Structure and Function of Mitochondrial Protein Kinases

描述（由申请人提供）：常驻线粒体蛋白激酶（mPKs）包括丙酮酸脱氢酶激酶（PDKs）和支链1-酮酸脱氢酶激酶（BCK）是控制碳水化合物和支链氨基酸降解的分子开关。线粒体PDKs（同种异构体1,2,3和4）通过可逆磷酸化下调线粒体丙酮酸脱氢酶复合物的活性，以响应激素和营养刺激。某些PDK亚型在2型糖尿病、肥胖和癌症等疾病状态下过度表达，导致葡萄糖氧化降低。为了理解这些pdk的结构和功能，pi他的实验室已经解决了PDK四种异构体中的三种（PDK1、PDK3和PDK4）和各种PDK抑制剂/活化剂复合物的晶体结构。在此基础上，作者建议对哺乳动物PDKs的结构、功能和调控进行进一步的研究。具体目的是：1)破译L2结构域和合成配体调节PDK活性的变构机制；2)为PDK4的高活性提供生化和结构基础，并确定该激酶同工异构体中的E1p底物结合位点；3)通过高通量筛选分离新一代PDK4特异性小分子抑制剂，并在体外和细胞培养中对这些新抑制剂进行表征。标准方法包括x射线晶体学，等温滴定量热法，激酶活性测定和高通量筛选方法将被用于实现这些特定目标。pdk4特异性抑制剂的可用性将促进新的策略来减轻肥胖和2型糖尿病中的葡萄糖氧化缺陷。公共卫生相关性：本项目研究的线粒体蛋白激酶是控制肝脏和骨骼肌中碳水化合物和氨基酸降解的分子开关。这些蛋白激酶的异常功能与肥胖和2型糖尿病有关。了解丙酮酸脱氢酶激酶（PDK）异构体的结构和功能以及PDK异构体#4特异性抑制剂的开发将促进减轻这些人类疾病中葡萄糖氧化缺陷的新策略。

DESCRIPTION (provided by applicant): The resident mitochondrial protein kinases (mPKs) comprising pyruvate dehydrogenase kinases (PDKs), and branched-chain 1-ketoacid dehydrogenase kinase (BCK) are the molecular switches that control carbohydrate and branched-chain amino acid degradation. Mitochondrial PDKs (isoforms 1, 2, 3 and 4) down-regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation, in response to hormonal and nutritional stimuli. Certain PDK isoforms are over-expressed in disease states such as type 2 diabetes, obesity and cancer, resulting in decreased glucose oxidation. Towards understanding the structure and function of these PDKs, the P.I.'s laboratory has solved the crystal structures for three (PDK1, PDK3 and PDK4) of the four PDK isoforms and various PDK-inhibitor/activator complexes. Based on these advances, the P.I. proposes to continue investigation into the structure, function and regulation of mammalian PDKs. The Specific Aims are: 1) To decipher the allosteric mechanisms by which the L2 domain and the synthetic ligands modulate PDK activities; 2) To offer biochemical and structural basis for the hyperactivity of PDK4 and to identify the E1p substrate-binding site in this kinase isoform; 3) To isolate a new generation of small-molecule inhibitors that are specific for PDK4 by high-through-put screening and characterize these novel inhibitors both in vitro and in cell culture. Standard methods including X-ray crystallography, isothermal titration calorimetry, kinase activity assays and the high-through-put screening method will be employed to achieve these Specific Aims. The availability of PDK4-specific inhibitors will foster new strategies to mitigate defective glucose oxidation in obesity and type 2 diabetes. PUBLIC HEALTH RELEVANCE: The mitochondrial protein kinases to be studied in this project are molecular switches that control carbohydrate and amino acid degradation in the liver and skeletal muscle. Aberrant functions of these protein kinases have been implicated in obesity and type 2 diabetes. Understanding the structure and function of pyruvate dehydrogenase kinase (PDK) isoforms and the development of PDK isoform #4-specific inhibitors will foster new strategies to mitigate defective glucose oxidation in these human diseases.