title: Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.
abstract: Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal-associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations. Ternary complex formation by synaptobrevin (green) and syntaxin/synaptosomal-associated protein of 25 kDa (red) is necessary for vesicle fusion, membrane trafficking, and cell homeostasis. Botulinum proteases cleave the three SNAREs proteins as indicated, resulting in a loss of cell viability. Lipofection reagents were used to deliver botulinum proteases or short SNARE peptides into neuroblastoma cells, revealing cytotoxic effects of SNARE fragments.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Jason
authors.initials: J
authors.lastname: Arsenault
authors.forename: Sabine A G
authors.initials: SA
authors.lastname: Cuijpers
authors.forename: Enrico
authors.initials: E
authors.lastname: Ferrari
authors.forename: Dhevahi
authors.initials: D
authors.lastname: Niranjan
authors.forename: Aleksander
authors.initials: A
authors.lastname: Rust
authors.forename: Charlotte
authors.initials: C
authors.lastname: Leese
authors.forename: John A
authors.initials: JA
authors.lastname: O'Brien
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
authors.forename: Bazbek
authors.initials: B
authors.lastname: Davletov
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D051379:Mice; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000188:drug therapy* / Q000473:pathology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000737:chemistry*; D050600:SNARE Proteins / Q000737:chemistry*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry; D050827:Syntaxin 1 / Q000737:chemistry; D014161:Transduction, Genetic; D014162:Transfection; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D010446:Peptide Fragments; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050988:Vesicle-Associated Membrane Protein 2; D010447:Peptide Hydrolases
keywords: SNARE; botulinum; cytotoxicity; neuro2A; syntaxin; transfection reagents