MODULATION OF CALPASTATIN-CALPAIN INTERACTIONS BY ETHANOL

乙醇对钙蛋白酶抑制素-钙蛋白酶相互作用的调节

• 批准号: 6097584
• 负责人: PAOLO B DE PETRILLO
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6097584
• 关键词: PC12 cells; calpain; cytotoxicity; drug withdrawal; endopeptidases; enzyme inhibitors; ethanol; immunochemistry; intermolecular interaction; neurotoxicology; phosphorylation; posttranslational modifications; protein binding; protein kinase C; protein sequence

Exposure of PC12 cells to ethanol results in large changes in calcium-stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol-mediated neural toxicity may result from calcium- activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions, and will determine what, if any, post-translational modifications occur to alter these interactions. A variety of techniques are used for these studies, including gel filtration, fluorescence-based protease assays, identification of protein-protein complexes using a variety of immunochemical techniques and mass-mapping of isolated protein complexes. Because of the hydrophobic nature of calpastatin-calpain interactions, the possibility that ethanol might modify protease-inhibitor complex stability is being entertained.

将PC 12细胞暴露于乙醇导致大的 钙刺激蛋白酶活性的变化。由于这些 蛋白酶是神经递质释放的关键调节剂， 以及细胞毒性和死亡，我们正在探索的假设， 乙醇介导的神经毒性可能是由钙引起的， 激活的蛋白酶失调。钙蛋白酶抑制剂， 钙蛋白酶是一种酸性疏水蛋白， μ-和m-钙蛋白酶疏水活性位点。一系列 已经描述了钙蛋白酶抑制蛋白的翻译后修饰 它们改变了与钙蛋白酶的结合亲和力，其中， PKC介导的磷酸化。使用PC 12模型，我们 研究酒精暴露和戒断对 蛋白质之间的相互作用，并将决定什么，如果有的话， 发生翻译后修饰以改变这些相互作用。一 各种技术用于这些研究，包括凝胶 过滤，荧光蛋白酶测定，鉴定 蛋白质-蛋白质复合物使用多种免疫化学 技术和分离的蛋白质复合物的质量作图。 由于钙蛋白酶抑制素-钙蛋白酶的疏水性 相互作用，乙醇可能会改变 蛋白酶-抑制剂复合物的稳定性受到关注。