Human Skeletal Muscle Proteome and Phosphoproteome in Obesity and Type 2 diabetes

描述（由申请人提供）：尽管骨骼肌胰岛素抵抗是代谢异常和慢性疾病（如2型糖尿病和心血管疾病）发展的主要因素，但其发病机制的分子机制仍然难以捉摸。胰岛素信号通路中的磷酸化和去磷酸化事件的缺陷，例如胰岛素受体底物-1（IRS-1）的丝氨酸/苏氨酸磷酸化，被认为是胰岛素抵抗的主要原因之一。大多数关于这些磷酸化事件的调节的研究都集中在激酶上，并在细胞培养或动物模型中进行。我们推测，丝氨酸/苏氨酸蛋白磷酸酶2A（PP 2A）复合物在人类骨骼肌过度激活和失调的胰岛素抵抗和2型糖尿病。这一假设是基于现有文献提出的，这些文献表明在细胞/动物模型中，PP 2A在糖脂毒性和糖尿病的胁迫下过度活化，以及我们自己的以下新的初步发现：1）。PP 2A的催化亚基（PP 2Ac）与瘦型健康受试者骨骼肌中的IRS-1相关，并且这种相互作用在肥胖/超重和T2 D肌肉中显著增加; 2）胰岛素抑制瘦型健康受试者肌肉中的PP 2Ac活性和Leu 309羧甲基化，但不抑制2型糖尿病患者。为了验证我们的假设，我们将评估 (Aim 1），翻译后修饰（目标2），和相互作用伙伴（目标3）的PP 2A骨骼肌瘦健康对照（LC），肥胖非糖尿病受试者（OC），2型糖尿病患者（T2 D），使用最先进的高效液相色谱-纳米喷雾-串联质谱（HPLC-ESI-MS/MS）。总体目标是解决磷酸酶功能和调节胰岛素抵抗和T2 D在人类的知识差距，使用我们实验室开发的临床，生物学和蛋白质组学方法的组合。对于目的1，我们将进行PP 2Ac共免疫沉淀实验，然后在基础和胰岛素输注下测定来自3组的肌肉活检中的PP 2A活性。对于目标2，我们将使用蛋白质印迹分析评估3组中的pTyr 307和mLeu 309。此外，我们将采用我们实验室开发的蛋白质组学方法进行磷酸化定量，以提供3组PP 2Ac磷酸化的全局视图。对于目标3，我们将使用针对PP 2Ac的抗体进行免疫共沉淀实验，然后使用我们实验室开发的蛋白质复合物的蛋白质组学方法评估3组中基础和胰岛素输注下的免疫共沉淀蛋白质量。这项研究的结果将是发现PP 2A活性异常，翻译后修饰和人类骨骼肌胰岛素抵抗的相互作用伙伴，促进新药的设计，以调节PP 2Ac翻译后修饰和蛋白质-蛋白质相互作用。这些药物可以恢复异常的PP 2Ac活性并改善胰岛素信号传导，从而治疗和/或预防T2 D以及其他慢性疾病。

DESCRIPTION (provided by applicant): Although skeletal muscle insulin resistance is a primary contributor to the development of metabolic abnormalities and chronic diseases, such as type 2 diabetes and cardiovascular disease, the molecular mechanisms of its pathogenesis remain elusive. Defects in the phosphorylation and dephosphorylation events in the insulin signaling pathway, such as serine/threonine phosphorylation of Insulin Receptor Substrate-1 (IRS-1), are considered to be one of the main causes of insulin resistance. Most research on the regulation of these phosphorylation events has been focused on kinases and is performed in cell culture or animal models. We hypothesize that serine/threonine protein phosphatase 2A (PP2A) complexes in human skeletal muscle are over-activated and dysregulated in insulin resistance and type 2 diabetes. This hypothesis is formulated based on the existing literature suggesting hyperactivation of PP2A under the duress of glucolipotoxicity and diabetes in cell/animal models, and the following novel preliminary findings of our own: 1). The catalytic subunit of PP2A (PP2Ac) associates with IRS-1 in skeletal muscle of lean healthy participants, and this interaction is significantly increased in obese/overweight and T2D muscle; 2), Insulin suppresses PP2Ac activity and Leu309 carboxylmethylation in muscle of lean healthy participants, but not type 2 diabetic patients. To test our hypotheses, we will assess the activity (Aim 1), post-translational modifications (Aim 2), and interaction partners (Aim 3) of PP2A in skeletal muscle from lean healthy controls (LC), obese nondiabetic subjects (OC), and type 2 diabetic patients (T2D), using state-of-the-art HPLC-nanospray-tandem mass spectrometry (HPLC-ESI-MS/MS). The overall goal is to address the knowledge gap of phosphatase function and regulation in insulin resistance and T2D in humans, using a combination of clinical, biological, and proteomic approaches developed in our laboratory. For Aim 1, we will perform PP2Ac co- immunoprecipitation experiments followed by assay of PP2A activity in muscle biopsies from the 3 groups under basal and insulin infusion. For Aim 2, we will assess pTyr307 and mLeu309 in the 3 groups using Western blot analysis. In addition, we will employ the proteomics approach for phosphorylation quantification developed in our laboratory to provide a global view of PP2Ac phosphorylation in the 3 groups. For Aim 3, we will perform co-immunoprecipitation experiments using antibodies against PP2Ac followed by assessment of co-immunoprecipitated protein quantities under basal and insulin infusion in the 3 groups using the proteomics approach for protein complexes developed in our laboratory. The outcome of this research will be the discovery of abnormalities in PP2A activity, post-translational modifications, and interaction partners in skeletal muscle insulin resistance in humans, facilitating the design of new drugs to modulate PP2Ac post-translational modifications and protein-protein interactions. These drugs may restore abnormal PP2Ac activity and improve insulin signaling, resulting in the treatment and/or prevention of T2D, as well as other chronic disease.