Identification of Speg Binding Proteins and Phosphorylation Targets in Skeletal and Cardiac Muscle

标题：骨骼肌和心脏中Speg结合蛋白和磷酸化靶点的鉴定 肌肉 摘要 重度危及生命的骨骼肌和/或心肌病，包括中枢性肌病（CNM） 和扩张型心肌病是由Speg基因突变和/或缺陷引起的（横纹肌优先 表达的Ser/Thr蛋白激酶）。Speg至少部分地位于三元/二元交界区域中， 骨骼肌和心肌。Speg激活的机制和Speg的功能作用，结合蛋白， 骨骼肌和心肌中的磷酸化靶点仍有待充分阐明。的当前方法 评估蛋白质相互作用伴侣缺乏可重复性和高水平的非特异性 交互.这对斯皮克来说尤其如此。我们创建了将V5/HA标签插入Speg的小鼠，用于 抗-HA-Speg免疫沉淀以鉴定Speg结合蛋白。在本申请中，我们将阐明Speg 相互作用组和确定Speg磷酸化的目标，在骨骼肌和心肌使用优化 最先进的质谱相关蛋白质组学技术与平行反应监测（PRM）。我们将 还鉴定了RyRs、Jph 2和Speg上的磷酸化位点，这些位点被Speg缺陷减少。我们的具体 目的是：SA 1：确定Speg缺乏对肌肉蛋白质组的影响。SA 2：鉴别Speg结合 使用具有V5/HA标记的Speg的小鼠的研究伙伴。SA 3.鉴定和定量RyRs、Jph 2上的磷酸化位点 和Speg，并评估Speg缺乏对这些网站的影响。成功完成本研究将 鉴定Speg结合蛋白并鉴定其主要靶点上的磷酸化位点。而此应用程序 中心的相互作用和目标的Speg，开发的技术将适用于其他骨骼和 心肌蛋白

Title: Identification of Speg Binding Proteins and Phosphorylation Targets in Skeletal and Cardiac Muscle Abstract Severe life-threatening skeletal muscle and/or cardiac myopathies including centronuclear myopathy (CNM) and dilated cardiomyopathy are caused by mutations and/or deficiency of Speg (striated muscle preferentially expressed Ser/Thr protein kinase). Speg is at least partially located in triadic/dyadic junctional regions in skeletal and cardiac muscle. The mechanisms of Speg activation and Speg’s functional roles, binding proteins, and phosphorylation targets in skeletal and cardiac muscle remain to be fully elucidated. Current methods of assessing protein interacting partners suffer from lack of reproducibility and high levels of nonspecific interactions. This is particularly true for Speg. We created mice with a V5/HA tag inserted into Speg to use for anti-HA-Speg immunoprecipitations to identify Speg binding proteins. In this application, we will elucidate Speg interactome and identify Speg phosphorylation targets in the skeletal and cardiac muscle using optimized state-of-the-art mass-spec-associated proteomics techniques with parallel reaction monitoring (PRM). We will also identify phosphorylation sites on RyRs, Jph2 and Speg that are reduced by Speg deficiency. Our specific aims are to: SA 1: Define the effects of Speg deficiency on the muscle proteome. SA2: Identify Speg binding partners using mice with V5/HA tagged Speg. SA 3. Identify and quantify phosphorylation sites on RyRs, Jph2 and Speg and assess the effects of Speg deficiency on these sites. Successful completion of this study will identify Speg binding proteins and identify phosphorylation sites on its major targets. While this application centers on interactions and targets of Speg, the techniques developed will be applicable to other skeletal and cardiac muscle proteins.