Investigation of the substrate recognition mechanisms of botulinum neurotoxin and its application in detection and therapy development

Abstract

Botulinum Neurotoxin(abbreviated as BTX or BoNT),produced by Clostridium botulinum,can lead to clinical syndrome of botulism by specifically targeting and blocking the release of neurotransmitter at nerve terminals.Among the 7 unambiguously identified serotypes(designated as BoNT/A,/B,/C,/D,/E,/F,and /G), BoNT/A, B, E,and/F (rarely) mainly cause human botulism, and BoNT/C and /D infect animals.Even though BoNTs are considered as the most potent protein toxins, they are approved by FDA as effective therapies for numerous neuronal disorders and cosmetics.No effective antitoxins or inhibitors,however,are available to date. A better understanding of the mechanism of substrate recognition and cleavage by BoNTs is the prerequisite to develop inhibitors or antidotes for BoNTs intoxication,novel therapeutic applications and rapid detection system.This thesis focuses on the investigation of the substrate recognition mechanisms employed by BoNT/D,/F5 and/F7 to dissect their step by step substrate binding and cleavage. In addition,based on previous understanding and comparison of the substrate recognition mechanisms utilized by BoNT/B and Tetanus Neurtoxins (TeNT),a BoNT/B derivative was engineered with elevated substrate cleavage activity, with the hope to minimize the immunoresistance impact from patients during BoNT/B therapy.As an extension of this work, a FRET peptide(designated as FVP-B) was synthesized to develop a rapid and sensitive detection assay for BoNT/B detection. In the last part of the thesis,a set of potent peptide inhibitors were developed to inhibit BoNT/A activity both in vitro and in murine model. In summary,the research works in this study will provide useful information for the development effective countermeasures to combat BoNT intoxication and for the development of nopvel BoNT based therapies.