BoNT Oyler IBRCC Thur 05
Transcript of BoNT Oyler IBRCC Thur 05
-
8/8/2019 BoNT Oyler IBRCC Thur 05
1/27
George A. Oyler MD PhD
President and Founder
Synaptic Research LLC
-
8/8/2019 BoNT Oyler IBRCC Thur 05
2/27
-
8/8/2019 BoNT Oyler IBRCC Thur 05
3/27
Two major proteolytic pathways in CellsTwo major proteolytic pathways in Cells
1. Proteasomes
2. Lysosomes
E2E1E3
ATP
Ubiquitin
AutophagyCell proteins& Organelles
-
8/8/2019 BoNT Oyler IBRCC Thur 05
4/27
Autophagy in BoNT RegulationAutophagy in BoNT Regulation
Ubiquitin like conjugation inautophagy:Apg8, Apg12, LC3, GATE16,GABARAP
BoNT/E Lc interacts with GABARAPon Y2H
Inhibition of autophagy by 3MAincreases levels of BoNT/E Lc in
transfected cells.
-
8/8/2019 BoNT Oyler IBRCC Thur 05
5/27
BoNT/A Lc appears to be trafficked from vacuoles andBoNT/A Lc appears to be trafficked from vacuoles and
induces large vacuoles when expressed in cellsinduces large vacuoles when expressed in cells
-
8/8/2019 BoNT Oyler IBRCC Thur 05
6/27
Ubiquitin proteasome systemUbiquitin proteasome system
90-100 DUBs inhuman genome
E1 =2
E2 =40
E3 =600
-
8/8/2019 BoNT Oyler IBRCC Thur 05
7/27
Model of Ubiquitination by an E3 and Deubiquitination by aModel of Ubiquitination by an E3 and Deubiquitination by a
DUB acting as a clock for presynaptic proteinsDUB acting as a clock for presynaptic proteins
Ubiquitination state of proteins in the presynaptic terminal is
dynamic.Model: BoNT/E interacts with an E3 (TRAF2) and BoNT/A with a
DUB (VCIP135) to shift the balance towards or away fromproteasome degradation.
-
8/8/2019 BoNT Oyler IBRCC Thur 05
8/27
BoNT/E Lc has much shorter half-life in cells dueBoNT/E Lc has much shorter half-life in cells due
to UPSto UPS
-
8/8/2019 BoNT Oyler IBRCC Thur 05
9/27
BoNT/E Lc has much shorter half-life in cells dueBoNT/E Lc has much shorter half-life in cells due
to UPSto UPS
-
8/8/2019 BoNT Oyler IBRCC Thur 05
10/27
Half-life of BoNT protease in M17 neuroblastoma cellsHalf-life of BoNT protease in M17 neuroblastoma cells
IB: CFP
BoNT/E Lc is turned over rapidly inneuronal cells
-
8/8/2019 BoNT Oyler IBRCC Thur 05
11/27
Stable MDCK cell lines expressing GFP-BoNT A, B, and E Lc
GFP-BoNT/A Lc
GFP-BoNT/B Lc
GFP-BoNT/E Lc
-
8/8/2019 BoNT Oyler IBRCC Thur 05
12/27
0
10
20
30
40
50
60
70
80
90
100
Increase in steady state BoNT fluorescence withIncrease in steady state BoNT fluorescence with
proteasome inhibitionproteasome inhibition
RelativeF
luor
escen
ce(%
Max)
-
8/8/2019 BoNT Oyler IBRCC Thur 05
13/27
BoNT/E Lc but not BoNT/A Lc Interacts with TRAF2BoNT/E Lc but not BoNT/A Lc Interacts with TRAF2
TRAF2 is a Ring E3 Ubiquitin LigaseTRAF2 is a Ring E3 Ubiquitin Ligase
TRAF2 is
associatedwithBoNT/E Lcand not
BoNT/A Lc
-
8/8/2019 BoNT Oyler IBRCC Thur 05
14/27
TRAF2 is a ubiquitin ligase involved in a numberof pathways
A20 =
DUB
TRAF2 =E3
TRAF2 andA20
compete fornet Ubi of
RIP
-
8/8/2019 BoNT Oyler IBRCC Thur 05
15/27
RIP regulates the TNF-R/NFkB signaling pathway by
cycles of Ubiquitination and Deubiquitination
RIPE3: TRAF2
+ Ubi
DUB: A20
- Ubi
E3: TRAF2
+ UbiDUB: VCIP135
- UbiBoNT/LC
-
8/8/2019 BoNT Oyler IBRCC Thur 05
16/27
BoNT/E Lc interacts with TRAF2BoNT/E Lc interacts with TRAF2
E3 ligase mutant TRAF2 stabilizes BoNT/E LcE3 ligase mutant TRAF2 stabilizes BoNT/E Lc
-
8/8/2019 BoNT Oyler IBRCC Thur 05
17/27
BoNT/E Lc degradation is blocked by siRNA Knock-BoNT/E Lc degradation is blocked by siRNA Knock-
down of TRAF2down of TRAF2
-
8/8/2019 BoNT Oyler IBRCC Thur 05
18/27
Y2H Screen Identified p47 as a BoNT/A Lc interactorY2H Screen Identified p47 as a BoNT/A Lc interactor
SNAP25
p47
Ezrin
SNAP25
p47
Ezrin
BoNT/A Lc BoNT/E Lc
Septin8
Septin 9 Septin8
Septin 9
-
8/8/2019 BoNT Oyler IBRCC Thur 05
19/27
Similarity between NSF and p97 (aka cdc48 or VCP)Similarity between NSF and p97 (aka cdc48 or VCP)
p97 with p47 bound motor of
ERAD (and more)
NSF motor of SNARE
fusion
-
8/8/2019 BoNT Oyler IBRCC Thur 05
20/27
Vesicular Cycle involving p97, p47, VCIP, SNAREs and UbiVesicular Cycle involving p97, p47, VCIP, SNAREs and Ubi
-
8/8/2019 BoNT Oyler IBRCC Thur 05
21/27The EMBO Journal Vol. 21 No. 21 pp. 5645-5652, 2002
VCIP135
p97 adaptors in ubiquitin-mediated processesp97 adaptors in ubiquitin-mediated processes
-
8/8/2019 BoNT Oyler IBRCC Thur 05
22/27
p47 Interaction with p97 can Recruit Ubiquitinatedp47 Interaction with p97 can Recruit Ubiquitinated
Proteins to ProteasomeProteins to Proteasome
-
8/8/2019 BoNT Oyler IBRCC Thur 05
23/27
p47 plays a role in BoNT/A persistencep47 plays a role in BoNT/A persistence
Short-lived BoNT/E Lc is highly ubiquitinated while BoNT/ALc is not
p47 forms a complex with p97 and VCIP135 (DUB) whichpromotes protein de-ubiquitination
p47 interacts with BoNT/A but not BoNT/E
Hypothesis:p47 is involved in promoting persistent cell
intoxication byACTIVELY
deubiquitinatingBoNT/A Lcp47 Knockdown
BoNT/A Lc halflife
-
8/8/2019 BoNT Oyler IBRCC Thur 05
24/27
BoNT/A Lc steady state level was reduced with p47 knock downBoNT/A Lc steady state level was reduced with p47 knock down
Control cellsp47 knock down7
-
8/8/2019 BoNT Oyler IBRCC Thur 05
25/27
DUBs are drugable targets and represent new
therapeutic targets for BoNT
-
8/8/2019 BoNT Oyler IBRCC Thur 05
26/27
Summary of Results for BoNT/A and /E interactorsSummary of Results for BoNT/A and /E interactors
BoNT/A has reduced ubiquitination in cells and appears to evade ubiquitination and
proteasome degradation.
BoNT/A Lc interacts with p47 which can result in binding of BoNT/A to p97.
p47 can recruit a DUB VCIP135.
siRNA knock-down of p47 shortens BoNT/A Lc half-life.
BoNT/E Lc is highly ubiquitinated in cells and appears to be degraded by
proteasome.
BoNT/E Lc interacts with E3 ligase TRAF2.
Mutation of of TRAF2 E3 domain or siRNA knock-down of TRAF2 stabilize BoNT/E
in cells.
Acknowledgements
-
8/8/2019 BoNT Oyler IBRCC Thur 05
27/27
Acknowledgements
Tufts:Tufts:Saul Tzipori
Charles ShoemakerChuehling KuoClaudia AbeijonHanping Feng
Synaptic Research:Synaptic Research:Yung-Nien ChangJulian RosenbergArchana KotiyaPilar Oyler
U Mass:U Mass:Bal Ram SinghCharlene Mello
NCI:NCI:Yien Che TsaiAllen Weissman
University of MarylandUniversity of Maryland::Paul Fishman
USAMRICD:USAMRICD:
Michael AlderBrain MollesPatrick McNutt
USAMRIID:USAMRIID:Robert WebbAshraf AhmedMartha Hale
BioSentinelBioSentinelWard Tucker
Funding: NIH NIAID N01-AI-30050,DTRA subcontracts SR Development