Characterization of protein and transcript levels in amisyn mutant mice

• Autor(a) principal: Ferreira, Joana Sofia Lopes
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/29009
• topic: Amisyn; Exocytosis; Synaptic vesicle; Negative regulation

Amisyn is a 24 kDa protein vertebrate-specific SNARE protein known to interact with key exocytic proteins syntaxin1A, syntaxin4, SNAP25 and phospholipid PI(4,5)P2. Based on the recently published work in neuroendocrine cells and amisyn’s high expression in brain tissue, we hypothesize that this protein has a role in the negative regulation of synaptic vesicle exocytosis. Mutations in amisyn (gene name: STXBP6) have been reported in the context of several diseases like ASD (autism spectrum disorders), cancer and diabetes, which emphasized the importance of further studies. In this thesis, various experimental approaches were performed such as recombinant protein expression, liposome co-sedimentation assay, Western Blot, and quantitative PCR analyses, to advance the characterization of the amisyn protein and its possible functions. Liposome co-sedimentation assay revealed that amisyn requires phospholipid PI(4,5)P2 to bind to the membranes through its PH domain. Mutations in the amisyn’s PH domain abolished protein’s interactions with the membrane. Further, we observed that amisyn knock-out (KO) mice are viable, and we preformed systematic analysis of protein and transcript levels of various proteins related to the process of neurotransmission. This analysis revealed that the lack of amisyn leads to a decrease in VAMP2 and PSD95 protein levels and to an increase in Rab3A and α-synuclein protein levels. It was also found that the transcript levels of EndoA1, PSD95, RhoA and CAMKIV decreased and the transcript levels of Rab7 increased when amisyn was not present. Further studies are needed to fully comprehend the functions of this small SNARE protein.