A molecular study linking cTnT dynamics to genetic cardiomyopathy

描述(由申请人提供)： 家族性肥厚型心肌病是一种常见且往往具有破坏性的遗传性心脏病。心脏蛋白的特定突变已被确定为该病的根本原因，但它们往往在远离突变的地方发挥其生物学效应。这种效应通常统称为变构，是蛋白质生物化学常用词汇的一部分，我们研究计划的实施将展示这种效应也是心脏马达的多蛋白质控制组件-细丝的一部分。特别是，我们专注于钙与cTnT的结合(长期以来，cTnT是控制心脏跳动的主要成分)和cTnI中已知重要位置的磷酸化。我们计划研究的突变(都在cTnT中)在某些情况下被证明对这两种控制机制都产生了显著的变化，这似乎证明了“远距离作用”的原理，但缺乏对这些变化如何从分子水平到整个动物生理学导致疾病的翻译理解。因此，以这种方式研究复杂的多组分机器中的变构对基础科学具有重大影响，对理解一种毁灭性和相对常见的人类疾病的根本原因具有最高的意义。为了解决这些问题，我们设计了一系列方法的研究策略，从对肌钙蛋白复合体、原肌球蛋白和肌动蛋白骨架的全原子模型的计算，到野生型和突变重组细丝性质的生物物理测量，再到纤维研究。这些方法产生了部分互补但重叠的信息，为这一复杂问题提供了全面的综合分析。为了更好地了解变构在这些生防剂在健康和疾病中的作用，我们将研究以下两个特定目标：特定目标1：评估钙结合转导到原肌球蛋白运动的分子机制，以及这如何调节野生型和已知FHC连锁TNT1突变的心功能细丝控制的生物物理学和生理学。特异性目的2：探讨野生型和已知FHC连锁TNT1突变中cTnI Ser23/24磷酸化调控肌丝激活的分子机制。 公共卫生相关性： 家族性肥厚型心肌病是一种常见的心脏遗传性疾病。心脏蛋白的特定突变已被确定为该病的根本原因，但它们往往在远离突变的地方发挥其生物学效应。这项申请旨在阐明这种“远距离行动”如何导致功能障碍和最终的人类疾病。

DESCRIPTION (provided by applicant): Familial Hypertrophic Cardiomyopathy is a common and often devastating genetic cardiac disease. Specific mutations in cardiac proteins have been identified as the root cause of this disease, but they often exert their biological effect far from the site of mutation. Such effects, usually known collectively as allostery are part of the common vocabulary of protein biochemistry, and implementation of our research program will demonstrate how such effects are also part of a multi-protein controlling component of the cardiac motor - the thin filament. In particular we focus on Ca2+ binding to cTnT (long known to be a major component in the control of a beating heart,) and phosphorylation at a known important location in cTnI. The fact that the mutations we plan to study (all in cTnT) have in some cases been shown to effect significant changes on both these control mechanisms seems to demonstrate the principle of "action at a distance" but what is lacking is a translational understanding of how these changes cause disease from the molecular level to whole animal physiology. Allostery in a complex multi-component machine investigated in this fashion is thus both of great impact in basic science and of the highest significance in understanding the root cause of a devastating and relatively common human disease. To address these questions we have devised a research strategy of methodologies that range from computation on an all atom model of the troponin complex, tropomyosin, and an actin backbone, to biophysical measurements of the properties of wildtype and mutated reconstituted thin filaments, to fiber studies. The methodologies yield partially complementary yet overlapping information that provides a fully integrated analysis of this complex question. In order to better understand allostery in the function of these biological control agents in both health and disease we will study the following 2 specific aims: Specific Aim 1: To evaluate the molecular mechanism of the transduction of Ca2+ binding to the movement of tropomyosin and how this regulates the biophysics and physiology of the thin filament control of cardiac function in wildtype and known FHC-linked TNT1 mutations. Specific Aim 2: To evaluate the molecular mechanism of the phosphorylation of Ser 23/24 of cTnI in regulating myofilament activation in wildtype and known FHC-linked TNT1 mutations. PUBLIC HEALTH RELEVANCE: Familial Hypertrophic Cardiomyopathy is an often devastating and common cardiac genetic disease. Specific mutations in cardiac proteins have been identified as the root cause of this disease, but they often exert their biological effect far from the site of mutation. This application is aimed at elucidating how this "action at a distance" causes dysfunction and eventual human disease.