BeyondSNO: Signalling beyond protein S-nitrosylation - determining the roles of nitroxyl and hydroxylamine

氮氧基(HNO)是在细胞内内源性形成的，在治疗心力衰竭的临床开发中，药物可以释放它。尽管如此，目前还不清楚一氧化氮(NO)的这种化学相关物质是如何发挥其生物学和治疗作用的--这是我们在这里要解决的一个重要问题。我们将使用氧化还原蛋白质组学来定义氮氧基翻译后修饰以调节心血管功能的蛋白质半胱氨酸。HNO修饰蛋白质硫醇的一个未被认识和尚未确定的结果是形成羟胺(NH_2OH)，它从蛋白质中去除了S棕榈酰化等修饰。因此，我们还将确定心脏和动脉中被HNO存在时形成的羟胺去除的蛋白质。识别被HNO和羟胺修饰的目标蛋白，将使人们能够通过制造目标半胱氨酸突变的氧化还原死亡蛋白并在功能上鉴定其功能，来确定氮氧基驱动的信号转导的细胞后果。缺乏这些特定调节半胱氨酸的新型“氧化还原死亡”小鼠的产生将使HNO和羟胺信号在心血管健康和疾病中的作用在体内得到确立。我们将通过从我们的亲电化合物库中识别硫醇活性药物来利用我们的发现进行治疗，这些药物以HNO或羟胺修饰的蛋白质为靶标并改变其活性。这类药物的副作用可能比硝基供体要少，因为除了那些调节其治疗作用的药物外，氮氧基供体还会引起广泛的非靶标氧化。相反，我们确定的亲电药物将选择性地修饰介导HNO治疗作用的蛋白质半胱氨酸残基。我们将产生的氧化还原死亡小鼠对于药物发现也将是无价的，因为它们将对亲电药物产生抗药性，因为它们缺乏关键的半胱氨酸，提供了强有力的治疗靶点验证。

Nitroxyl (HNO) is formed endogenously in cells, with drugs that release it in clinical development for heart failure. Despite this, it remains relatively unclear how this chemical relative of nitric oxide (NO) exerts its biological and therapeutic actions - an important issue we will address here. We will define the protein cysteines that nitroxyl post-translationally modifies to modulate cardiovascular function using redox proteomics. An underappreciated and yet undefined consequence of HNO modifying protein thiols is formation of hydroxylamine (NH2OH), which removes modifications such as S-palmitoylation from proteins. Consequently, we will also identify the proteins in heart and arteries that are removed by hydroxylamine formed when HNO is present. Identifying the target proteins modified by HNO and hydroxylamine will enable the cellular consequences of nitroxyl-driven signalling to be determined by making and functionally characterising 'redox dead' mutant proteins with the target cysteine mutated. Generation of novel 'redox dead' mice that lack these specific regulatory cysteines will allow the role of HNO and hydroxylamine signaling in cardiovascular health and disease to be established in vivo. We will leverage our findings therapeutically by identifying thiol reactive drugs, from our electrophilic compounds library, that target and alter the activity of the proteins modified by HNO or hydroxylamine. Such drugs are likely to have less side effects than nitroxyl donors that cause widespread off-target oxidations in addition to those that mediate their therapeutic actions. In contrast, the electrophilic drugs we identify will selectively modify the protein cysteine residue that mediates the therapeutic actions of HNO. The 'redox dead' mice we will generate will also be invaluable for drug discovery, as they will be resistant to the electrophilic drug because they lack the critical cysteine, providing robust therapeutic target validation.