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: 6097573
• 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/6097573
• 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 interaction of these inhibitors with the target proteases is, in part, mediated by strong hydrophobic interactions. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. Activity of these proteases appears to be strongly affected by alcohol exposure. We have established that exposure of PC12 cells to ethyl alcohol for 96 hours results in: a) a decrease in calcium-stimulated protease activity, which is evident at exposure concentrations of 20 mM and, b) a decrease in both isoforms of calpain, which is evident at 40 mM and 80 mM. The results of this work were published (J Neurochem 1997;68:1863-9). We are working towards defining a mechanism for this alcohol-induced inhibition of calcium-activated protease activity. We have generated a polyclonal antibodies to Domain IV of calpastatin, a cellular inhibitor of protease activity, as well as the subunit of the the 5HT-3 receptor. Using quantitative RT-PCR, we are determining whether ethanol exposure and withdrawal changes the expression of calpastatin, m-calpain. mu-calpain, and 5HT3 receptor message. We have found that corticosterone interacts with alcohol exposure to modulate both message levels and protein levels of relevant target proteins, and are working to determine the mechansims for these observed effects.

某些类型的细胞半胱氨酸蛋白酶可能发挥作用， 在调节G蛋白偶联的活性中起重要作用， 受体。这类酶的活性受到严格的调节 细胞质和细胞核抑制剂。这些因素的相互作用 与靶蛋白酶的抑制剂，部分是由强介导的 疏水相互作用酒精暴露对 半胱氨酸蛋白酶活性已经在细胞培养物中利用 PC 12细胞系。这些蛋白酶的活性似乎强烈 受酒精影响。我们已经确定， PC 12细胞与乙醇接触96小时导致： 钙刺激的蛋白酶活性，这在暴露时是明显的 和，B）在20 mM的浓度下， 钙蛋白酶，在40 mM和80 mM下明显。 发表了工作（J Neurochem 1997;68：1863-9）。我们 致力于确定这种酒精诱导的 抑制钙激活蛋白酶活性。我们已经生成 a抗钙蛋白酶抑制蛋白结构域IV的多克隆抗体， 蛋白酶活性的抑制剂，以及蛋白酶活性的亚基， 5 HT-3受体。使用定量RT-PCR，我们确定 酒精暴露和戒断是否改变了 钙蛋白酶抑制素，m-钙蛋白酶μ-钙蛋白酶和5 HT 3受体信息。 我们发现皮质酮与酒精接触相互作用 以调节相关蛋白质的信息水平和蛋白质水平， 目标蛋白质，并正在努力确定机制， 这些观察到的效果。