UMass Dartmouth Botulinum Research Center
Introduction Symposium
Insights into the mechanism of BoNT/A neuronal
persistence and avenues for novel therapies
George A. Oyler MD, PhD
Friday August 24, 2007
Proposed Mechanisms of Persistence
• Cleavage product of SNAP25 by BoNT/A is stable and acts as
dominant negative for synaptic transmission. This requires cleavage
products from different serotypes to have different recycling time.
• The catalytic subunit is stable and persists in an active form. This
requires the different serotypes to have different stability.
• Differential compartmentalization of the catalytic subunits of different
serotypes:
YFP-BoNT/A LC is trafficked through multiple vesicle
compartments in neuronal cells
GFP-BoNT/A LC is trafficked in a polarized fashion and
accumulates in specific sites of neuronal cells
YFP-BoNT/E LC is also trafficked to plasma
membrane
Differential compartmentalization alone cannot account
for differences in persistence
YFP-LCE
RFP-LCA
N18 neuroblastoma
Merged
BoNT/A and /E LC stability in SH-SY5Y cells
YFP-LCE
CHX:
anti-GFP
anti-actin
0
1 2 4 6 8
YFP-LCA
0
1 2 4 6 8
Ubiquitin proteasome system
YFP-LCA
YFP-LCE
YFP
YFP-LCA
YFP-LCE
YFP
250
100
75
50
25
IP: anti-GFP
IB: anti-HA
IP: anti-GFP
IB: anti-GFP
+ HA-Ub
YFP-BoNT/E LC is ubiquitinated more extensively than
YFP-BoNT/A LC in N18 cells
a-YFP
a-Ubi
Ubin YFPBoNT LC
YFP-LC
Myc-LC/A
Myc-LC/E
HectD2
TRIM63
TRIP12
Cbl-b
E4A
TEB4
Triad3
DZIP3
Designer E3 ligases that target toxins for
proteasome degradation
Natural proteasomal turnover of BoNT LC
Ub
Ub
Cellular E3 ligase
Ubiquitin
E3-ligase
Target
Ub
Ubiquitin
E3-ligase
Target
bindin
g
domai
n
Ub
bindin
g
domai
n
ubiquitination
BoNT LC
“slow”
BoNT lc
Proteasome
recognition
Ub
Ub
slow BoNT
degradation
Ub
Ub
Ub
Ub
BoNT lc
Degraded BoNT lc
Proteasome
Complex
Ub
Ub
Ub
Ub
Designer E3 ligases that target toxins for
proteasome degradation
Enhanced proteasomal turnover of BoNT LC
Ub
Ub
“Designer E3 ligase”
E3-ligase
Ub
E3-ligase
Ub
“fast”
Ub
Ub
Ub
Ub
LC binding
agent
Therapeutic
fusion protein
LC binding
agent
ubiquitination
BoNT lc
BoNT LC
Proteasome
recognition
Ub
Ub
Accelerated
BoNT
degradation
Ub
Ub
Ub
Ub
BoNT lc
Degraded BoNT lc
Proteasome
Complex
Antidotes that accelerate turnover of
intraneuronal BoNT LC
Background:
•
The concept of targeted proteolysis of cellular proteins has been
demonstrated several times in the literature.
•
SNAP25/nc based “proof of concept” for a designer E3-ligase strategy.
•
For potential therapeutic applications, we are currently developing:
1.
Camelid antibodies as more effective LC targeting domains.
2.
Optimal E3-ligase domain (e.g. F-box proteins).
3.
Neuronal delivery vehicle.
BIR1
BIR2
BIR3
C
C
Zn
C
C
C
C Zn
C
H
XIAP BIR1-3 domains
recognizes and binds caspase
substrate for ubiquitination
XIAP RING is
Catalytic E3 domain
SNAP-25/NC
SNAP25 replaces XIAP BIR1-3
domains and recognizes BoNT
as substrate for ubiquitination
BoNT/A and E
noncleavable Cterminus of SNAP25
C
C
Zn
C
C
C
C Zn
C
H
Designer E3 ligase accelerates BoNT/A LC turnover
in N18 cells
Relative amount of 35S
labelled YFP-LC
SNAP-25/NC-RING +
proteasome inhibitor
(MG132)
cells alone
SNAP-25/NC Control
SNAP-25/NC-RING
0
5
10
15
Time (hours)
20
25
SNAP-25/NC-RING “designer E3 ligase”
substantially accelerates proteasomemediated degradation of recombinant
BoNT/A in transfected neurons
Camelid VHH forms a compact well-folding
single peptide structure
Spinelli S,
Desmyter
A, Frenken L,
Domain swapping of a llama VHH
Ver r ips T, Tegoni M,
Cambillau C.
. FEBS Lett.
2 004 ;5 64(1 - 2 ):3 5 - 40.
VHHs as targeting domains
Background:
•
VH domains of camelid HcAbs (VHHs) are easy to produce as recombinant
proteins in E. coli and have excellent hydrodynamic properties.
•
These antibodies are also generally superior for enzyme neutralization as they
bind better into “pockets” such as found in enzyme active sites.
Progress:
•
We hyper-immunized two alpacas in New Zealand with A-LC and prepared a
VHH phage display library.
•
We obtained five unique A-LC binding positives screening at high stringency,
three with particularly high apparent affinity.
Elisa analysis of Anti-BoNT/A Lc VHH clones
SDS-PAGE (Coomassie)
A6 E3 D4 G6 B8
A6 E3 D4 G6 B8
VHH
ELISA
on BoNT/A
LC
ELISA
vs BoNT/A
LC
350 ng each
Absorbance
3.5
SDS-PAGE (Coomassie)
350 ng each
3
2.5
Series1
A6
A6
E3
E3
Series2
D4
D4
Series3
G6
G6
Series4
B8
B8
Series5
2
1.5
1
0.5
0
2.5e6
5e5
300
10e4
60
2e4
12.5
4000
2.5
nM VHH
800
0.5
160
0.1
32
0.02
6.4
VHH-B8 selected as having the
highest affinity for BoNT/A LC
YFP-SNAP25-CFP
cleavage activity (%)
GST-VHH-B8 potently inhibits BoNT/A Lc
100
100% inhibition of 2.5 pM
of BoNT/A LC by 0.4 ug,
or ~10 pM, of VHH-B8
75
50
25
0
0
0.1
0.2
0.3
0.4
0.5
GST-VHH-B8 (ug)
0.6
0.7
0.8
VHH-B8 was expressed as a GST fusion protein (GST-VHH-B8). Assays were
conducted with 0.2 ug BoNT/A LC in 100 ul reaction volume (25 nM), 0.5 ug YFPSNAP25-CFP substrate (80 nM), and increasing concentrations of GST-VHH (B8).
Inhibition of BoNT/A LC activity by GST-VHH (B8) was near stoichiometric.
0.9
Anti-A-LC VHH co-localizes with A-LC in cells
YFP Channel
+ YFP-VHH-RING
- CFP-BoNT/A LC
+ YFP-VHH-RING
+ CFP-BoNT/A LC
- YFP-VHH-RING
+ CFP-BoNT/A LC
CFP Channel
Anti-A-LC VHH
localizes to cytosol in
transfected Neuro2a
cells.
When co-expressed
with BoNT/A LC, the
VHH localizes with ALC at the plasma
membrane.
Western Blot for Steady State level of CFP-BoNT/A
LC with YFP-VHH-RING Designer ligases
a-BoNT/A LC
N2a cells Expressing Yes-SNAP25-Cer FRET Indicator
YesFP
CerFP
YesFP
FRET
CerFP
SNAP25 (1-206)
FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media
VHH-B8 inhibits A-LC co-expressed in cells
Transfected BoNT/A LC activity in N2a cell lysates is inhibited when
co-transfected with VHH-B8 constructions using YFP/SNAP25/CFP
FRET reduction assay
Cer BoNT LC
Cer BoNT LC
Y B8 only
Cer BoNT LC
Y B8 Ring
Cer BoNT LC
Y B8 TrCP
TrCP designer ligases themselves turnover rapidly
MG135
treatment
M
-
4 hr
o/n
Inhibition of proteasomes with
MG135 stabilizes TrCP fusion
protein and leads to accumulation of
poly-ubiquitinated forms
YFP/VHH-B8/TrCP
Anti XFP 1:5000
VHH based designer ligases prevent YFP-SNAP25CFP cleavage in intoxicated M17 cells.
Anti XFP 1:5000
1: YFP-B8 +/A24+24
2: YFP-B8 +/control
250
YFP-B8
YFP-VHH
B8-Trcp
YFP-VHH
B8-RING
3: Indicator only +/A24+24
150
4: YFP-VHH B8-Trcp +/A24+24
100
75
NC YFP-SNAP25-CFP 5: YFP-VHH B8-Trcp+/control
YFP-VHH B8-RING
6:YFP-VHH B8-Trcp +/A24
50
C YFP-SNAP25-CFP
YFP-B8
37
1
2
3
4
5
6
7
8
M
9
10
11 12
7: Indicator only +/A24
8: Indicator only +/control
25
9: YFP-VHH B8-RING+/A24+24
10: YFP-VHH B8-RING +/control
11: No transfection +/A24+24
Anti SNAP 1:5000
1
2
3
4
5
6
7
12: No transfection +/control
8
M
9
10
11 12
Jun. 11th-15th.2007
Designer E3 ligases that target toxins for
proteasome degradation
Preferred strategy for targeted destruction of BoNT:
a smaller, modular “designer E3 ligase”
E3 ligase targeting domain,
e.g. minimal TrCP (F-box)
E3-ligase
VHH-LC targeting domain
LC binding
agent
Delivery vehicle to neuronal cytosol
BoNT LC
Note that the
targeting domain
can be
interchanged to
create botulism
therapeutics for
each serotype
once an A-LC
prototype has
been developed.
BoNT Lc
BoNT Hc-N
BoNT Hc-C
BoNT/A Heavy Chain can be used for trafficking cargo to
hippocampal organotypic neurons
a.
b.
c.
d.
Conclusions:
1. BoNT/A and /E LC are plasma membrane localized.
2. BoNT/E is degraded much more rapidly than BoNT/A LC in cells.
3. BoNT/E is ubiquitinated and degraded by the proteasome rapidly.
4. Designer E3 ligases can be constructed to accelerate BoNT/A
degradation.
5. VHH camelid antibodies have been generated against BoNT/A LC.
6. VHH-based designer E3 ligases are effective in degrading BoNT/A LC.
7. Delivery to intoxicated neurons of VHH-based designer E3 ligases may
offer novel post-exposure therapies for BoNT intoxication.
Synaptic Research:
Acknowledgements:
George A Oyler MD PhD
Tufts Team:
James R Oyler
Chuck Shoemaker PhD
Saul Tzipori DVM PhD
USAMRICD:
Chueh-Ling Kuo
Michael Adler PhD
Jong Beak Park PhD
James Eric Keller PhD (now FDA)
Ira Herman PhD
Metabiologics:
University of Maryland:
Michael Goodnough PhD
Paul Fishman MD PhD
Yien Che Tsai PhD (now NCI)
University of Wisconsin:
Eric Johnson PhD
Johns Hopkins:
Daniel Drachman MD
UMass Dartmouth:
Michael Betenbaugh PhD
Bal Ram Singh PhD
This w o rk w a s supp o rted b y co n tract N O1 -AI3 0050 from the N a tio n al Inst itut e s o f Healt h (N IH) and the National
of Allergy and Inf
e ctio u s Di sease s (NI A ID ) an d Bioshiel d N IA ID 1R 0 1AI
Inst itu te
67504 -01 to Geor ge A. O yler M D, PhD