MODULATION OF CALPASTATIN-CALPAIN INTERACTIONS BY ETHANOL

PC12细胞暴露于乙醇会导致钙刺激蛋白酶活性的巨大变化。由于这些蛋白酶是神经递质释放以及细胞毒性和死亡的关键调节剂，我们正在探索乙醇介导的神经毒性可能是由钙激活的蛋白酶失调引起的假设。Calpastatin是一种钙蛋白酶抑制剂，是一种酸性疏水蛋白，可与mu-和m-钙蛋白酶的疏水活性位点相互作用。calpastatin的一系列翻译后修饰已经被描述，这些修饰改变了calpastatin与calpain的结合亲和力，其中包括pkc介导的磷酸化。使用PC12模型，我们正在研究乙醇暴露和退出对蛋白质-蛋白质相互作用的影响。由于钙pastatin-calpain相互作用的疏水性，我们研究了乙醇可能改变蛋白酶-钙pastatin（抑制剂）复合物稳定性的可能性。我们发现PC12细胞暴露于乙醇中导致钙蛋白酶和钙pastatin- containing蛋白复合物的分子量增加，并且这与蛋白酶活性的变化有关。我们正在使用免疫细胞化学技术扩展这些观察，这些复合物的性质和它们的细胞位置将被检查。-酒精中毒、神经毒性、PC12、翻译后修饰、蛋白-蛋白相互作用、钙蛋白酶、钙pastatin、蛋白酶

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. Because of the hydrophobic nature of calpastatin-calpain interactions, we examined the possibility that ethanol might modify protease-calpastatin (inhibitor) complex stability. We found that exposure of PC12 cells to ethanol results in an increase in the molecular weight of calpain and calpastatin- containing protein complexes, and that this is associated with a change in protease activity. We are extending these observations by use of immunocytochemical techniques, where the nature of these complexes and their cellular locations will be examined. - alcoholism, neurotoxicity, PC12, post-translational modifications, protein-protein interactions, calpain, calpastatin, proteases