S-亚硝基化(SNO)是氧化还原信号保护细胞免受心脏氧化应激的新兴范例。我们以前的研究表明,含有valosin的蛋白质(VCP),一种ATP酶相关蛋白,是保护心脏免受心脏压力和缺血性损伤的重要介质。然而,尚不完全了解VCP在心脏中赋予的分子调控。在这项研究中,我们使用多种心脏特异性转基因小鼠模型和包括生物素开关测定在内的各种分析技术,探索了VCP在心脏蛋白SNO中的潜在作用。液相色谱法,质谱,和西方印迹。我们的结果表明,VCP的心脏特异性过表达导致转基因(TG)中SNO修饰的心脏蛋白水平整体增加。野生型(WT)小鼠。质谱分析确定了参与呼吸的线粒体蛋白,新陈代谢,和解毒作为过表达VCP的小鼠心脏中SNO修饰的主要目标。特别是,我们发现VCP本身在其N域的特定半胱氨酸残基处经历了SNO修饰.此外,我们的研究表明,甘油醛3-磷酸脱氢酶(GAPDH),糖酵解的关键酶,也经历了响应VCP过表达的SNO增加。而VCPTG小鼠中诱导型一氧化氮合酶(iNOS)的缺失不影响VCPSNO,它确实废除了线粒体复合蛋白中的SNO修饰,提示涉及iNOS依赖性和独立途径的双重调节机制。总的来说,我们的发现揭示了心脏中VCP的翻译后修饰,揭示了VCP在调节心脏蛋白SNO方面以前未被认可的作用,并为其在心脏保护中的功能提供了新的见解。
S-nitrosylation (SNO) is an emerging paradigm of redox signaling protecting cells against oxidative stress in the heart. Our previous studies demonstrated that valosin-containing protein (VCP), an ATPase-associated protein, is a vital mediator protecting the heart against cardiac stress and ischemic injury. However, the molecular regulations conferred by VCP in the heart are not fully understood. In this study, we explored the potential role of VCP in cardiac protein SNO using multiple cardiac-specific genetically modified mouse models and various analytical techniques including biotin switch assay, liquid chromatography, mass spectrometry, and western blotting. Our results showed that cardiac-specific overexpression of VCP led to an overall increase in the levels of SNO-modified cardiac proteins in the transgenic (TG) vs. wild-type (WT) mice. Mass spectrometry analysis identified mitochondrial proteins involved in respiration, metabolism, and detoxification as primary targets of SNO modification in VCP-overexpressing mouse hearts. Particularly, we found that VCP itself underwent SNO modification at a specific cysteine residue in its N-domain. Additionally, our study demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, also experienced increased SNO in response to VCP overexpression. While deletion of inducible nitric oxide synthase (iNOS) in VCP TG mice did not affect VCP SNO, it did abolish SNO modification in mitochondrial complex proteins, suggesting a dual mechanism of regulation involving both iNOS-dependent and independent pathways. Overall, our findings shed light on post-translational modification of VCP in the heart, unveiling a previously unrecognized role for VCP in regulating cardiac protein SNO and offering new insights into its function in cardiac protection.