Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States.
Nitric Oxide. 2018 Apr 1;74:56-64. doi: 10.1016/j.niox.2018.01.007. Epub 2018 Jan 31.
S-nitrosothiols derived from nitric oxide are known to regulate cell signaling through thiol modification. Since small G protein RhoA contains cysteine residues in the GTP-binding domain which is critical for its function, modification these thiols may alter RhoA activity and lead to changes in the downstream signaling such as myosin light chain phosphorylation. However, it is still unclear that if RhoA activity and its downstream signals might be modulated by S-nitrosothiols and if the two cysteine residues located in the GTP-binding domain are critical for the regulation. In this study we show that S-nitroso-L-cysteine (CSNO) blocked RhoA activation as determined by either GDP/GTP exchange, active RhoA binding to rhotekin or RhoA translocation. CSNO was shown to lead to RhoA nitrosylation and RhoA thiol oxidation status was found to be consistent with loss of its activity. Mutation of all 6 single cysteine residues to serine showed that purified recombinant C20S mutant and C26/20S mutant were resistant to CSNO, but interestingly, in the intact cells only the double C16/20S mutant was resistant to CSNO. Moreover, inhibition of RhoA activation led to Rho-kinase inhibition and inhibition of Rho pathway signaling by CSNO. In both smooth muscle cells and aortic tissue, the outcome was inhibition of agonist-stimulated MYPT1 phosphorylation and reduced levels of myosin light chain phosphorylation. These effects of CSNO on MYPT1 and myosin light chain phosphorylation appear to be cGMP-independent since they were unaffected by inhibition of guanylyl cyclase. In contrast to CSNO, spermine NONOate did not alter RhoA GDP/GTP exchange and the effects of this compound on myosin light chain phosphorylation were blocked by guanylyl cyclase inhibition. And importantly, in C16/20S overexpressed smooth muscle cells, MYPT1 phosphorylation was resistant to the inhibitory effect of CSNO. Together, these data suggest that S-nitrosothiols regulate myosin light chain phosphorylation by inhibiting RhoA/Rho-kinase signaling through modification of RhoA cysteine residues at 16 and 20 in its GTP-binding domain, which might be an important therapeutic target for diseases with imbalanced vascular resistance.
S-亚硝基硫醇来源于一氧化氮，已知可通过硫醇修饰来调节细胞信号转导。由于小 G 蛋白 RhoA 的 GTP 结合域中含有半胱氨酸残基，这些残基对于其功能至关重要，因此修饰这些硫醇可能会改变 RhoA 的活性，并导致下游信号如肌球蛋白轻链磷酸化的变化。然而，目前尚不清楚 S-亚硝基硫醇是否可以调节 RhoA 活性及其下游信号，以及位于 GTP 结合域中的两个半胱氨酸残基对于调节是否至关重要。在这项研究中，我们表明 S-亚硝基-L-半胱氨酸（CSNO）通过 GDP/GTP 交换、活性 RhoA 与 rhotekin 的结合或 RhoA 易位来阻断 RhoA 的激活。CSNO 导致 RhoA 亚硝基化，并且 RhoA 硫醇氧化状态与失去其活性一致。将所有 6 个单个半胱氨酸残基突变为丝氨酸表明，纯化的重组 C20S 突变体和 C26/20S 突变体对 CSNO 具有抗性，但有趣的是，在完整细胞中，只有双 C16/20S 突变体对 CSNO 具有抗性。此外，抑制 RhoA 激活导致 Rho 激酶抑制和 Rho 通路信号转导被 CSNO 抑制。在平滑肌细胞和主动脉组织中，结果是激动剂刺激的 MYPT1 磷酸化和肌球蛋白轻链磷酸化水平降低。CSNO 对 MYPT1 和肌球蛋白轻链磷酸化的这些作用似乎与 cGMP 无关，因为它们不受鸟苷酸环化酶抑制的影响。与 CSNO 相反，精脒 NONOate 不会改变 RhoA 的 GDP/GTP 交换，并且该化合物对肌球蛋白轻链磷酸化的作用被鸟苷酸环化酶抑制所阻断。重要的是，在 C16/20S 过表达的平滑肌细胞中，CSNO 的抑制作用使 MYPT1 磷酸化不受影响。总之，这些数据表明，S-亚硝基硫醇通过修饰 RhoA 的 GTP 结合域中的第 16 和 20 位半胱氨酸残基来抑制 RhoA/Rho 激酶信号转导，从而调节肌球蛋白轻链磷酸化，这可能是血管阻力失衡相关疾病的重要治疗靶点。
