Citrullination of myofilament proteins in heart failure
Citrullination of myofilament proteins in heart failure
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DOI：
10.1093/cvr/cvv185
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发表时间：
2015-11-01
影响因子：
10.8
通讯作者：
Van Eyk, Jennifer E.
中科院分区：
文献类型：
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作者：
Fert-Bober, Justyna;Giles, John T.;Van Eyk, Jennifer E.
瓜氨酸化是肽基精氨酸脱亚胺酶 (PAD) 家族将精氨酸翻译后转化为瓜氨酸的过程，与多种疾病相关，并且特定的瓜氨酸化蛋白已被证明会改变功能，而其他蛋白则充当自身抗原。在这项研究中，我们鉴定了来自健康和心力衰竭患者的人类心肌样本中的瓜氨酸蛋白，并确定了几种潜在的功能后果。我们进一步研究了心脏中 PAD 亚型细胞特异性表达。使用数据独立 (SWATH) 采集方法的瓜氨酸靶向蛋白质组学策略用于鉴定修饰的心脏蛋白。使用二维凝胶电泳验证瓜氨酸诱导的肌节蛋白，并使用生化和功能测定进行研究。通过 RT-PCR 证实了心肌 PAD 亚型，其中 PAD2 是心肌细胞中的主要亚型。总共鉴定出 304 个瓜氨酸位点，映射到三个研究组中的 145 种蛋白质：正常组、缺血组和扩张型心肌病组。肌球蛋白的瓜氨酸化（使用 HMM 片段）降低了其内在 ATP 酶活性并抑制了 acto-HMM-ATP 酶活性。瓜氨酸 TM 导致更强的 F-肌动蛋白结合并抑制 acto-HMM-ATPase 活性。瓜氨酸化 TnI 不会改变与 F-肌动蛋白的结合或 acto-HMM-ATPase 活性。总体而言，肌节蛋白的瓜氨酸化导致带皮心肌细胞的 Ca2+ 敏感性降低，但最大钙激活力或希尔系数没有变化。确定了心脏蛋白质组特有的瓜氨酸化。我们的数据表明心脏肌节的重要结构和功能改变以及蛋白质瓜氨酸化对此过程的贡献。
Citrullination, the post-translational conversion of arginine to citrulline by the enzyme family of peptidylarginine deiminases (PADs), is associated with several diseases, and specific citrullinated proteins have been shown to alter function while others act as auto-antigens. In this study, we identified citrullinated proteins in human myocardial samples, from healthy and heart failure patients, and determined several potential functional consequences. Further we investigated PAD isoform cell-specific expression in the heart.A citrullination-targeted proteomic strategy using data-independent (SWATH) acquisition method was used to identify the modified cardiac proteins. Citrullinated-induced sarcomeric proteins were validated using two-dimensional gel electrophoresis and investigated using biochemical and functional assays. Myocardial PAD isoforms were confirmed by RT-PCR with PAD2 being the major isoform in myocytes. In total, 304 citrullinated sites were identified that map to 145 proteins among the three study groups: normal, ischaemia, and dilated cardiomyopathy. Citrullination of myosin (using HMM fragment) decreased its intrinsic ATPase activity and inhibited the acto-HMM-ATPase activity. Citrullinated TM resulted in stronger F-actin binding and inhibited the acto-HMM-ATPase activity. Citrullinated TnI did not alter the binding to F-actin or acto-HMM-ATPase activity. Overall, citrullination of sarcomeric proteins caused a decrease in Ca2+ sensitivity in skinned cardiomyocytes, with no change in maximal calcium-activated force or hill coefficient.Citrullination unique to the cardiac proteome was identified. Our data indicate important structural and functional alterations to the cardiac sarcomere and the contribution of protein citrullination to this process.