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

• 批准号: 10749281
• 负责人: Angela Greenman
• 金额: $ 6.91万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10749281
• 关键词: Address; Adrenergic Agents; Affect; Alanine; Biological Assay; Calcium; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cardiac health; Cell Membrane Permeability; Cells; Contractile Proteins; Cyclic AMP-Dependent Protein Kinases; Cysteine; Data; Development; Diamond; Disulfides; Down-Regulation; Etiology; Functional disorder; Gel; Glutathione; Glutathione Disulfide; Heart; Heart Diseases; Heart failure; In Situ; In Vitro; Incubated; Individual; Ischemia; Kinetics; Knock-out; Knockout Mice; Lead; Location; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Microfilaments; Modeling; Modification; Mus; Muscle Cells; Muscle function; Myocardial Ischemia; N-terminal; Oxidation-Reduction; Oxidative Stress; Paste substance; Peptide Hydrolases; Permeability; Pharmaceutical Preparations; Phosphorylation; Post-Translational Protein Processing; Pro-Q aerosol foam; Proteins; Publishing; Rattus; Reactive Oxygen Species; Recombinants; Regulation; Relaxation; Research Proposals; Role; Sarcomeres; Signal Transduction; Site; Site-Directed Mutagenesis; Skin; Stains; Stress; Sulfhydryl Compounds; Tertiary Protein Structure; Testing; Therapeutic; Tobacco use; Viral; Western Blotting; cardioprotection; experimental study; fighting; functional improvement; hypertensive; improved; mechanical force; mouse model; myosin-binding protein C; novel; prevent; protein function; ranolazine; response

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 在心脏病中，氧化应激增加与心脏细胞功能障碍有关。不平衡的氧化还原 状态导致修饰半胱氨酸的S-谷胱甘肽的翻译后修饰增加 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)具有心脏保护作用。然而，在衰竭的心脏中，cMyBP-C的磷酸化水平降低，这与 S-谷胱甘肽基化的cMyBP-C的增加。氧化谷胱甘肽与心肌细胞孵育的实验研究 (GSSG)，肌丝钙敏感性增加，跨桥动力学减慢。之前的实验是 无法分离出S-谷胱甘肽修饰的cMyBP-C的特异性作用(即没有其他S的作用- 谷胱甘肽基化蛋白)，也不是导致功能变化的特定部位。磷酸化与S- CMyBP-C的谷胱甘肽基化可能具有拮抗作用。CMyBP-C三个N‘末端结构域的孵育 用药后S谷胱甘肽结合的cMyBP-C显著升高，cMyBP-C表达下调 磷酸化。这些结果表明，cMyBP-C磷酸化水平的降低和随之而来的 在衰竭心脏中发现的磷酸化cMyBP-C的心脏保护作用可能是由于 S-谷胱甘肽-cMyBP-C。此外，抗脑缺血药物雷诺嗪已经被证明是有效的。 舒张期功能恢复至假手术水平，与S-谷胱甘肽结合的cMyBP-C相关。这些数据表明， 目前可用的心脏疗法可能有助于调节cMyBP-C、S-谷胱甘肽的水平。 因此，这项提议将确定磷酸化和S-谷胱甘肽基化之间的相互作用如何影响 在正常、升高和治疗条件下的心肌细胞功能 粘贴“SpyC3鼠标模型。CMyBP-C的蛋白结构域C0C7将被从肌节上用 烟草蚀刻病毒蛋白酶，洗涤步骤后，经修饰和未修饰的重组C0C7sc蛋白 半胱氨酸残基将取代它在肌节内源性位置的位置。站点定向 突变将被用来产生半胱氨酸取代构建体，防止S-谷胱甘肽在特定的 每一种结构的残基和功能效应将使用“剪切和粘贴”模型进行测量。双人 PKA和GSSG在雷诺嗪处理前后的孵育将决定这一功能的影响 互动。ProQ钻石染色、免疫印迹、PHOS-TAG凝胶和质谱学的组合 将用于测量总修改级别并确定特定于站点的修改。由此产生的结果 提案将首次确定单个cMyBP-C S-谷胱甘肽基化残基的功能效应和 的磷酸化和S-谷胱甘肽的cMyBP-C串扰。这项研究建议将导致一个更好的 了解氧化应激对cMyBP-C功能的影响及其对磷酸化的影响 如果目前可用的治疗方法可能使受氧化应激影响的心脏受益。