Ubiquitin Protein
Ligase E3
Regulation of Protein Expression
Widespread Cancer Implications
John Prodromo
Regulatory Proteins

Protein expression is regulated on many levels
 Transcription

operons, histones
 Translation

Modification of mRNAs, excision of introns
 Post


translation
Turnover time of proteins
Degradative pathways
Ubiquitination and the Proteasome


Ubiquitination is a common method of marking a
protein for degradation.
Ubiquitous in nature (hence the name)
 Small
polypeptide of 76 a.a. attaches to amino
terminal lysine residue
 Targets to proteasome
 Can mono or poly-ubiquinate
 Lys-48 linked poly-ubiquitin chains
are common, others are also known.

Lys6, Lys11, Lys29, Lys63
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteasome.html
Ubiquitin with purple
Lysine Residues
The E1, E2, E3 system



Ubiquitin-activating enzyme
(E1) activates Ub by
adenylating C-terminus,
binding at its own Cys.
Ub transferred to an E2
With help of E3, transferred
to a lysine residue of
substrate protein.


E3 helps in the transfer, exact
mechanism is varied, and
often confers specificity
E3 is not ALWAYS used
Target; Proteasome

Addition of Ubiquitin -> regulatory particle
binds to the complex-> hydrolyzes ATP to
unfold protein-> moves protein to
proteasome-> cuts protein into smaller units
 These
units are either completely broken down to
amino acids
 Or presented to the immune system as antigens


Immune system activity releases cytokine interferon
gamma, which modifies proteasome and makes it
release antigenic determinants
Transporter associated with antigen processing (TAP)
proteins move the antigens to ER, they pair to class I
MHC and then are taken to the cell membrane for
recognition by T cells
 Ubiquitin
is released and reused.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteasome.html
Signals for Degradation
Not completely known
 Some motifs recur

 N-terminal

BUT- Also applies to bacteria (no ubiquitin)
 PEST

(proline-glutamic acid-serine-threonine)
5 minute-50 minute half life
 Misfolded

residue- the N degron
proteins
Exposing core sequences
http://homepages.bw.edu/~mbumbuli/cell/ublec
E3 ubiquitin protein ligase structure

Split into two distinct groups
 Based
on the presence of a HECT
domain or a RING finger domain
HECT- catalytic contribution
 RING finger- mostly acts as a scaffolding

 Other
Domains variable
 May contain a cullin domain

In vitro activation of E2s
Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic
mechanisms of protein ubiquitylation”
HECT
350 aa sequence
 Two lobes form an L shape and transfer
Ub using a conserved Cysteine and
transfers to a lysine via an undetermined
general base deprotonation

Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic
mechanisms of protein ubiquitylation”
RING Finger



Really Interesting New Gene
Also structurally related to U Box domain
15th most common domain in human genome
 Uncertain

how many RING fingers act as E3s
Conserved Zn++ with cross brace arrangement
 Divided

 All
into RING-HC or RING-H2
based on Cys or His
have 2 Zn sites
http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&Itemid=64&id=176
The RING-finger Domain

Binding of Zinc atoms- cross bridge
x x x
x x x
x
xx
x
x
x
x
x
xx
x
C
C C
C
x \
/x x \ / x
x Zn x x Zn x
C/
\C H/ \C
x
x x
x
xxxxxx
x
xxxxxx
http://pfam.sanger.ac.uk/family?acc=PF00097
Zn: Zinc
C: Cysteine
H: Histidine
X: unspecified amino acid
Ubox domain replaces Zn with H bonds
RING Finger
May allosterically activate, or stabilize
transfer
 Example: APC is a multisubunit E3 with 13
individual subunits.
 Scaffolding;

 Bring
donor Ub closer to Lys residues
Passmore, Lori A. and Barford, David. Biochem J. “Getting Into position; the catalytic
mechanisms of protein ubiquitylation”
C-CBL/UBCH7 Complex: RING
domain function in Ub-protein ligases


UBCH7- activity with
ZAP70
steroid hormone
receptors
ZAP70-zeta chain
associated protein
kinase
 Role
in T-cell
signaling
C-CBL
Ubiquitin
conjugating
enzyme
Common Domains/patterns
N- terminal 4 helix bundle
 EF Hand calcium binding domain
 Src homology-2 (SH2)-like domain
 The key is that the E3 has either the RING
Finger domain or HECT domain, and aids
an E2 in binding ubiquitin to a target
protein

http://www.rcsb.org/pdb/explore/explore.do?structureId=1FBV
The Immune System

Major Histocompatability complex (MHC)

Outside of cell membranes


Determine immune response


Self/nonself recognition
Class I




Lymphocyte recognition and antigen presentation
Marks cells as self to immune system
Presents to T cells
Endogenous antigen presentation to cytotoxic T cellspart of viral protein and tumor protein recognition
T cells


Lymphocytes that mature in the thymus
T cell receptors recognize antigens and can start
immune response
 Used in labeling, can terminate cells that are
infected

B cells

Lymphocytes that mature in the spleen and
produce antibodies
 Positive recognition- destroyed if paired to self
http://www.cehs.siu.edu/fix/medmicro/mhc.htm
http://youtube.com/watch?v=E2e5J1dfiE0
Viral Immunoevasion






Poxvirus and gamma-2 herpesvirus use E3s
with RING domain
Downregulates their cell surface glycoproteins
(MARCH proteins)
Catalyze ubiquitin conjugation to surface
proteins in vitro
Ubiquitination leads to protein endocytosis
This makes the cells avoid recognition by MHC I
Avoids detection by the immune system
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=321412
Receptor Tyrosine Protein Kinases



E3 proteins help to recognize and degrade receptor protein
tyrosine kinases (PTKs)
Receptor dimerizes when it binds to a growth factor ligand, then
phosphorylation of a Tyrosine, and signal transduction.
PTKs are involved in:


Mitosis
Differentiation
 Apoptosis
 70% of onco and proto-oncogenes are PTKs



Activated kinases are ubiquinated to terminate the signal
E3 protein recruits an E2, and the complex with the activated
PTK transfers a ubiquitin to destroy the protein.
Deactivates PTK.
http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Key_Resources/Protein_Kinase_Explorer/Tyrosine_Kinase_Ove
rview.html
Cbl-c
TKB (tyrosine
kinase binding)
 RING finger
domains
 a short proline-rich
region

http://www.rcsb.org/pdb/explore.do?structureId=3BUO
Cell Cycle Regulation



Cyclin dependent kinases (Cdks) regulate transitions in the cell
cycle
E1 uses ATP to bind and transfer ubiquitin to an E2, which can
sometimes transfer directly, or often uses an E3 for specificity in
ubiquitin transfer to the protein.
This pathway is necessary to

enter S phase

Destroy Sic1p- by CDC 4, CDC53, CDC34- ubiquitin conjugation

separate sister chromatids- destruction of kinetochores in Anaphase via
Ub
 end mitotic division.

Cyclins have a destruction box motif

Cyclin A and B have similar boxes, but A is degraded first
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWMD&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=
c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381001&md5=4ff0df5d107483b178ceb6b6e808ba4e
Cyclin levels
 Cyclins
are used to regulate and control the
transition between checkpoints of the cycle.
 Cyclin dependent kinases (Cdks)
phosphorylate proteins of the cell cycle to
change activity and aid in the progress of the
cycle.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWMD&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=
c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381001&md5=4ff0df5d107483b178ceb6b6e808ba4e
•
The Cell Cycle
•
•
•
•
•
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195BWMD&_coverDate=07%2F26%2F1996&_alid=471901450&_rdoc=1&_fmt=&_orig=search&_qd=1
&_cdi=7051&_sort=d&view=c&_acct=C000052510&_version=1&_urlVersion=0&_userid=1381
001&md5=4ff0df5d107483b178ceb6b6e808ba4e
Rising levels of cyclin D in the G1
phase bind to their corresponding
Cdks, which then begin the
process of chromosomal
replication
S phase- DNA duplication occurs
as cyclin A bound to Cdk2 enters
the nucleus. This leads to the
destruction of cyclin E and the end
of S phase.
Cyclins B and A rise.
As mitosis occurs, metaphase
marks the central chromosomal
alignment, which activates the
anaphase promoting complex, and
ubiquitination of cyclin B
the anaphase promoting complex
(APC) degrades cohesins and
causes separation of sister
chromatids and degradation of
cyclin B
This causes a rise in cyclin D to
begin the cycle all over again.
E3 activity and (more) Cancer


P53- the “guardian of the
genome”
HDM2 is a ubiquitin E3 ligase
that is a key negative
regulator of the tumor
suppressor p53
 Has
3(10) helix followed by four
beta-strands
 Zinc finger motif
http://www.rcsb.org/pdb/explore/pubmed.do?structureId=2C6A
The key points


RING Finger is very common in the genome, and often
acts as an E3
Widespread cancer implications on many levels





Cell cycle regulation, tyrosine kinases, immune system
E3s help with specificity and transfer of Ub to target
proteins to Proteasome
Ub targeting varies
Zn++ helps to stabilize the transfer
E3 may be a scaffold for E2 specificity

Exact mechanism is undetermined
Questions?/Comments