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 氧化应激增加与心脏病中心脏细胞功能障碍有关。不平衡的氧化还原 状态导致S-Glutathione的翻译后修饰增加，这会改变半胱氨酸 关键肌丝蛋白上的残基，例如心脏肌球蛋白结合蛋白-C（CMYBP-C）。 CMYBP-C 调节肌节的收缩和放松。蛋白激酶A对CMYBP-C的磷酸化 （PKA）是心脏保护。然而，在心脏失败的心脏中，CMYBP-C的磷酸化水平降低，与 S-glutathioneyplated CMYBP-C的增加。当心肌细胞与氧化的谷胱甘肽孵育时 （GSSG），肌丝钙敏感性增加，跨桥动力学减慢。先前的实验是 无法隔离s-谷胱甘肽化的cmybp-c的特定作用（即，没有其他s-的影响 谷胱甘肽蛋白）或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'-末端域 随着GSSG，S-谷胱甘肽化的CMYBP-C显着增加，CMYBP-C下调 磷酸化。这些结果表明，CMYBP-C磷酸化的减少以及随之而来的损失 在失败的心脏中看到的磷酸化CMYBP-C的心脏保护作用可能是由于增加 s-谷胱甘肽化的CMYBP-C。此外，抗缺血药物Ranolazine已被证明可以改善 舒张功能到假水平，与S-谷胱甘肽化的CMYBP-C相关。这些数据表明 当前可用的心脏疗法可能有助于调节CMYBP-C S-谷酰二酰化的水平。 这是该建议将确定磷酸化和S-谷二酰化之间的相互作用如何影响 通过我们的小说“切割和 粘贴” spyc3小鼠模型。蛋白质结构域CMYBP-C的蛋白质结构域C0C7将使用肌膜“切割” 烟草蚀刻病毒蛋白，并在洗涤步骤后，有或没有修饰的重组C0C7SC蛋白 半胱氨酸的保留将取代其在肌膜中内源性位置内的位置。站点定向 诱变将用于产生半胱氨酸的替代构建体，以防止特异 残留物和每个构建体的功能效应将使用“切割和糊状”模型测量。双重的 在有或没有雷诺嗪治疗的情况下，在PKA和GSSG中孵育将决定这一点的功能效应 相互作用。 ProQ钻石染色，免疫印迹，PHOS-TAG凝胶和质谱法的组合 将用于衡量总修改水平并确定特定地点的修改。结果 提案将是第一个确定单个CMYBP-C s-谷胱甘肽的残差和 磷酸化和s-谷胱甘肽化cmybp-c串扰。这项研究建议将导致更好 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，如果当前可用的治疗可能使受氧化应激影响的心脏受益。