Novel Regulation of Renal Function by S-Nitrosylation

项目摘要/摘要 肾小管一氧化氮合酶(ENOS)在肾脏产生一氧化氮合酶(NO)，具有肾脏保护作用。 效果。然而，人们对NO的作用机制仍知之甚少。一氧化氮生物活性 主要由S传递-蛋白质的亚硝化，蛋白质硫醇的氧化修饰由NO转化为 形式S-亚硝硫醇(SNOS)。蛋白质S亚硝化反应受酶机制可逆调节 包括S-亚硝酸酶和反硝酸酶，并积累证据牵连调节失调的S- 疾病中的亚硝酸化。在这里，我们鉴定了一种新的酶机制，即为蛋白质S提供服务的亚硝化。 肾脏，包括一种新的代谢中间体S亚硝基辅酶A(SNO-CoA)，它传递NO 生物活性及其同源还原酶SNO-CoA还原酶(SCoAR)--一种未知的酶 在肾脏近端小管高度表达的功能。SCoAR调解SNO-CoA的崩溃 从而降低SNO-蛋白质的稳态水平(与SNO-CoA平衡)。SCoAR淘汰赛在 小鼠(Scoar-/-)被证明增加肾脏内S的亚硝化并保护肾脏免受 缺血/再灌注(I/R)损伤，而eNOS(SCoAR-/-/eNOS-/-)的缺失取消了这一保护作用。 因此，经典的eNOS肾保护作用与SNO-CoA/SCoAR系统相一致。使用新陈代谢 图谱和基于质谱仪(MS)的SNO-蛋白质鉴定，我们发现通过 SNO-CoA/SCoAR系统可能通过肾脏内的代谢重新编程来调节。从机械上讲， 我们的数据表明，丙酮酸激酶(PKM2)是SNO-CoA/SCoAR的主要调节点 系统在急性肾损伤(AKI)期间。因此，我们的资助探索了PKM2的S-亚硝化是 通过代谢重新编程来保护肾脏，这需要SCoAR调节的燃料协调 利用和抗氧化剂/再生防御。为了为新疗法奠定基础，我们还 筛选SCoAR抑制剂并成功开发出一类新型纳米分子候选药物 效力，这将在这里进行研究。因此，我们的发现不仅揭示了细胞的第一个生理功能 哺乳动物中的SNO-CoA/SCoAR系统，但有望开启我们对AKI的理解的新篇章 具有立竿见影的疗效。 好了！

PROJECT SUMMARY/ABSTRACT Nitric oxide (NO), generated in the kidney by renal tubular NO synthase (eNOS), can exert renoprotective effects. However, the mechanisms by which NO does so remain poorly understood. Nitric oxide bioactivity is principally conveyed by S-nitrosylation of proteins, the oxidative modification of protein thiols by NO to form S-nitrosothiols (SNOs). Protein S-nitrosylation is reversibly regulated by enzymatic mechanisms including S-nitrosylases and denitrosylases and accumulating evidence implicates dysregulated S- nitrosylation in disease. Here, we identify a novel enzymatic machinery subserving protein S-nitrosylation in the kidney, including a novel metabolic intermediate S-nitroso-coenzyme A (SNO-CoA) that conveys NO bioactivity and its cognate reductase, SNO-CoA reductase (SCoAR), an enzyme of previously unknown function that is highly expressed in kidney proximal tubules. SCoAR mediates the breakdown of SNO-CoA thereby lowering steady-state levels of SNO-proteins (in equilibrium with SNO-CoA). Knockout of SCoAR in mice (SCoAR-/-) is shown to increase S-nitrosylation within the kidney and to protect against ischemia/reperfusion (I/R) injury, whereas deletion of eNOS (SCoAR-/-/eNOS-/-) abrogates this protection. Thus classic renoprotection by eNOS is identified with the SNO-CoA/SCoAR system. Using metabolic profiling and mass spec (MS)-based SNO-protein identification, we have found that renoprotection by the SNO-CoA/SCoAR system is likely mediated by metabolic reprogramming within the kidney. Mechanistically, our data suggest that pyruvate kinase (PKM2) is a major locus of regulation by the SNO-CoA/SCoAR system during acute kidney injury (AKI). Our grant thus explores the idea that S-nitrosylation of PKM2 is renoprotective through metabolic reprogramming that entails SCoAR-regulated coordination of fuel utilization and antioxidant/regenerative defenses. To lay the groundwork for new therapies, we have also screened for SCoAR inhibitors and successfully developed a new class of drug candidate with nanomolar potency, which will be studied herein. Thus, our discovery not only reveals the first physiological function of the SNO-CoA/SCoAR system in mammals, but promises to open a new chapter in our understanding of AKI with immediate therapeutic implications. !