Top Down Proteomics Of Myofilaments In Heart Failure

描述(申请人提供)：心力衰竭是美国男性和女性的主要死亡原因。心力衰竭的潜在分子和细胞机制非常复杂，而且知之甚少。关键的肌丝调节蛋白(KMRPs)包括心肌肌钙蛋白(CTn)、原肌球蛋白(TM)、肌球蛋白调节轻链2(RLC2)和心肌肌球蛋白结合蛋白C(cMyBP-C)，在心肌收缩能力中起重要作用。假说是外在和内在的应激都会触发分子信号传递过程，导致KMRPs的改变，导致收缩功能障碍，最终导致心力衰竭。最近的研究令人信服地表明，cTnI和cMyBP-C的改变与心功能障碍直接相关。对KMRP进行公正和系统的分析，以全面检测蛋白质修饰的变化，识别哪些位点被修改或改变，并阐明这些改变如何在心力衰竭的过渡过程中协同作用，对于理解潜在的分子机制至关重要。然而，这仍然是一个重大挑战。为了应对这一挑战，我们建议建立一个简单而强大的基于自上而下的质谱学(MS)的疾病蛋白质组学平台来检测从正常组织和疾病组织中提取的KMRPs，以建立KMRPs的改变与心功能障碍之间的相关性。自上而下的MS直接分析完整的蛋白质，提供了在一个光谱中同时观察所有可能的修饰的“鸟瞰”，这比用传统的自下而上的方法测量蛋白质分解的多肽要可靠得多。集成的自上而下的蛋白质组学平台将提供一个全面的工具，以有效地分离从心肌组织中提取的完整的KMRP，在全球范围内检测反映外部和内在压力的所有修改，3)识别和量化(新的)修改，并识别KMRP中的多个协同变化以及在向终末期心力衰竭过渡期间多个靶点之间PTM的分布的变化。具体目标包括：1)建立一种高效、灵敏、简便的完整KMRP分离鉴定的自上而下完整的疾病蛋白质组学技术。2)从肥厚和衰竭的猪心肌中确定KMRP的蛋白修饰。3)测定蛋白激酶A(PKA)和蛋白激酶A(PKC)介导的蛋白激酶A(PKA)和蛋白激酶A(PKC)在正常和病变猪心肌KMRP中的功能效应。4)确定KMRP的一个新的改变，例如cTn，在调节心脏收缩能力方面的功能后果。这项整合了蛋白质组学和功能研究的研究项目的成功，将提供KMRP在正常和疾病条件下发生的蛋白质修饰的全球图谱，并为心力衰竭中收缩功能障碍的分子机制提供新的见解。 公共卫生相关性：心力衰竭仍然是美国死亡率和发病率的主要原因，并正在接近老龄化人口的流行水平。本研究旨在通过蛋白质组学和功能的综合研究，为心力衰竭患者心功能障碍的分子机制研究提供新的见解。这些研究发现可能会促进开发新的治疗靶点，以更好地诊断和治疗心脏病。

DESCRIPTION (provided by applicant): Heart failure is the leading cause of death for both men and women in the United States. The underlying molecular and cellular mechanisms of heart failure are very complex and poorly understood. Key Myofilament Regulatory Proteins (KMRPs), which include cardiac troponin (cTn), tropomyosin (Tm), myosin regulatory light chain 2 (RLC2) and cardiac myosin binding protein C (cMyBP-C), play essential roles in cardiac contractility. The hypothesis is that both extrinsic and intrinsic stresses trigger the molecular signaling processes that result in altered modifications to KMRPs leading to contractile dysfunction and eventually heart failure. Recent studies show convincingly that altered modifications in cTnI and cMyBP-C are directly linked to cardiac dysfunction. An unbiased and systematic analysis of the KMRPs to globally detect the changes in protein modifications, identify which sites are modified or altered, and elucidate how these alterations act in concert during the transition to the heart failure is of paramount importance for the understanding of the underlying molecular mechanisms. However, this remains a major challenge. To address this challenge, we propose to establish a simple and robust top-down mass spectrometry (MS)-based disease proteomics platform to examine KMRPs extracted from both normal and diseased tissues to establish a correlation between altered modifications of KMRPs and cardiac dysfunction. Top-down MS directly analyzes intact proteins providing a "bird's eye view" to observe all possible modifications simultaneously in one spectrum, which is much more reliable than measuring the proteolytically-digested peptides in the conventional bottom-up approach. The integrated top-down proteomics platform will provide a comprehensive tool to effectively separate the intact KMRPs extracted from myocardial tissues, globally detect all modifications that reflect extrinsic and intrinsic stresses, 3) identify and quantify (novel) modifications, and identify multiple concerted alterations in KMRPs and the changes in the distribution of PTMs among multiple targeted sites during the transition to the end- stage heart failure. The specific aims include: 1) Establish an integrated top-down disease proteomics technology for the separation and characterization of intact KMRPs with high efficiency, sensitivity and simplicity. 2) Determine altered protein modifications in KMRPs from hypertrophied and failing swine myocardium. 3) Determine the functional effects of protein kinase A (PKA) and protein kinase A (PKC)- mediated phosphorylation in KMRPs of normal and diseased swine myocardium. 4) Determine the functional consequence of one novel alteration in KMRPs, e.g. cTn, in regulating cardiac contractility. The success of this research project, which integrates proteomics and functional studies, will provide a global map of protein modifications occurring to the KMRPs under normal and diseased conditions and shed new insights into the molecular mechanism of contractile dysfunction in heart failure. PUBLIC HEALTH RELEVANCE: Heart failure remains a leading cause of mortality and morbidity in the United States and is approaching epidemic levels in the aging population. This proposal aims to provide the new insights into the molecular mechanism of cardiac dysfunction in heart failure through an integrated proteomics and functional study. The research discoveries could foster the development of new therapeutic targets for better diagnosis and treatment of heart diseases.