Date

2021-2-15

Author

Şen, Edanur

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Botulinum neurotoxins (BoNTs), known as the most potent bacterial toxins, cause potentially lethal disease botulism. BoNTs are classified as category A bioterror agents by many countries because they are easy to produce and have extreme toxicities. On the other hand, BoNTs are considered a therapeutic marvel in medicine because they have been extensively utilized not only for cosmetic purposes to get rid of wrinkles but also to treat various conditions in clinics, including movement disorders. These toxins specifically target cholinergic nerve terminals and block acetylcholine release by cleaving Soluble N-ethylmaleimidesensitive Factor Attachment Protein Receptor (SNARE) complex proteins, which are crucially important for neuroexocytosis. There are eight serologically distinct serotypes of BoNTs (BoNT/A–G, BoNT/X); however, only serotypes A, B, E, and F lead to human botulism. Among these, BoNT/A is the most widely used serotype in clinics and also the most studied serotype as more than half of botulism cases are due to BoNT/A poisoning. BoNT/A has the longest-lasting effect as compared to other serotypes. For example, both BoNT/A and BoNT/E serotypes cleave the same protein, SNAP-25, and the half-life of BoNT/E is limited to days in cells while BoNT/A can be active for up to 6 months in the neuronal cytosol. The underlying mechanisms for the half-life differences between different serotypes have not been well understood. However, there has been heightened research interest in understanding how BoNT/A manages to escape destructive protein degradation machinery in neurons. Recent studies have identified the E3 ligases and deubiquitinases (DUBs) critical for the destruction of BoNT/A. More specifically, one specific E3 ligase, HECTD2, leads to ubiquitination of the enzymatic component of BoNT/A Light Chain (LC) in cells, but the dominant DUB activity of VCIP135 inhibits the proteolytic degradation of the LC. In addition to this, another DUB, USP9X, indirectly affects the stability of BoNT/A in cells. Therefore, the persistence of BoNT/A in the cell can be potentially modified by affecting these factors using small molecule modulators. Modulation of BoNT half-life in cells by small molecules can be important for research purposes to understand intoxication/recovery mechanisms, as well as for the generation of effective countermeasures against botulism. In this study, the main goal was to screen a focused ubiquitin-proteasome system (UPS) inhibitor library to reveal compounds modulating BoNT/A activity and half-life in cells. Our screen utilizing mouse embryonic stem cell-derived motor neurons identified 10 potential lead compounds affecting BoNT/A mediated SNAP-25 in neurons. Then, tested dosedependent effects of the selected compounds and further tested their potential toxic effects in the cell. Following, we explored the effects of the lead compounds on the stability of LC in cells. Small molecules WP1130, b-AP15, NSC632839, PR-619, Celastrol, MDBN, PYR-41, and SL-01 exhibited efficacy against BoNT/A LC action and half-life in cells. Among these, PR-619 appears to be highly crucial as it is an inhibitor of VCIP135 that has been identified as a crucial DUB affecting the half-life of BoNT/A LC. To the best of my knowledge, this is the first study in the literature showing that UPS targeting small molecules can modulate BoNT/A LC action and half-life in cells.

Subject Keywords

Botulism, Botulinum neurotoxin (BoNT), Synaptosomal-associated protein 25 (SNAP25), Motor neuron, Synaptic transmission

URI

https://hdl.handle.net/11511/89615

Collections

Graduate School of Natural and Applied Sciences, Thesis