Top Down Proteomics Of Myofilaments In Heart Failure

描述（由申请人提供）：心力衰竭是美国男性和女性死亡的主要原因。心力衰竭的潜在分子和细胞机制非常复杂且知之甚少。关键肌丝调节蛋白（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（RLC 2）和心肌肌球蛋白结合蛋白C（cMyBP-C），它们在心肌收缩性中起重要作用。该假说认为，外在和内在应激均触发分子信号传导过程，导致KMRP的改变修饰，从而导致收缩功能障碍并最终导致心力衰竭。最近的研究令人信服地表明，cTnI和cMyBP-C的改变与心功能不全直接相关。对KMRPs进行公正和系统的分析，以全面检测蛋白质修饰的变化，确定哪些位点被修饰或改变，并阐明这些改变在向心力衰竭转变期间如何协同作用，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自上而下的质谱（MS）为基础的疾病蛋白质组学平台，检查从正常和患病组织中提取的KMRPs，以建立KMRPs的改变修饰和心功能不全之间的相关性。自上而下的MS直接分析完整的蛋白质，提供“鸟瞰图”，以在一个光谱中同时观察所有可能的修饰，这比在传统的自下而上的方法中测量蛋白水解消化的肽可靠得多。整合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提取的完整KMRP，全局检测反映外在和内在应力的所有修饰，3）识别和量化（新的）修饰，并确定在向终末期心力衰竭过渡期间KMRP的多个协同改变和PTM在多个靶位点中分布的变化。具体目标包括：1）建立一种整合的自上而下的疾病蛋白质组学技术，用于高效、灵敏和简单地分离和表征完整的KMRP。2)确定肥厚和衰竭猪心肌KMRP中改变的蛋白质修饰。3)确定蛋白激酶A（PKA）和蛋白激酶A（PKC）介导的磷酸化在正常和患病猪心肌KMRPs中的功能作用。4)确定KMRP（例如cTn）中一种新的改变在调节心肌收缩力方面的功能后果。该研究项目的成功，整合了蛋白质组学和功能研究，将提供一个全球地图的蛋白质修饰发生在正常和疾病条件下的KMRPs，并揭示了新的见解收缩功能障碍的分子机制在心力衰竭。

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.