umass dartmouth botulinum research center introduction symposium insights into the mechanism of...

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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 George A. Oyler MD, PhD Friday August 24, 2007 Slide 2 Slide 3 Slide 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: Slide 5 YFP-BoNT/A LC is trafficked through multiple vesicle compartments in neuronal cells Slide 6 GFP-BoNT/A LC is trafficked in a polarized fashion and accumulates in specific sites of neuronal cells Slide 7 YFP-BoNT/E LC is also trafficked to plasma membrane Slide 8 YFP-LCERFP-LCAMerged N18 neuroblastoma Differential compartmentalization alone cannot account for differences in persistence Slide 9 anti-actin anti-GFP CHX:012468012468 YFP-LCE YFP-LCA BoNT/A and /E LC stability in SH-SY5Y cells Slide 10 Ubiquitin proteasome system Slide 11 250 100 75 50 25 IB: anti-HA IP: anti-GFP IB: anti-GFP IP: anti-GFP YFPYFP-LCEYFP-LCAYFPYFP-LCEYFP-LCA + HA-Ub Slide 12 Ubi n YFP- BoNT LC -Ubi -YFP YFP-LC YFP- LC/A YFP- LC/E YFP- LC/A YFP- LC/E YFP-BoNT/E LC is ubiquitinated more extensively than YFP-BoNT/A LC in N18 cells Slide 13 Myc-LC/AMyc-LC/E HectD2TRIM63 TRIP12Cbl-b E4A TEB4 Triad3 DZIP3 Slide 14 Proteasome Complex Degraded BoNT lc BoNT LC Ub ubiquitination Proteasome recognition slow BoNT degradation Natural proteasomal turnover of BoNT LC Designer E3 ligases that target toxins for proteasome degradation Ub BoNT lc Ub Target bindin g domai n Ubiquitin E3-ligase Cellular E3 ligase Target bindin g domai n Ubiquitin E3-ligase slow Ub BoNT lc Ub Slide 15 Degraded BoNT lc Therapeutic fusion protein BoNT LC Ub Designer E3 ligases that target toxins for proteasome degradation Enhanced proteasomal turnover of BoNT LC Ub Designer E3 ligase fast Proteasome Complex Ub BoNT lc Ub E3-ligase LC binding agent E3-ligase LC binding agent BoNT lc ubiquitination Proteasome recognition Accelerated BoNT degradation Ub Slide 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: 1.Camelid antibodies as more effective LC targeting domains. 2.Optimal E3-ligase domain (e.g. F-box proteins). 3.Neuronal delivery vehicle. Slide 17 XIAP RING is Catalytic E3 domain SNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitination SNAP-25/NC BoNT/A and E noncleavable C- terminus of SNAP25 BIR1BIR2BIR3 C C C C C H C C Zn XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitination C C C C C H C C Zn Slide 18 SNAP-25/NC-RING cells alone SNAP-25/NC Control SNAP-25/NC-RING + proteasome inhibitor (MG132) Relative amount of 35 S labelled YFP-LC Time (hours) 5101502025 SNAP-25/NC-RING designer E3 ligase substantially accelerates proteasome- mediated degradation of recombinant BoNT/A in transfected neurons Designer E3 ligase accelerates BoNT/A LC turnover in N18 cells Slide 19 Slide 20 Camelid VHH forms a compact well-folding single peptide structure Slide 21 Background: V H 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. VHHs as targeting domains Slide 22 2.5e66.43216080040002e410e45e5 ELISA vs BoNT/A LC SDS-PAGE (Coomassie) A6E3D4G6B8 VHH ELISA on BoNT/A LC 0 0.5 1 1.5 2 2.5 3 3.5 3006012. nM VHH Absorbance A6 E3 D4 G6 B8 Series1 Series2 Series3 Series4 Series5 A6 E3 D4 G6 B8 350 ng each SDS-PAGE (Coomassie) A6E3D4G6B8 350 ng each VHH-B8 selected as having the highest affinity for BoNT/A LC Elisa analysis of Anti-BoNT/A Lc VHH clones Slide 23 GST-VHH-B8 (ug) YFP-SNAP25-CFP cleavage activity (%) 25 50 75 100 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. 0 100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B8 GST-VHH-B8 potently inhibits BoNT/A Lc Slide 24 YFP ChannelCFP Channel + YFP-VHH-RING - CFP-BoNT/A LC + YFP-VHH-RING + CFP-BoNT/A LC - YFP-VHH-RING + CFP-BoNT/A LC Anti-A-LC VHH co-localizes with A-LC in cells Anti-A-LC VHH localizes to cytosol in transfected Neuro2a cells. When co-expressed with BoNT/A LC, the VHH localizes with A- LC at the plasma membrane. Slide 25 Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases -BoNT/A LC Control (Y-SNAP25-C) YFP-B8-RING YFP-D4-RING Slide 26 N2a cells Expressing Yes-SNAP25-Cer FRET Indicator YesFP CerFPFRETYesFP CerFP SNAP25 (1-206) FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media Slide 27 Cer BoNT LCCer BoNT LC Y B8 only Cer BoNT LC Y B8 Ring Cer BoNT LC Y B8 TrCP 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 Slide 28 TrCP designer ligases themselves turnover rapidly Anti XFP 1:5000 YFP/VHH-B8/TrCP M - 4 hr o/n MG135 treatment Inhibition of proteasomes with MG135 stabilizes TrCP fusion protein and leads to accumulation of poly-ubiquitinated forms Slide 29 75 50 37 100 150 25 250 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 1 2 3 4 5 6 7 8 M 9 10 11 12 Jun. 11 th -15 th.2007 Anti XFP 1:5000 Anti SNAP 1:5000 1 2 3 4 5 6 7 8 M 9 10 11 12 YFP-B8 NC YFP-SNAP25-CFP C YFP-SNAP25-CFP YFP-VHH B8-RING VHH based designer ligases prevent YFP-SNAP25- CFP cleavage in intoxicated M17 cells. YFP-B8YFP-VHH B8-Trcp YFP-VHH B8-RING Slide 30 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) LC binding agent VHH-LC targeting domain Delivery vehicle to neuronal cytosol E3-ligase 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. Slide 31 BoNT Lc BoNT Hc- N BoNT Hc- C Slide 32 a.b. c.d. BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons Slide 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. Slide 34 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 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