Ranek Mark J, Kost Curtis K, Hu Chengjun, Martin Douglas S, Wang Xuejun
Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, SD 57069, USA.
Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Vermillion, SD 57069, USA.
J Mol Cell Cardiol. 2014 Apr;69:43-51. doi: 10.1016/j.yjmcc.2014.01.017. Epub 2014 Feb 5.
Proteasome function insufficiency and inadequate protein quality control are strongly implicated in a large subset of cardiovascular disease and may play an important role in their pathogenesis. Protein degradation by the ubiquitin proteasome system can be physiologically regulated. Cardiac muscarinic 2 (M2) receptors were pharmacologically interrogated in intact mice and cultured neonatal rat ventricular myocytes (NRVMs). Proteasome-mediated proteolysis was measured with a surrogate misfolded protein, proteasome peptidase assay, and by characterizing key proteasome subunits. Successful M2 receptor manipulation in cardiomyocytes was determined by measuring an endogenous protein substrate, and in mice, the cardiovascular physiological response. M2 receptor stimulation was associated with increased proteasome-mediated proteolysis and enhanced peptidase activities, while M2 receptor inhibition yielded opposing results. Additionally, M2 receptor manipulation did not alter abundance of the key proteasome subunits, Rpt6 and β5, but significantly shifted their isoelectric points. Inhibition of protein kinase G abrogated the stimulatory effects on proteasome-mediated proteolysis from M2 receptor activation. We conclude that M2 receptor stimulation enhances, whereas M2 receptor inhibition reduces, proteasome-mediated proteolysis likely through posttranslational modifications. Protein kinase G appears to be the mediator of the M2 receptors actions.
蛋白酶体功能不足和蛋白质质量控制不当与很大一部分心血管疾病密切相关，可能在其发病机制中起重要作用。泛素蛋白酶体系统介导的蛋白质降解可受到生理调节。在完整小鼠和培养的新生大鼠心室肌细胞（NRVMs）中对心脏毒蕈碱2（M2）受体进行了药理学研究。用替代错误折叠蛋白、蛋白酶体肽酶测定法并通过表征关键蛋白酶体亚基来测量蛋白酶体介导的蛋白水解。通过测量内源性蛋白质底物以及在小鼠中测量心血管生理反应来确定心肌细胞中M2受体的成功操纵。M2受体刺激与蛋白酶体介导的蛋白水解增加和肽酶活性增强相关，而M2受体抑制则产生相反的结果。此外，M2受体操纵并未改变关键蛋白酶体亚基Rpt6和β5的丰度，但显著改变了它们的等电点。蛋白激酶G的抑制消除了M2受体激活对蛋白酶体介导的蛋白水解的刺激作用。我们得出结论，M2受体刺激增强，而M2受体抑制降低蛋白酶体介导的蛋白水解，这可能是通过翻译后修饰实现的。蛋白激酶G似乎是M2受体作用的介质。
