The role of S-glutathione in regulating cardiac myosin binding protein-C function

S-谷胱甘肽在调节心肌肌球蛋白结合蛋白-C功能中的作用

基本信息

  • 批准号:
    10749281
  • 负责人:
  • 金额:
    $ 6.91万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-09-30 至 2026-09-29
  • 项目状态:
    未结题

项目摘要

Project Summary Increased oxidative stress is associated with cardiac cell dysfunction in heart disease. An unbalanced redox state leads to an increase in the post-translational modification of S-Glutathione, which modifies cysteine residues on key myofilament proteins, such as cardiac myosin binding protein-C (cMyBP-C). cMyBP-C regulates contraction and relaxation of the sarcomere. The phosphorylation of cMyBP-C by Protein Kinase A (PKA) is cardioprotective. Yet, in the failing heart, phosphorylation levels of cMyBP-C are reduced, contrary to an increase in S-glutathionylated cMyBP-C. When cardiomyocytes were incubated with oxidized glutathione (GSSG), myofilament calcium sensitivity increased and cross-bridge kinetics slowed. Prior experiments were unable to isolate the specific effects of S-glutathionylated cMyBP-C (i.e., without the effects of other S- glutathionylated proteins) nor the specific sites responsible for the functional change. Phosphorylation and S- glutathionylation of cMyBP-C may have antagonistic effects. Incubating three N’-terminal domains of cMyBP-C with GSSG led to a significant increase in S-glutathionylated cMyBP-C and a downregulation in cMyBP-C phosphorylation. These results indicate that the decrease in cMyBP-C phosphorylation and consequent loss of the cardioprotective effect of phosphorylated cMyBP-C seen in the failing heart could be due to an increase in S-glutathionylated cMyBP-C. In addition, the anti-ischemic drug, ranolazine, has been shown to improve diastolic function to sham levels, which correlated with S-glutathionylated cMyBP-C. These data indicate that a currently available cardiac therapy might be useful in moderating the levels of cMyBP-C S-glutathionylation. Thus, this proposal will identify how the interaction between phosphorylation and S-glutathionylation affects cardiomyocyte function under normal, elevated, and therapeutically treated conditions using our novel “cut and paste” SpyC3 mouse model. Protein domains C0C7 of cMyBP-C will be “cut” from the sarcomere using the tobacco etch viral protease and, after washing steps, recombinant C0C7sc protein with and without modified cysteine residues will replace its location within its endogenous location in the sarcomere. Site-directed mutagenesis will be used to generate cysteine substitution constructs preventing S-glutathionylation at specific residues and the functional effects of each construct will be measured using the “cut and paste” model. Dual incubation of PKA and GSSG with and without ranolazine treatment will determine the functional effects of this interaction. A combination of ProQ Diamond staining, immunoblotting, Phos-tag gels, and mass spectrometry will be used to measure total modification levels and identify the site-specific modifications. Results from this proposal will be the first to identify the functional effects of individual cMyBP-C S-glutathionylated residues and of phosphorylation and S-glutathionylation cMyBP-C crosstalk. This research proposal will lead to a better understanding of the effects of oxidative stress on cMyBP-C function, its potential to affect phosphorylation in the heart, and if a currently available therapeutic might benefit hearts affected by oxidative stress.
项目摘要 氧化应激增加与心脏病中的心脏细胞功能障碍有关。不平衡的氧化还原 状态导致S-谷氨酸的翻译后修饰增加,S-谷氨酸修饰半胱氨酸 关键肌丝蛋白上的残基,如心肌肌球蛋白结合蛋白-C(cMyBP-C)。cmybp-C 调节肌节的收缩和松弛。蛋白激酶A对cMyBP-C的磷酸化作用 (PKA)有心脏保护作用然而,在衰竭的心脏中,cMyBP-C的磷酸化水平降低,与正常人相反。 S-谷胱甘肽化cMyBP-C增加。当心肌细胞与氧化型谷胱甘肽孵育时, (GSSG),肌丝钙敏感性增加,跨桥动力学减慢。先前的实验是 不能分离S-谷胱甘肽化cMyBP-C的特异性作用(即,没有其他S的影响- 谷胱甘肽化的蛋白质),也不是负责功能变化的特定位点。磷酸化和S- cMyBP-C的谷胱甘肽化可能具有拮抗作用。孵育cMyBP-C的三个N '末端结构域 GSSG导致S-谷胱甘肽化cMyBP-C显著增加,cMyBP-C下调, 磷酸化这些结果表明,cMyBP-C磷酸化的减少和随之而来的cMyBP-C磷酸化水平的丧失, 在衰竭心脏中观察到的磷酸化cMyBP-C的心脏保护作用可能是由于 S-谷胱甘肽化cMyBP-C。此外,抗缺血药物雷诺嗪已被证明可以改善 舒张功能与假手术水平相比,与S-谷胱甘肽化cMyBP-C相关。这些数据表明,A 目前可用的心脏疗法可能用于调节cMyBP-CS-谷胱甘肽化的水平。 因此,该建议将确定磷酸化和S-谷胱甘肽化之间的相互作用如何影响 使用我们的新的“切割和切割”技术, 粘贴”SpyC 3小鼠模型。cMyBP-C的蛋白质结构域C 0 C7将被从肌节上“切割”下来, 烟草蚀纹病毒蛋白酶,以及洗涤步骤后的经修饰和未经修饰的重组C 0 C7 sc蛋白 半胱氨酸残基将取代其在肌节中内源性位置内的位置。定点 诱变将用于产生半胱氨酸取代构建体,其防止在特定的位置S-谷胱甘肽化。 将使用“剪切和粘贴”模型测量每个构建体的残基和功能效应。双 PKA和GSSG与雷诺嗪处理和不与雷诺嗪处理的孵育将确定这种作用的功能效应。 互动结合ProQ Diamond染色、免疫印迹、Phos标记凝胶和质谱 将用于测量总的修改水平,并确定特定于站点的修改。结果从这个 该提案将是第一个确定单个cMyBP-C S-谷胱甘肽化残基的功能效应, 磷酸化和S-谷胱甘肽化cMyBP-C串扰。这项研究计划将带来更好的 了解氧化应激对cMyBP-C功能的影响,其影响磷酸化的潜力, 心脏,如果目前可用的治疗方法可能有益于受氧化应激影响的心脏。

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