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

描述(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发展的主要因素，如2型糖尿病和心血管疾病，但其发病的分子机制仍不清楚。胰岛素受体底物-1(IRS-1)的丝氨酸/苏氨酸磷酸化等信号通路中的磷酸化和去磷酸化缺陷被认为是胰岛素抵抗的主要原因之一。大多数对这些磷酸化事件的调节的研究都集中在激酶上，并且是在细胞培养或动物模型中进行的。我们假设人类骨骼肌丝氨酸/苏氨酸蛋白磷酸酶2A(PP2A)复合体在胰岛素抵抗和2型糖尿病中过度激活和调节失调。这一假说是基于现有文献提出的在细胞/动物模型中糖毒和糖尿病胁迫下PP2A的过度激活，以及我们自己的以下新的初步发现：1)瘦肉型健康受试者骨骼肌中PP2A催化亚单位(PP2Ac)与IRS-1结合，这种相互作用在肥胖/超重和T2D肌肉中显著增加；2)胰岛素抑制瘦肉型健康受试者肌肉中PP2Ac活性和Leu309羧甲基化，但不抑制2型糖尿病患者肌肉中PP2Ac活性和Leu309羧甲基化。为了检验我们的假设，我们将评估活动 (Aim 1)、翻译后修饰(Aim 2)和相互作用伙伴(Aim 3)来自瘦健康对照组(LC)、肥胖的非糖尿病受试者(OC)和2型糖尿病患者(T2D)，使用最先进的高效液相-纳米级联质谱(LC-ESI-MS/MS)。总体目标是利用我们实验室开发的临床、生物学和蛋白质组学方法相结合的方法，解决人类胰岛素抵抗和T2D中磷酸酶功能和调节的知识差距。对于目标1，我们将进行PP2Ac免疫共沉淀实验，然后在基础和胰岛素输注下测定三组肌肉活检组织中PP2A的活性。对于目标2，我们将使用Western印迹分析来评估3组中的pTyr307和mLeu309。此外，我们将使用我们实验室开发的蛋白质组学方法进行磷酸化量化，以提供三个组中PP2Ac磷酸化的全局视图。对于目标3，我们将使用PP2Ac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体蛋白质组学方法评估基础和胰岛素输注下三组患者的免疫共沉淀蛋白质数量。这项研究的结果将是发现人类骨骼肌胰岛素抵抗中PP2A活性、翻译后修饰和相互作用伙伴的异常，促进设计新的药物来调节PP2Ac翻译后修饰和蛋白质-蛋白质相互作用。这些药物可能会恢复异常的PP2Ac活性，改善胰岛素信号转导，从而治疗和/或预防T2D以及其他慢性病。

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.