INTERACTION OF ETHYL ALCOHOL WITH CELLULAR CYSTEINE PROTEASES

乙醇与细胞半胱氨酸蛋白酶的相互作用

• 批准号: 6431369
• 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/6431369
• 关键词: PC12 cells; antibody formation; antireceptor antibody; calcium flux; cysteine endopeptidases; enzyme activity; ethanol; physical chemical interaction; polymerase chain reaction; protease inhibitor; serotonin receptor

Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. We have previously established that exposure of PC12 cells to ethyl alcohol for 96 hours results in a decrease in calcium-stimulated protease activity. We developed assays to measure GSH, GSSG, and NO and we have determined that both GSH and GSSG are important modulators of calpain activity. GSH is an uncompetitive inhibitor while GSSG is a competitive inhibitor of calpains, with inhibition constants well within range of concentrations of GSH and GSSG found in cells. We have also determined that nitric oxide also strongly inhibits protease activity. These results may relate to the previous reports that both ischemia and oxidative stress increase calpain activity in several mammalian cell types, and both processes are known to decrease both GSH and GSSG. We are now examining how alcohol exposure alters GSH, GSSG and NO as possible mechanisms for alcohol induced alterations in protease activity.

某些类型的细胞半胱氨酸蛋白酶可能在调节 G 蛋白偶联受体的活性中发挥重要作用。这类酶的活性受到细胞质和核抑制剂的密切调节。已经使用 PC12 细胞系在细胞培养物中研究了乙醇暴露对半胱氨酸蛋白酶活性的影响。我们之前已经确定，PC12 细胞暴露于乙醇 96 小时会导致钙刺激的蛋白酶活性降低。我们开发了测定 GSH、GSSG 和 NO 的方法，并且我们已经确定 GSH 和 GSSG 都是钙蛋白酶活性的重要调节剂。 GSH 是一种非竞争性抑制剂，而 GSSG 是钙蛋白酶的竞争性抑制剂，其抑制常数完全在细胞中发现的 GSH 和 GSSG 浓度范围内。 我们还确定一氧化氮也会强烈抑制蛋白酶活性。这些结果可能与之前的报道有关，即缺血和氧化应激都会增加几种哺乳动物细胞类型中的钙蛋白酶活性，并且已知这两个过程都会降低 GSH 和 GSSG。我们现在正在研究酒精暴露如何改变 GSH、GSSG 和 NO，作为酒精诱导蛋白酶活性改变的可能机制。