A new post-translational modification, citrullination, changes in heart failure

描述（由申请人提供）：蛋白质瓜氨酸化（脱胺化）是一种改变氨基酸残基精氨酸（Arg）的翻译后修饰（PTM），由于其在人类自身免疫疾病中的新作用，最近成为一个感兴趣的领域。我们是第一个证实PTM存在于人类心脏的研究小组。具体来说，我们的初步数据显示瓜氨酸化发生在肌丝蛋白中，如原肌球蛋白（Tm）、肌球蛋白（重链和轻链）和肌球蛋白结合蛋白c。此外，我们发现与对照心脏相比，心力衰竭（HF）患者心肌中去亚胺化蛋白的程度有所增加。基于这些结果，我们假设瓜氨酸化在HF的发病机制中起作用，特别是影响心肌收缩。此外，我们假设瓜氨酸化在HF中加剧，特别是当存在自身免疫成分时。这项探索性资助旨在建立蛋白瓜氨酸化、肽基精氨酸脱亚胺酶（PAD）（负责这种PTM的酶）与衰竭心脏的心脏和心肌功能障碍之间的关系。在Specific Aim 1中，研究了三种HF、慢性主动脉缩窄和两种炎症扩张型心肌病小鼠模型中PAD异构体的表达和瓜氨酸化心肌蛋白。使用最近开发的基于质谱的方法，瓜氨酸化蛋白质和修饰的氨基酸残基将被识别和定量。此外，利用分子生物学和第二种创新的质谱方法的结合，我们将明确地定义和量化心肌细胞中的PAD亚型。具体目的2侧重于评估PAD和蛋白瓜氨酸化在心肌细胞中的功能作用。结合分子生物学和药理学方法对PAD异构体活性进行调控，蛋白修饰程度与心肌收缩功能相关。用Ala（不可修饰）或Gln（潜在的Arg- cit模拟物）取代可修饰的瓜氨酸Arg残基的Tm突变体将进行功能改变收缩测试。这个程序是高度新颖的，因为蛋白质瓜氨酸化和PAD系统从未在心脏中被探索过，但我们的新证据支持其调节肌肉收缩力的潜力。这项工作将导致对心脏翻译后蛋白调控的全新机制的理解。

DESCRIPTION (provided by applicant): Protein citrullination (deimination), a post-translational modification (PTM) altering the amino acid residue arginine (Arg), has recently become an area of interest due its emerging role in human auto-immune diseases. We are the first group to establish that this PTM is present in the human heart. Specifically, our preliminary data shows citrullination occurs in myofilament proteins such as tropomyosin (Tm), myosin (heavy and light chain) and myosin binding protein C. Furthermore, we find an increase in the extent of deiminated proteins in myocardium obtained from patients with heart failure (HF) compared to control hearts. Based on these results, we hypothesize that citrullination plays a role in the pathogenesis of HF, particularly affecting myocardial contraction. Furthermore, we hypothesize that citrullination is exacerbated in HF especially when there is an autoimmune component. This exploratory grant is aimed to establish the relation between protein citrullination, peptidyl arginine deiminase (PAD), the enzyme responsible for this PTM, and cardiac and myocardial dysfunction in the failing heart. In Specific Aim 1, PAD isoform expression and citrullinated myocardial proteins are explored in three mouse models of HF, chronic aortic constriction and two inflammatory dilated cardiomyopathies. Using a recently developed mass spectrometry-based method, citrullinated proteins and the modified amino acid residues will be identified and quantified. In addition, using a combination of molecular biology and a second innovative mass spectroscopy approach, we will unambiguously define and quantify the PAD isoforms in cardiac myocytes. Specific aim 2 focuses on the assessment of the functional role of PAD and protein citrullination in cardiac myocytes. Combing molecular biology and pharmacological methods to manipulate PAD isoform activity, the extent of modified proteins is correlated to myocardial contractile function. Tm mutants with Ala (non modifiable) or Gln (a potential mimetic of Arg-Cit) replacing the modifiable citrullinatable Arg residues, will be tested for functional alterations contraction. This program is highly novel as protein citrullination and PAD system has never been explored in the heart, yet our new evidence supports its potential to modulate muscle contractility. This work will lead to understanding of an entirely new mechanism for post-translational protein regulation in the heart.