well and incubated for 1 hour at 37 'C and 5% CO 2. The
radioactivity was removed and the cells were washed three times with PBS and suspended in
scintillation fluid. Scintillation counting was used to measure the amount of 3H-Leu present,
which is indicative of DTA activity (i.e. fraction of protein synthesis). For each sample, the
scintillation counts were normalized to a PA only control. The data were fitted with a sigmoidal
Boltzmann fit using OriginLab software.
5.4.6. Translocation and Western Blot Analysis with X-LFN-DTAm.t Constructs
For western blot analysis, 200,000 CHO-Ki cells were plated per well in 12-well plates
16 hours prior to treatment. Cells were treated with 100 nM X-LFN-DTAmut construct in the
presence of 20 nM PA in serum-containing F-12K for 6 hours at 37 'C and 5% CO2 . In select
experiments, lactacystin was used to inhibit the proteasome. For this treatment, cells were preincubated with 20 pM lactacystin for 1 hour at 37 0C and 5% CO 2 and then subsequently treated
with the X-LFN-DTAmut constructs in the presence of PA. After translocation, the medium was
removed and 0.25% trypsin-EDTA was added to each well for 5 minutes at 37 'C and 5% CO2 to
remove and non-specifically bound material from the cell surface as well as lift the cells from the
228
plate. The cells were washed twice with PBS at 500 g for 2 minutes at room temperature. In
order to obtain the cytosolic fraction, cells were lysed according to the conditions previously
reported. In brief, the cells were lysed using 50 pg mL' digitonin in buffer containing 75 mM
NaCl, 1 mM NaH2PO 4, 8 mM Na2HPO 4, 250 mM sucrose and protease inhibitor cocktail
(Roche) for 10 minutes on ice then spun down at 4 'C for 10 minutes.
The extracted lysates were analyzed by western blot. Nitrocellulose membrane and filters
were soaked in buffer containing 48 mM Tris-HC, 39 mM glycine, 0.0375% SDS (v/v), and
20% methanol (v/v). Proteins from the gel were transferred to the membrane at 17 V for 1 hour
using a TE 70 Semi-Dry Transfer Unit (GE Healthcare). After transfer, the membrane was
blocked for two hours at room temperature with blocking buffer (LI-COR) and then incubated
with the appropriate primary antibody (LF, Erkl/2, or Rab5) in TBST (50 mM Tris-HCI, 150
mM NaCl, 0.1% Tween 20 (v/v)) overnight at 4 'C. The membranes were washed three times
with TBST then stained with a secondary antibody and imaged using an Odyssey Infrared
Imaging System (LI-COR). The efficiency of lysis was analyzed by anti-Erkl/2 (cytosolic
protein) and anti-Rab5 (early endosome) immunostaining.
5.4.7. Native Chemical Ligation of Native X-LFN-DTAmut
The following hydrazide peptides were synthesized in preparation for native chemical
ligation of native LFN-DTAmut: XGGHGDVGMHVKEKEK, where X represents LA,
DW.
DA,
LW, or
Additionally, 17C-LFN-DTAmut was expressed and purified from E coli using the expression
and purification protocol described above. For native chemical ligation, each peptide hydrazide
(LA
DA, LW,
and
DW-NHNH 2)
was converted to a mercaptophenylacetic acid (MPAA) thioester
using the following protocol.36 Each peptide hydrazide (5 mM; 1 mg) was dissolved in 0.2 M
229
phosphate buffer pH 3.0 then 1:10 dilution 0.2 M sodium nitrite (in 0.2 M phosphate buffer pH
3.0) was added to a final concentration of 20 mM sodium nitrite. The reaction was incubated for
10 minutes at -10 'C while stirring to form the peptide azide. After 10 minutes, the reaction was
warmed to room temperature and the pH was adjusted to pH 7.0. A 1:1 dilution of 0.1 M MPAA
(in 0.2 M phosphate buffer pH 7.0) was added to the reaction for a final concentration of 50 mM
MPAA for 20 minutes at room temperature. Formation of the MPAA thioester peptides was
confirmed by LC-MS.
In one pot, 1 eq
17C-LFN-DTAmut
(1 mg, 79.5 pM) was added to 28 eq of MPAA thioester
(1 mg, 2.0 mM) and the pH was adjusted to pH 7.0 and incubated for 10 hours at room
temperature to allow the NCL to go to completion. The reactions were monitored by LC-MS.
Upon completion of the reactions, 50 mM DTT was added to the samples for 20 minutes at room
temperature to reduce any disulfides. The samples were buffer exchanged twice into PBS pH 7.0
to remove excess starting materials. The ligated proteins (LA,
DA, LW,
and 'W-LFN-DTAmUt)
were alkylated at the cysteine residue to form pseudoglutamine at position 17, which is
asparagine in native LF. The cysteine residue was alkylated using 20 mM 2-bromoacetamide (in
0.2 M phosphate buffer pH 7.0) and pH was adjusted to pH 7.0 then incubated for 15 min then
quenched with 50 mM sodium 2-mercaptoethane sulfonate (MESNa in 0.2 M phosphate buffer
pH 7.0). The alkylated ligated proteins were desalted into 20 mM Tris pH 7.5, 150 mM NaCL
using a HiTrap desalt column (GE Healthcare) then concentrated and analyzed by LC-MS.
5.4.8. In Vitro Stability of X-LFN-DTAm.t Constructs
The in vitro stability of X-LFN-DTAmt constructs (where X represents
LW,
230
or
DW)
LV DV LA, DA,
was analyzed in rabbit reticulocyte lysate (RRL). Each X-LFN-DTAmut construct (4
ng) was incubated in a 70% RRL solution for up to 120 minutes. Time points were pulled at 0,
10, 60, and 120 minutes. At each time point, 2 jiL of each sample was added to 20 pL IX
loading dye and flash frozen. Time points were analyzed by western blot, which was
immunostained with LF and P-actin antibodies. The bands were quantified using LI-COR Image
Studio software. The rate of degradation graph was plotted according to normalized values.
5.4.9. Streptavidin Pulldown of Ubiquitinated Constructs
In order to analyze the ubiquitination of the biotinylated constructs, 1 pM X-K(bio)-LFNDTAmut in 70% RRL (20 pL total volume) at 37 'C for 10 minutes. The samples were then
incubated with 20 piL Dynabeads MyOne Streptavidin C1 beads (LifeTechnologies; washed
twice with 200 uL 50 mM HEPES pH 7.1, 200 mM KCl, 10% glycerol, 0.02% NP-40) for 1 hour
at room temperature. After incubation, the beads were washed twice with the same HEPES
buffer then eluted in 20 pl 2X loading dye for 10 minutes at 95 'C. Samples were analyzed by
western blot, which was stained with streptavidin and ubiquitin antibody.
5.4.10. Stabilization of X-DTAmut or X-DARPin after Translocation
The hindered disulfide conjugates (1-X, 2-X, and 3-X) were synthesized through Cterminal penicillamine (C*) on LFN (LFN-C*) and a C-terminal cysteine (C) on X-DTAmut, XDARPin, or X-RRSPc 2. A three-step ligation strategy was optimized for the synthesis of the
hindered disulfide conjugates: 1, sortagging to form LFN-C*; 2, sortagging to form X-DTAmut-CEllman's, X-DARPin-C-Ellman's, or X-RRSPc 2-C-Ellman's; and 3, oxidation to form 1-X, 2-X
or 3-X conjugates, where X is any amino acid on the N-terminus of DTAmut, DARPin, or
RRSPc 2 . According to the ligation scheme in Figure 5.6.7, the peptide G3LRLAC* was
synthesized and purified then sortagged onto LFN-LPSTGG under standard sortagging conditions
231
to yield LFN-C*. Protein cargos like DTAmut, DARPin, and RRSPc
2
were expressed with a C-
terminal cysteine (G 5-cargo-C) for oxidation with C*. Each G 5-cargo-C protein was expressed
and purified then activated with Ellman's reagent to form G 5-cargo-C-Ellman's. In order to
obtain the desired N-terminus, XALPSTGG peptides, where X represents
and
DW,
LV, DV,
'A,
D
LW,
were sortagged onto G 5-cargo-C-Ellman's using optimized conditions, giving a product
yield between 90-95%. Finally, LFN-C* and X-G 5-cargo-C-Ellman's were combined to form the
hindered disulfide then purified to give the desired conjugates, 1-X, 2-X, or 3-X. The hindered
disulfide comprised of cysteine and penicillamine was found to be 2-fold more stable to
reduction than a cysteine-cysteine disulfide (Figure 5.6.8).
As a first measure of X-DTAmut 's protein stability, we used the protein synthesis
inhibition assay with 1-X conjugates. CHO-Ki cells were treated with 10-fold dilutions of 1-X
conjugates for 6 hours in the presence of 20 nM PA for 6 hours at 37 'C and 5% CO 2. After 6
hours, the cells were washed three times with PBS then treated with leucine-free F-12K medium
containing 3H-leucine (1 pCi mL-, PerkinElmer) for 1 hour at 37 'C and 5% CO 2. The cells
were washed three times then resuspended in scintillation fluid and 3H radioactivity was counted.
For each sample, the scintillation counts were normalized to a PA only control.
Both the protein stability of X-DTAmut and X-DARPin was determined using western
blot analysis. CHO-KI cells were treated with 100 nM 1-X or 2-X (where X represents
'A,
DA,
LV, DV,
LW, or DW) in the presence of 20 nM PA for 6 hours at 37 'C and 5% CO 2. After 6
hours, the medium was removed and 0.25% trypsin-EDTA was added to each well for 5 minutes
at 37 'C and 5% CO 2 . The cells were washed twice with PBS at 500 g for 2 minutes at room
temperature. The cytosolic fraction was extracted using the digitonin lysis conditions and
232
analyzed by western blot as previously described. The membrane was stained with LF, DTA or
streptavidin, Erkl/2, and Rab5 antibodies then stained with the appropriate secondary antibodies
prior to imaging.
5.4.11. EGFR-Targeted Translocation of RRSPC 2
The bioactivity of stabilized RRSPC 2 was analyzed after translocation through EGFR into
AsPC-I pancreatic cancer cells (ATCC). AsPC-1 cells were treated with 100 nM 3-X (where X
represents LF or
DF)
for 9 hours in the presence of 10 nM mPA-EGF in serum-free RPMI
medium at 37 'C and 5% CO2 . Additionally, samples were treated with 100-fold excess EGF (1
ptM; LifeTechnologies) to outcompete mPA-EGF binding or 10 nM PA[F427H]-EGF to block
translocation. The following samples were used as controls: 100 nM 3DF only, 100 nM LA-LFNRRSPC 2 (N-terminal Ala), 100 nM LF-LFN-RRSPC
2
(N-terminal Phe), 100 nM wild-type LFN,
and 10 nM mPA-EGF only. After treatment, the medium was removed and 0.25% trypsin-EDTA
was added to each well for 5 minutes at 37 'C and 5% CO 2 . The cells were washed twice with
PBS at 500 g for 2 minutes at room temperature. The cytosolic fraction was extracted using the
digitonin lysis conditions and analyzed by western blot as previously described. The membrane
was stained with streptavidin, anti-Erkl/2, and anti-Rab5. The total cell lysate was extracted
using RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM sodium chloride, 0.1% SDS, 0.5% sodium
deoxycholate, 1% NP-40) plus protease inhibitor cocktail and PhosStop phosphatase inhibitor
(Roche) for 30 minutes on ice then spun at 4'C for 10 minutes. The total cell lysate was analyzed
by western blot, which was immunostained using pErkl/2, KRas, and Erkl/2 antibodies then
stained with the appropriate secondary antibodies prior to imaging. The bands were quantified
using LI-COR Image Studio software and normalized to Erkl/2. The PA only sample was set to
1.
233
5.5. Acknowledgements
This research was generously supported by MIT start-up funds, the MIT Reed Fund, NSF
CAREER Award (CHE-1351807), and a Damon Runyon Cancer Research Foundation award for
B.L.P, and a National Science Foundation Graduate Research Fellowship for A.E.R. We would
like to thank Prof. R. John Collier (Harvard) for his continued support. We thank the NERCE
facility (grant: U54 A1057159) for expressing some toxin proteins and Prof. Douglas
Lauffenburger (MIT) for the use of the Odyssey Infrared Imaging System. We also thank Prof.
Alexander Varshavsky (CalTech), Dr. Jang-Hyun Oh (CalTech), Dr. Xiaoli Liao (MIT), Dr.
Faycal Touti (MIT), and Mr. Ethan Evans (MIT) for helpful conversations.
234
5.6. Appendix
b.
a.
1.6-
(n
-
U)
-C 1.4-
1.2-
LFN-DTA.,
LD-LFN-DTA
-A -D-LF
F
N-DT
A..
.2
LF-LFN-DTA
C0
1.2-
c
C
0
1.0-
-4- DF-LFN-DTA"'"
-+-LL-LFN Uk.
-4-L-LFN
0
C:
>1
-
0.6
0.4-
LF N-DT
--
LR-LFN-DTA
A DR -LFN-DTA
0.8-
u-DT
4.
0.6-
-!-LLFN-DTA
0
- - DM-LF -DTA
-*-'P-LF N-DTAmt
-P -LFN t
0.8-
1.0-
--
T-LF N-DTA d
' V-LF NJ-TA.
DV-LFN-DTA
-
CC-
0
0.4.
0.2.
0.20.0.
-
0.0
-15
-14
-13
-12 -11 -10 -9
Log[Concentration(M)]
-15
-6
-7
-8
-14
-13
-12 -11 -10 -9
Log[Concentration(M)]
-8
-6
-7
d.
C.
1.4-
--
LF-DTA,,
t
S
-A-
1.6-
U)
1.2-
-C
1.0-
-V- LW-LF N-DTk
-4- DW-LF N-DT A.,
1.4LFN-DTA
- -H-LFTN
At
-
,
1.2
ADH-LFN-DTA
C
_
C
LE-LFN-DTA
4- DE-LFN-DTA
-
--0
-
9C 0.6-.
LI-LF N-D
0
T
C
0L
,
.0
~-0.8
D4j-LFN-DTA
O LY-LFN-DTA
0.8-
-
0 - Y-LFN-DTA,,
LA-LFN
-0-DA-LF
0.6-
N-DTA,,
LN-LFN
--
DTA,.
N-LFN-DT A
,
U)
-LFN-DTA,
0.4-
0.4-
I
0.2-
0.2-
0.0-
-
0.0
-15
-14
-13
-6
-7
-8
-12 -11 -10 -9
Log[Concentration(M)]
-15
-14
-13
-12 -11 -10 -9
Log[Concentration(M)]
-8
-7
-6
e.
,
1.21.0U)
-
C- 0.8
0
0.6
Ca
U)
0.4-
--- LF N-DTA ,,
-- KLFN-DTA.
-A-K-LF N-DTA
,
-
0C
0.2-
0.0
-15
-14
-13
-12
-11
-10
-9
-8
-7
-6
Log[Concentration(M)]
Figure 5.6.1. Translocation of X-LFN-DTAmut constructs were analyzed by the protein synthesis
inhibition assay in CHO-KI cells treated for 6 hours in the presence of 20 nM PA.
235
Table 5.6.1. EC50 values for X-LFN-DTAmut constructs translocated in CHO-Ki cells from
Figure 5.6.1.
X-LFN-DTA, X represents:
Dy
DW
0.077
0.078
DM
DF
DH
Di
Dp
Lp
DK
D
DL
DR
DE
DT
DV
LV
DA
0.084
0.085
0.089
0.092
0.094
0.094
0.099
0.10
0.11
0.11
0.11
0.11
0.12
LM
0.13
i
0.13
0.13
DD
DN
LT
DS
LA
LL
LS
LD
LN
LR
LE
LH
LF
K
Ly
LW
236
EC5 0 (nM)
0.074
0.15
0.16
0.17
0.21
0.21
0.56
0.96
1.6
1.6
1.9
2.4
2.5
3.7
3.8
5.5
DV
+
+
Bafilomycin Al
anti-LF
DA
LW
DW
+
+
alMZ,
4
anti-Erk/2
LA
-
LV
+
DW
-
LW
+
DA
+
LA
+
+
+
+
DV
+
+
LV
+
+
+
LD, LD,
+
1 ng
X-LFN-DTAmt
PA
w
OW*O*
two*
anfi-Rab5
1 ng
LA
DA
LW
+
+
+
+
+
4*C
400
400
4*C
4*C
DW
LV
DV
LA
+
+
+
DA
LW
DW
+
DV
+
+
PA[F427H]
LV
+
PA
LDt LDmt
+
X-LFN-DTA,
4*C
anti-LF
10OW
IPPW
fpow
&
-
APIWO
anti-Erk1/2
anti-Rab5
+
Figure 5.6.2. Western blot analysis of X-LFN-DTAmut constructs in CHO-Ki cells in the
presence of 200 nM Bafilomycin A1, 20 nM PA[F427H], or incubated at 4'C over 6 hours.
237
a. HEK-293T
20 [tM lactacystin
1 ng
X-LFN-DTAt
LD,
LV
DV
LA
DA
LW
DW
LV
DV
DA
LA
1ng
X-LFN-DTAt LD,
DW
= It
=
b. HeLa
LW
20 [M lactacystin
LV
DV
LA
DA
LW
DW
LV
DV
LA
DA
LW
DW
I
Figure 5.6.3. Western blot analysis of X-LFN-DTAmUt constructs delivered into HEK-293T
(a) or
HeLa (b) cells in the presence of 20 nM PA over 6 hours.
238
NH,
X-ALPSTG
1 ng
X-LF N-DTAmUt
LDmut
LD
LA
DA
X/\\17C INJ
5 --
LW
DW
LA
DA
LW
DW
anti-LF
anti-Erki /2
tsa emp Mne4mi
M
emstee tnt
aitwo
anti-Rab5
Figure 5.6.4. Sortagged (left) and native (right) X-LFN-DTAmut constructs (where X represents
LA, DA, LW, and DW) were translocated in CHO-Ki cells in the presence of 20 nM PA for 6
hours then analyzed by western blot.
239
t =O0min
X-Kbio-LFN-DTAmat
Streptavidin
anti-p-actin
LV LW DW LR
.%
t =10 min
DR
LV
LW DW LR
t = 60 min
DR
LV
LW DW
DR
two 6".. 6" 6 No W b" 0. *0%
y~~
Figure 5.6.5. In vitro degradation of X-K(bio)-LFN-DTAmut, where X represents
and DR, in rabbit reticulocyte lysate over time.
240
LR
WooM44
LV, LW, DW, LR,
20 [M lactacystin
1 ng
PA
LFNX-Kbio-LFN-DTAmut DTAmUt only
LV
LW
DW
LR
DR
LV
LW
DW
LR
DR
anti-LF
Streptavidin
anti-Erk1/2
anti-Rab5
Figure 5.6.6. LV-, LDW-, and LDR-K(bio)-LFN-DTAmut were translocated into CHO-Ki cells in
the presence of 20 nM PA for 6 hours then analyzed by western blot. As a proteasomal inhibitor,
20 [M lactacystin was used.
241
G,,LRLA- TANH2
HS-T-
LPSTGG
I-
H
-
0
LPSTG LRLA-NyNH
oxidation
HSI
-
0
LPSTG LRLA-
iNANH2
2
S
G5
os
c,CO
5
)
X-ALPSTGG
G
X-ALPSTG,
X-ALPSTG,
--
OH
LFN-C* X-cargo-C
Figure 5.6.7. LFN-C*_X-cargo-C is the oxidation product of LFN-C* and X-G -cargo-C5
Ellman's.
242
a.
LF N-C G 5-DTAmu-$
Time (min) 0
5
LF N-C* G5 -DTAmut-C
15 30 60 120 240 0 5 15 30 60 120 240 420
ft"O * %-wow
-FN-C(*) G5-DTAmut-C (53 kDa)
-
,~~~. 4"**-4
_FN-C(*) (32 kDa)
35 -DTA-C (21 kDa)
b.
0.50.0-
-C
*
LFN-C G -DTA
*
LFN-C* G5 -DTAmut-
-0.5-
<.E
-1.0-
)
I--
U.
-
S-2.0-
LL
C: -2.5-3.0-3.5-20
0
20 40 60 80 100 120 140 160 180 200 220 240 260
Time (minutes)
Figure 5.6.8. Rate of reduction for 1-X. a. Coomassie-stained SDS Page gel of 1 JAM 1-X
(disulfide) or 1-X (hindered disulfide) incubated with 1 mM DTT in 20 mM Tris pH 7.5 and 150
mM NaCl incubated at 37 'C. b. The first order reaction rates were determined by plotting ln([1X]) over time. The rate of reduction (kb,) for 1-X (disulfide) was 0.0225 min-' (R2 = 0.972) and
1-X (hindered disulfide) was 0.0116 min-1 (R2 = 0.995).
243
a.
- SGLRLA-N",.NH
LA
-LPST
5
NH
s
X-ALPST
G5
N f 4OH
H
0
1_x
b.
1.6-
1.4A)
Uf)
1.2-
UI) 1.0-
0.8-
LFNDTAmu
1-G5
-0
CT3
a> 0.6-
A
ILV
-y
1 -"V
-4--
LA
..
q-
0.40.2-
----
0.0-15
-1
-14
1 -DA
1 LW
-DW
-13
I
I
-12
-11
-10
-9
-8
-7
-6
Log[Concentraion(M)]
Figure 5.6.9. Delivery of 1-X. a. Molecular composition of 1-X. b. CHO-Ki cells were treated
with ten-fold dilutions of 1-X in the presence of 20 nM PA then analyzed by protein synthesis
inhibition assay. The results indicated that X-DTAmut was stabilized by one N-terminal D-amino
acid after cleavage from LFN-
244
-
1.0-
LF -DTA
- LFN-DTA-R RSP C2
C/)
0.8H
Cl)
C
0.6+-P
0
0.40
L-
0.2-
LL
0.0
-15
-14
-13
-12
-11
-10
-9
-8
-7
-6
Log (Concentration[M])
Figure 5.6.10. Translocation of LFN-DTA-RRSPc2 was analyzed by the protein synthesis
inhibition assay in CHO-KI cells treated for 30 minutes in the presence of 20 nM PA.
245
t=0
LFN-
3-
LF
WT
RRSPLFN
DF
t=10min
LFN-
-
WT
RRSP LFN
LF
DF
LFN
LF
WT
RRSPLFN
DF
kDa
98
62
3-X (79 kDa)
LFN-RRSPC 2(77 kDa)
49
38
WT LFN (30 kDa)
28
KRas (20 kDa)
KRas, (17 kDa)
14
6
3
KRasN (3.5 kDa)
Figure 5.6.11. In vitro cleavage of 10 pM KRas by 0.4 pM RRSPc 2 conjugates in 20 mM Tris
pH 7.5 and 150 mM NaCl at 37 *C. Time points were taken and quenched with 2x non-reducing
loading dye after 0 and 10 minutes then analyzed by coomassie-stained SDS-PAGE gel.
246
a.
la
la
20608.5 Da
KRas only
KRas on1v
18000 20000 22000
I:
2
lb
3a
1b
881.7 m/
17103.4 Da
79594.8 Da
1175.3
3a
2
KRas + 3'F
16000 18000 20000
800 1000 1200 1400
2
C
881.7 m/z
lb
75000
80000
85000
3b
lb
17103.4 Da
79595.3 Da
1175.3
m/z
800 1000 1200 1400
3c
2
KRas + 3-DF
2
d.
881.7 mz
16000
1800
20000
lb
80000
7500
3c
85000
77464.7 Da
lb
17103.5 Da
1175.3
3c
2
KRas + 'A-LFN-RRSPC2
L_
800
1000 1200 14D0
16000
18000 20000
la
e.
75000
3d
80000 85000
300631.6
Da
20608.8 Da
1a
3d
KRas + LFN
1
2
4
5
6
Time
7
Imin]
8
9
10
11
18000 20000 22000
30000
3200
Figure 5.6.12. In vitro cleavage of 10 pM KRas by 0.4 pM RRSPC 2 conjugates in 20 mM Tris
pH 7.5 and 150 mM NaCl at 37 'C after 10 minutes. Time points were taken (from the same
reactions as in Figure 5.6.11) and quenched with 1:1 water:acetonitrile with 0.1% TFA after 10
minutes then analyzed by LC-MS. Peak la is uncleaved KRas (calc 20608.2 Da), peak lb is Cterminal portion of cleaved KRas (calc 17103.1 Da), peak 2 is N-terminal portion of cleaved
KRas (calc 3523.1 Da), peak 3a is LFNC* LF-RRSPC 2 C (calc 79582.5 Da), peak 3b is
LFNC* _DF-RRSPC 2C (calc 79582.5 Da), peak 3c is LFN-RRSPc2 (calc 77459.7 Da), and peak 3d
is wild-type LFN (calc 30631.2 Da).
247
--
1.8 -
LF N-DTA + PA
SLFN-DTA + PA-EGF
~LFN-DTA + PA +1 pM EGF
- LFN-DTA + PA-EGF + 1 sM EGF
.In 1.6
1.4C
0.I
" 0.8C
.2) 0.60.
0.2
-15 -14 -13 -12 -11 -10 -9 -8
Log[Concentration(M)]
-7
-6
Figure 5.6.13. Targeted delivery of LFN-DTA through EGFR was analyzed by the protein
synthesis inhibition assay in AsPC-1 treated for 30 minutes in the presence of 10 nM PA or PAEGF. For competition controls, 1 pM EGF was added to 100 nM LFN-DTA plus 10 nM PA
or
PA-EGF.
248
LA
-
LF
LA
-
-
-
3-X
-
-
DF
-
-
-
LF
DF
LF DF LF DF
LFN
-
-
-
mPA-EGF
+
-
-
+
+
+
+
+
+
+
-
-
mPA[F427H]-EGF
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
+
X-LFN-RRSPC2
-
-
+
1 [M EGF
anti-pErkl/2
S1.2.
a) 1 .0
30.8
c" .2~0.6-
-
-J. 4
set to 1.
249
5.6.1. LC-MS Traces
NH2
*
obs.52052.4
0.4
ca. 52051.2
LA \\17C
500.0 52500
1
2
3
4
5
6
7
Time [min]
8
10
9
11
LA-LFN-DTA NCL
obs.52053.2
Ca. 52051.2
DA /\^17C
50000
1
2
3
4
5
1
3
4
5
DA-LFN-DTA
6
7
6
7
Time [min]
8
9
0.4
52500
10
11
NCL
NHW
obs.52168.5 * 0.4
ca. 52166.3
LW /\/\17C
3
50000 52500
1
2
3
5
4
6
Time
(min]
7
8
10
9
11
LW-LFN-DTA NCL
NH
<
obs.52168.8*
ca. 52166.3
DW^^YX1 7C
50000
1
2
DW-LFN-DTA
250
3
NCL
4
5
6
Time [min]
7
8
9
52500
10
11
0.4
*
obs.52829.9
LA-ALPSTG5
0.4
ca. 52830.0
52000 54000
2
1
5
4
3
6
Time [mini
7
8
9
10
11
LA-LFN-DTA
*
obs.52830.1
0.4
cCa. 52830.0
A-ALPSTG5
52000 54000
2
1
3
4
5
6
Time [min]
7
8
9
10
11
DA-LFN-DTA
obs.52874.6
ca. 52874.0
LD-ALPSTG
0.4
52000 5400
1
2
3
4
5
6
Time [min]
7
8
9
10
11
LD-LFN-DTA
obs.52874.2 * 0.4
ca.
Dr"
L/-ALPSTG
52874.0
52000 54"0
1
2
3
4
5
6
Time
[min]
7
8
9
10
11
DD-LFN-DTA
251
obs.52888.1
ca. 52888.0
LE-ALPSTG 5
0.4
52000 54000
I
1
2
3
--~
T
4
5
8
7
8
ime [min]
9
10
11
LE-LFN-DTA
DE-ALPSTG
obs.52888.1
*
G
ca. 52888.0
52000
1
2
3
4
5
6
8
7
lime [min]
9
0.4
54000
10
11
DE-LFN-DTA
obs.52906.8 0.4
ca. 52906.1
LFALPSTG
52000 54000
1
2
3
4
5
6
ime [min]
8
i
9
0
11
LF-LFN-DTA
obs.52906.6
ca. 52906.1
DFALPSTG 5
52000 54000
1
2
3
4
5
6
ime [mini
DF-LFN-DTA
252
7
8
9
10
11
0.4
obs.52896.0 *t 0.4
ca. 52896.1
LH-ALPSTG
52000
8
8
7
6
Time [min]
5
4
3
2
1
9I
54000
1
11
1
10
LH-LFN-DTA
DH-ALPSTG
5
obs.52895.9
ca. 52896.1
.-
52000
6
Time [mini
5
4
3
2
1
7
54000
11
10
9
8
0.4
DH-LFN-DTA
obs.52872.3
0.4
ca. 52872.1
L 1 -ALPSTG
52000 54000
1
2
3
4
5
7
6
Time [mini
8
9
10
11
LI-LFN-DTA
obs.52871.9
0.4
ca. 52872.1
DI -ALPSTG
52000 54000
1
1
I
2
I
3
I
4
I
5
7
6
Time [mini
8
9
10
11i
DI-LFN-DTA
253
obs.52887.1
LK-ALPSTG
0.4
ca. 52887.1
..
50000 55000
1
2
3
4
8
Time [min]
9
10
11
LK-LFN-DTA
*
obs.52887.2
DK-ALPSTG 5
52000
1
T8
2
0.4
ca. 52887.1
3
4
9
i
ime [min)
54000
10
11
DK-LFN-DTA
obs.52872.7
ca. 52872.1
LL -ALPSTG
0.4
52000 54000
1
2
3
4
5
6
Time [min]
7
8
9
10
11
LL-LFN-DTA
obs.52872.8 0.4
ca. 52872.1
DL-ALPSTG
I
1
2
DL-LFN-DTA
254
3
4
5
6
Time [min)
8
5200054000
9
10
11
0.4
obs.52890.1
ca. 528 90.4
I
LM-ALPSTG,
52000 54W0
1
5
4
3
2
6
ime [min]
8
7
9
10
11
LM-LFN-DTA
*
obs.52890.1
ca. 52890.3
DM-ALPSTG.
52000
2
4
3
5
8
Time [min]
9
0.4
54000
10
11
DM-LFN-DTA
LN-ALPSTG 5
ca. 52873.0
Imam
54000
_]i200
2
1
3
0.4
obs.52873.4
4
5
6
Time [min]
7
8
9
10
11
LN-LFN-DTA
*
obs.52873.3
0.4
52873.0
D N-ALPSTG 5ca.
52000 54"0
1
2
3
6
lime
[mini
7
i
9
10
11
DN-LFN-DTA
255
obs.52856.4
ca. 52856.0
LP-ALPSTG 5
0.4
52000 54000
1
2
3
4
5
6
Time [minj
8
7
9
10
11
LP-LFN-DTA
obs.52856.3
ca. 52856.0
DRALPSTG
0.4
52000 54000
---- 1
2
v
4
3
5
T
Time [min)
7
8
9
10
11
DP-LFN-DTA
obs.52915.7
ca. 52915.1
0.4
.
LR-ALPSTG 5
52000 54000
3
4
5
6
Time [mini
7
8
9
10
11
LR-LFN-DTA
5
obs.52915.6 * 0.4
Ca. 52915.1
-
DR-ALPSTG
52000 54000
1
2
DR-LFN-DTA
256
3
4
5
6
Time [min
7
8
9
10
11
*
obs.52845.5 0.4
Ca. 52844.9
LS-ALPSTG5
52000
I
I
I
1
I
1
2
3
4
5
I
i
6
7
Time [min]
8
9
40
10
11
LS-LFN-DTA
obs.52845.0
D
*0.4
ca. 52844.9
S-ALPSTG
52000 54000
3
7
lm2
Time [min]
4e
8
9
1
1
DS-LFN-DTA
obs.52860.0
0.4
ca. 52860.0
LT-ALPSTG5
52000 54000
1
2
3
4
5
6
7
8
9
10
11
Time [min]
LT-LFN-DTA
obs.52860.1 0.4
DT-ALPSTG
ca. 52860.0
5
52000 54000
7
Time
[min]
8
9
10
11
DT-LFN-DTA
257
obs.52858.3
ca. 52858.0
LV-ALPSTG
0.4
52000 54000
1
2
3
4
5
6
7
8
9
10
11
Time [mini
LV-LFN-DTA
obs.52858.2
ca. 52858.0
DV-ALPSTG
0.4
52000 54000
1
2
3
4
5
6
Time
[mini
7
8
9
10
11
DV-LFN-DTA
obs.52944.7
ca. 52945.1
LW -ALPSTG
5
0.4
152000 64000
1
2
3
4
5
6
Time
[min]
7
8
9
10
11
LW-LFN-DTA
obs.52944.8
ca. 52945.1
*
DW-ALPSTG
52000 54000
1
2
3
4
6i
Time
DW-LFN-DTA
258
[min[
9
10
11
0.4
*
obs.52922.2
0.4
LY-ALPSTG
.
ca. 52922.1
52000 54000
1
2
3
4
5
6
Time
[mini
7
8
9
10
11
LY-LFN-DTA
obs.52922.3
ca. 52922.1
DYALPSTG
0.4
52000 54000
51
5
6
Time
[mini
7
8
9
10
11
DY-LFN-DTA
obs.53500.9 * 0.4
a. 53499.0
biotin
LV -AG KLPSTG
50000 58000
4
6
Time [min]
7
8
9
10
11
LV-K(bio)-LFN-DTA
obs.53587.6
ca. 53584.6
biotin
LWAGKPSTG
..
50000
2
3
4
5
6
Time [mini
7
8
9
0.4
500
10
11
LW- K(bio)-LFN-DTA
259
*
obs.53587.6
biotin
0.4
DWAGKLPSTG
ca. 53584.6
50000 55000
1
2
3
4
5
1
2
3
4
5
6
6
7
Time [min]
8
9
10
11
DW-K(bio)-LFN-DTA
biotin
obs.53558.3
LR -AGKLPSTG
0.4
ca. 53554.6
5
50000 55000
1
2
3
4
5
6
7
8
Time [mini
9
10
11
LR-K(bio)-LFN-DTA
biotin
obs.53557.6
ca. 53554.6
DR-AG5LPsTG
0.4
50000 55000
1
2
3
4
5
6
Time [mini
7
8
9
10
11
D RK(bio)LFDTA
LPSTGLRLA-Nfl
I.
NH,
obs.53089.1
ca. 53087.5
XOH
G
GWO
52500 55000
1
2
3
LFN-C*_G 5 -DTA-C
260
4
5
6
Time [mini
7
8
9
10
11
0.4
LPSTGLRA
NEW
obs.53629.1
53628.2
'ca.
0.4
.NX .OH
A-ALPSTG
52500 55000
3
2
6
5
4
Time [mini
9
8
7
11
10
LFN- *_LA-DTA-C
M
LPSTGLRLA-N
NH,
DA-ALPSTG,
obs.53629.3
ca. 53628.2
0.4
OH
52500 55000
6
5
4
3
2
1
Time [mini
7
8
10
9
11
LFNC* DA-DTA-C
0 NH
LPSTG,LRL-A-N
obs.53657.1 0.4
s
ca.
1
'V-ALPSTG,
53656.2
OH
w
52500 55DOO
- - -L
2
1
6
5
4
3
Time [mini
7
I8
9
10
11
LFN-C _LV-DTA-C
NV-ALPSTG 1
obs.53656.4
H
MLSGRA-sJ
.OH
ca.
H
*0.4
53656.2
52500 55000
1
2
3
5
6
7
8
i
1o
i1
Time [mini
LFN-C*_DV-DTA-C
261
0
OEN=LPSTGLRLAgNH
OH
.N
W-ALPSTG,
obs.53744.6 0.4
ca. 55743.3
52500 55000
1
2
4
3
5
6
Time
[min]
7
8
I',
LFN-C* LW-DTA-C
0
-
LPSTGLLA- N
NH
obs.53744.5
ca. 55743.3
sA
0.4
lW-ALPSTGWN
52500 55000
'
2
'3
'
'
'
4
5
6
7
'
I
1
ime [min]
8
9
10
11
LFN-C* DW-DTA-C
-LPSTGLRLA-N
obs.46637.4
*
0.4
ca. 46634.2
biotin
A-AGLPSTG
os
45000 47500
1
2
3
4
5
6
Time
[mini
7
8
9
10
11
LFN-C* L A-DARPin-C
4LPSTGLRLA64
6.4
4
ca.
biotin
46634.2
osf
-A-AG,KLPSTG
46000 47500
1
LFNC*
262
2
3
4
DA-DARPin-C
5
6
Time [min]
7
89
10
11
LVAG 5
obs.46665.4
KLILPSTGLRLA-
os,
-
LV-AG,KLPSTG
0.4
ca. 46662.3
4500047500
~~1 ~~
3
2
5
4
6
Time [min]
10
9
8
7
11
LFN-C* LV-DARPin-C
LPSTGLALA-
obs.46664.7
NH
ca.
bioinT
0.4
46662.3
X
-V-AGKLPSTG,
4.7500
2
1
3
7
6
5
4
Time [min]
8
9
10
11
LFNC _ DV-DARPin-C
-LPSTGLRLAbiotin
LW-AGKLPSTG,G
obs.46751.9
ca. 46749.4
0.4
,&-
5
45000 47500
2
1
3
4
5
6
Time
[mini
7
8
9
10
11
LFN-C* LW-DARPin-C
MLPSTGLRLA
obs.46751.5
<O,
0.4
ca. 46749.4
biotin
OW-AG KLPSTGo,
45000 47500
1
1
2
3
4
5
2
3
4
5
6
6
Time
[min]
7
8
9
7
8
9
10
11
LFNC* DW-DARPin-C
263
obs.77456.1
LPSTG, RRSP
ca. 77450.2
75000
1
2
3
4
5
0.4
6
Time [min[
8
7
9
80000
10
11
LA-LFN-RRSPC2
obs.77534.7 0.4
'FU
LPSTG, RRSP
ca. 77526.3
75000 80000
1
2
3
4
5
6
7
Time [mini
8
9
10
11
F-LFN-RRSPC2
LPSTGLRLA
obs.79591.2
ca. 79582.5
biotin
LF-AGKPSTG
1
LFN-C*
2
3
RRSPJ
4
5
os
L_
0
6
7
Time [min]
75000
8
9
80000
10
11
F-RRSPc 2 -C
obs.79590.4
T
ca. 79582. 5
biinITDF-AGKLPSTGjR sf
pOH
75000
....... oj
Time [min]
LFN-C* DF-RRSPC2-C
264
0.4
8
9
80000
10
11
0.4
5.7. References
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