Structure and Function of Mitochondrial Protein Kinases

描述（由申请人提供）：包含丙酮酸脱氢酶激酶（PDK）和支链1-酮酸脱氢酶激酶（BCK）的固有线粒体蛋白激酶（mPK）是控制碳水化合物和支链氨基酸降解的分子开关。线粒体PDK（亚型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同种型在疾病状态如2型糖尿病、肥胖症和癌症中过表达，导致葡萄糖氧化减少。为了了解这些PDK的结构和功能，P.I.的实验室已经解决了四种PDK亚型中的三种（PDK 1，PDK 3和PDK 4）和各种PDK抑制剂/激活剂复合物的晶体结构。基于这些进步，P.I.建议继续研究哺乳动物PDKs的结构、功能和调节。具体目标是：1）阐明L2结构域和合成配体调节PDK活性的变构机制：2）为PDK 4的高活性提供生物化学和结构基础，并鉴定该激酶亚型中的E1 p底物结合位点; 3）通过高通量筛选分离新一代PDK 4特异性小分子抑制剂，在体外和细胞培养中筛选和表征这些新的抑制剂。将采用标准方法，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，以实现这些特定目的。PDK 4特异性抑制剂的可用性将促进减轻肥胖和2型糖尿病中葡萄糖氧化缺陷的新策略。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）亚型的结构和功能以及PDK亚型#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.