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UMass Dartmouth Botulinum Research Center Introduction Symposium Insights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapies.

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Presentation on theme: "UMass Dartmouth Botulinum Research Center Introduction Symposium Insights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapies."— Presentation transcript:

1 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

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4 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:

5 YFP-BoNT/A LC is trafficked through multiple vesicle compartments in neuronal cells

6 GFP-BoNT/A LC is trafficked in a polarized fashion and accumulates in specific sites of neuronal cells

7 YFP-BoNT/E LC is also trafficked to plasma membrane

8 Differential compartmentalization alone cannot account for differences in persistence
YFP-LCE RFP-LCA Merged N18 neuroblastoma

9 BoNT/A and /E LC stability in SH-SY5Y cells
YFP-LCE YFP-LCA CHX: 1 2 4 6 8 1 2 4 6 8 anti-GFP anti-actin

10 Ubiquitin proteasome system

11 + HA-Ub IP: anti-GFP IP: anti-GFP IB: anti-HA IB: anti-GFP YFP YFP-LCE
YFP-LCA YFP YFP-LCE YFP-LCA + HA-Ub 250 100 75 50 25 IP: anti-GFP IP: anti-GFP IB: anti-HA IB: anti-GFP

12 YFP-BoNT/E LC is ubiquitinated more extensively than YFP-BoNT/A LC in N18 cells
Ubin YFP-BoNT LC a-Ubi a-YFP YFP-LC YFP-LC/A YFP-LC/E YFP-LC/A YFP-LC/E

13 Myc-LC/A Myc-LC/E HectD2 TRIM63 TRIP12 Cbl-b E4A TEB4 Triad3 DZIP3

14 Natural proteasomal turnover of BoNT LC
Designer E3 ligases that target toxins for proteasome degradation Natural proteasomal turnover of BoNT LC Ub Ub Target binding domain Ubiquitin E3-ligase Cellular E3 ligase Target binding domain Ubiquitin E3-ligase Ub Ub Ub Ub “slow” Ub ubiquitination BoNT lc Ub BoNT LC Proteasome recognition Ub Ub BoNT lc Proteasome Complex slow BoNT degradation Ub Degraded BoNT lc

15 Enhanced proteasomal turnover of BoNT LC Therapeutic fusion protein
Designer E3 ligases that target toxins for proteasome degradation Enhanced proteasomal turnover of BoNT LC Ub Ub “Designer E3 ligase” E3-ligase LC binding agent E3-ligase LC binding agent Ub Ub “fast” Ub Ub Ub BoNT lc Ub ubiquitination BoNT LC Therapeutic fusion protein Proteasome recognition Ub Ub BoNT lc Accelerated BoNT degradation Proteasome Complex Ub Degraded BoNT lc

16 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: Camelid antibodies as more effective LC targeting domains. Optimal E3-ligase domain (e.g. F-box proteins). Neuronal delivery vehicle.

17 Zn Zn Zn C H C C C C C C C H BIR1 BIR2 BIR3 SNAP-25/NC
XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitination XIAP RING is Catalytic E3 domain SNAP-25/NC C C Zn C C C SNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitination C Zn C H BoNT/A and E noncleavable C-terminus of SNAP25

18 Relative amount of 35S labelled YFP-LC
Designer E3 ligase accelerates BoNT/A LC turnover in N18 cells SNAP-25/NC-RING + proteasome inhibitor (MG132) cells alone Relative amount of 35S labelled YFP-LC SNAP-25/NC Control SNAP-25/NC-RING 5 10 15 20 25 Time (hours) SNAP-25/NC-RING “designer E3 ligase” substantially accelerates proteasome-mediated degradation of recombinant BoNT/A in transfected neurons

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20 Camelid VHH forms a compact well-folding single peptide structure

21 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.

22 Elisa analysis of Anti-BoNT/A Lc VHH clones
SDS-PAGE (Coomassie) 350 ng each SDS-PAGE (Coomassie) A6 E3 D4 G6 B8 ELISA vs BoNT/A LC VHH ELISA on BoNT/A LC A6 E3 D4 G6 B8 3.5 3 2.5 A6 E3 D4 G6 B8 Series1 Series2 Series3 Series4 Series5 Absorbance 2 1.5 1 0.5 2.5e6 6.4 32 160 800 4000 2e4 10e4 5e5 300 60 12.5 2.5 0.5 0.1 0.02 nM VHH VHH-B8 selected as having the highest affinity for BoNT/A LC

23 GST-VHH-B8 potently inhibits BoNT/A Lc
100 YFP-SNAP25-CFP cleavage activity (%) 100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B8 75 50 25 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 GST-VHH-B8 (ug) 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 YFP-SNAP25-CFP substrate (80 nM), and increasing concentrations of GST-VHH (B8). Inhibition of BoNT/A LC activity by GST-VHH (B8) was near stoichiometric.

24 Anti-A-LC VHH co-localizes with A-LC in cells
YFP Channel CFP Channel Anti-A-LC VHH localizes to cytosol in transfected Neuro2a cells. + YFP-VHH-RING - CFP-BoNT/A LC When co-expressed with BoNT/A LC, the VHH localizes with A-LC at the plasma membrane. + YFP-VHH-RING + CFP-BoNT/A LC - YFP-VHH-RING + CFP-BoNT/A LC

25 Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases
Control (Y-SNAP25-C) YFP-B8-RING YFP-D4-RING -BoNT/A LC

26 N2a cells Expressing Yes-SNAP25-Cer FRET Indicator
YesFP CerFP FRET YesFP CerFP SNAP25 (1-206) FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media

27 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

28 TrCP designer ligases themselves turnover rapidly
MG135 treatment M 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

29 VHH based designer ligases prevent YFP-SNAP25-CFP cleavage in intoxicated M17 cells.
Anti XFP 1:5000 1: YFP-B8 +/A24+24 2: YFP-B8 +/control 3: Indicator only +/A24+24 4: YFP-VHH B8-Trcp +/A24+24 5: YFP-VHH B8-Trcp+/control 6:YFP-VHH B8-Trcp +/A24 7: Indicator only +/A24 8: Indicator only +/control 9: YFP-VHH B8-RING+/A24+24 10: YFP-VHH B8-RING +/control 11: No transfection +/A24+24 12: No transfection +/control YFP-B8 YFP-VHH B8-Trcp YFP-VHH B8-RING 250 150 100 NC YFP-SNAP25-CFP 75 YFP-VHH B8-RING C YFP-SNAP25-CFP 50 YFP-B8 37 M 25 Anti SNAP 1:5000 M Jun. 11th-15th.2007

30 Designer E3 ligases that target toxins for proteasome degradation
Preferred strategy for targeted destruction of BoNT: a smaller, modular “designer E3 ligase” Note that the targeting domain can be interchanged to create botulism therapeutics for each serotype once an A-LC prototype has been developed. E3 ligase targeting domain, e.g. minimal TrCP (F-box) E3-ligase LC binding agent VHH-LC targeting domain BoNT LC Delivery vehicle to neuronal cytosol

31 BoNT Lc BoNT Hc-N BoNT Hc-C

32 BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons

33 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.

34 Acknowledgements: Synaptic Research: George A Oyler MD PhD
James R Oyler USAMRICD: Michael Adler PhD James Eric Keller PhD (now FDA) Metabiologics: Michael Goodnough PhD University of Wisconsin: Eric Johnson PhD UMass Dartmouth: Bal Ram Singh PhD Acknowledgements: Tufts Team: Chuck Shoemaker PhD Saul Tzipori DVM PhD Chueh-Ling Kuo Jong Beak Park PhD Ira Herman PhD University of Maryland: Paul Fishman MD PhD Yien Che Tsai PhD (now NCI) Johns Hopkins: Daniel Drachman MD Michael Betenbaugh PhD


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