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


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Two major proteolytic pathways in CellsTwo major proteolytic pathways in Cells

1. Proteasomes

2. Lysosomes

E2E1E3

ATP

Ubiquitin

AutophagyCell proteins& Organelles


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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.


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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


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Ubiquitin proteasome systemUbiquitin proteasome system

90-100 DUBs inhuman genome

E1 =2

E2 =40

E3 =600


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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.


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BoNT/E Lc has much shorter half-life in cells dueBoNT/E Lc has much shorter half-life in cells due

to UPSto UPS


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BoNT/E Lc has much shorter half-life in cells dueBoNT/E Lc has much shorter half-life in cells due

to UPSto UPS


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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


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


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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)


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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


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TRAF2 is a ubiquitin ligase involved in a numberof pathways

A20 =

DUB

TRAF2 =E3

TRAF2 andA20

compete fornet Ubi of

RIP


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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


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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


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BoNT/E Lc degradation is blocked by siRNA Knock-BoNT/E Lc degradation is blocked by siRNA Knock-

down of TRAF2down of TRAF2


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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


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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


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Vesicular Cycle involving p97, p47, VCIP, SNAREs and UbiVesicular Cycle involving p97, p47, VCIP, SNAREs and Ubi


21/27The EMBO Journal Vol. 21 No. 21 pp. 5645-5652, 2002

VCIP135

p97 adaptors in ubiquitin-mediated processesp97 adaptors in ubiquitin-mediated processes


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p47 Interaction with p97 can Recruit Ubiquitinatedp47 Interaction with p97 can Recruit Ubiquitinated

Proteins to ProteasomeProteins to Proteasome


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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


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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


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DUBs are drugable targets and represent new

therapeutic targets for BoNT


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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


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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

bont oyler ibrcc thur 05

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