Molecular Regulation of Titin Elasticity by Post-Translational Modification

 描述（由申请人提供）：在心脏充盈期间或在相对肌群收缩期间，肌节经历被动拉伸，使其总长度增加数百纳米。在过去，人们认为细胞外结构，主要是胶原蛋白，负责肌节的完整性，并提供阻力，以防止过度拉伸的肌节。在过去的30年中，肌小节的第三丝肌联蛋白（titin）在肌肉组织的被动牵张过程中承担大部分力，并且负责设定肌小节的最佳工作长度，这一点已经变得清楚。直到2012年，在对数百人进行基因测序后，才发现肌联蛋白突变是遗传性扩张型心肌病的主要原因。既然肌联蛋白突变和疾病之间存在明确的联系，人们的注意力已经转向确定肌联蛋白的正常生理作用。除了组织粗丝外，肌联蛋白的I-带段变形以适应肌节的拉伸。富含脯氨酸的肌联蛋白的非结构区域像分子弹簧一样延伸。另一方面，结构化的IG结构域在几个微微牛顿的力下展开，以显示可以进行翻译后修饰的隐藏残基。肌联蛋白的I-带异常富含隐蔽的半胱氨酸残基，当被强制暴露时，其可以与氧化和亚硝基化物质反应。因此，肌联蛋白被认为是骨骼肌和心肌中重要的氧化还原传感器。我建议研究翻译后修饰对肌联蛋白弹性和折叠的影响。这些研究对心肌梗死后或糖尿病、高血压和动脉粥样硬化等疾病的发生过程中心肌力学的变化具有重要意义。目的1研究活性氧是如何通过阻断肌联蛋白IG结构域的折叠或诱导二硫键的形成来改变其稳定性的。目的2是确定活性氮物质与肌联蛋白IG结构域中的残基反应是否改变肌联蛋白力学以及力学稳定性如何取决于IG折叠内修饰的位置。目的3寻求利用在肌联蛋白中含有遗传编码标签的新型小鼠模型来测量肌联蛋白IG在肌肉组织中展开的程度， 超分辨率和电子显微镜。这种单分子和单肌原纤维实验的独特组合将证明分子水平的变化如何转化为“机械表型”。“这些研究提供了关于氧化损伤如何影响肌肉细胞骨架，改变心肌力学，启动导致重塑和进一步损害心脏功能和舒张性能的信号通路的见解。

 DESCRIPTION (provided by applicant): During the filling of the heart or during the contraction of opposing muscle groups, the sarcomere experiences passive stretch that increases its overall length by several hundred nanometers. In the past, it was believed that extracellular structures, mainly collagen, were responsible for the integrity of the sarcomere and provided resistance to prevent over-stretching of the sarcomere. Within the past thirty years, it has become clear that titin, the third filament of the sarcomere, bears the majority of the force during passive stretch f muscle tissue, and is responsible for setting the optimal working length of the sarcomere. Only in 2012, after genetic sequencing of hundreds of individuals, was it shown that mutations in titin are the leading cause of inherited dilated cardiomyopathy. Now that there exists a clear link between titin mutations and disease, attention has turned towards identifying the normal physiological role of titin. Besides organizing the thick filament, the I-band segment of titin deforms to accommodate stretching of the sarcomere. Unstructured regions of titin rich in proline extend like molecular springs. Structured Ig domains, on the other hand, unfold at forces of several piconewtons to reveal cryptic residues that can undergo post-translational modification. The I-band of titin is unusually rich in cryptic cysteine residues, which can react with both oxidative and nitrosylative species when exposed by force. Hence, titin is thought to be an important redox sensor in skeletal and cardiac muscle. I propose to study the effects of post-translational modifications on titin elasticity and folding. These studies have important implications for how myocardial mechanics change after myocardial infarction, or in the setting of diseases such as diabetes, hypertension, and atherosclerosis. Aim 1 will study how reactive oxygen species alter the stability of titin Ig domain by blocking folding or inducing disulfide formation. Aim 2 is to determine if reactive nitrogen species react with residues in titin Ig domains alter titin mechanics and how mechanical stability depends on the location of the modification within the Ig fold. Aim 3 seeks to utilize a novel mouse model containing a genetically encoded tag in titin to measure the extent of titin Ig unfolding in muscle tissue using super-resolution and electron microscopy. This unique combination of single molecule and single myofibril experiments will demonstrate how changes at the molecular level translate into a "mechanical phenotype." These studies provide insights into how oxidative insults, such as those present in ischemia/reperfusion tend to affect the cytoskeleton of muscle, altering myocardial mechanics, and initiating signaling pathways that lead to remodeling and further impairment of cardiac function and diastolic performance.