Characterization of a new family of protein kinases

描述（由申请人提供）： 哺乳动物丙酮酸脱氢酶复合物的活性通过磷酸化（无活性）和去磷酸化（活性）形式的相互转化来调节。这种调节对于控制丙酮酸和碳水化合物燃料的代谢命运非常重要。通过去磷酸化激活复合物增加了乙酰辅酶A的可用性，用于外周组织中柠檬酸循环的完全氧化和肝脏中的脂肪酸合成。通过磷酸化使复合物失活限制了外周组织对葡萄糖的利用，并保留了肝脏和肾脏合成葡萄糖的三碳化合物。重要的是，丙酮酸脱氢酶复合物的失活也发生在糖尿病中。这通过在面对大量血糖水平时不适当地节省丙酮酸而加剧了糖尿病状态。该项目的长期目标是了解在正常情况下以及糖尿病中负责丙酮酸脱氢酶活性调节的分子机制。 丙酮酸脱氢酶激酶在这种调节中起关键作用。该激酶是复合物的组成部分，与转乙酰酶组分紧密相关。一般认为，由于这种紧密结合，激酶功能性在很大程度上由位于复合物的转乙酰酶组分上的激酶和硫辛酰承载结构域之间的蛋白质-蛋白质相互作用决定。在本申请中，我们建议使用X射线晶体学和结构/功能分析的组合来测试这一假设。 该建议的具体目的是：1）确定具有内部硫辛酰基结构域的复合物中激酶分子的三维结构; 2）鉴定决定不同激酶同工酶识别硫辛酰基结构域的特异性的分子特征; 3）建立负责硫辛酰基结构域介导的激酶活性调节的分子机制;和4）阐明新的激酶特异性抑制剂的作用机制，所述激酶特异性抑制剂被认为是通过激酶分子的硫辛酰基承载结构域结合位点起作用。本研究过程中产生的结构信息对于设计新一代激酶特异性药物是必不可少的。 这些化合物可能被证明是有益的，在减轻一些症状与糖尿病，以及代谢性酸中毒，缺血性心脏病和败血症。

DESCRIPTION (provided by applicant): The activity of mammalian pyruvate dehydrogenase complex is regulated through the interconversion of phosphorylated (inactive) and dephosphorylated (active) forms. This regulation is of great importance to the control of the metabolic fate of pyruvate and carbohydrate fuels. Activation of the complex by dephosphorylation increases the availability of acetyl-CoA for complete oxidation by the citric acid cycle in peripheral tissues and for fatty acid synthesis in the liver. Inactivation of the complex by phosphorylation limits the use of glucose by peripheral tissues and conserves three carbon compounds for glucose synthesis by the liver and kidney. Importantly, the inactivation of pyruvate dehydrogenase complex also occurs in diabetes. This exacerbates the diabetic state by inappropriately sparing pyruvate in the face of abundant levels of blood glucose. The long-term goal of this project is to understand the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase activity under normal circumstances as well as in diabetes. The key role in this regulation belongs to pyruvate dehydrogenase kinase. This kinase is an integral part of the complex, tightly associated with the transacetylase component. It is generally believed that due to this tight association, the kinase functionality is largely determined by protein-protein interactions between kinase and lipoyl-bearing domains located on the transacetylase component of the complex. In this application, we propose to test this hypothesis using a combination of X-ray crystallography and structure/function analysis. The Specific Aims for this proposal are: 1) to determine the three-dimensional structure of the kinase molecule in a complex with the inner lipoyl-bearing domain; 2) to identify the molecular features determining the specificity of recognition of lipoyl-bearing domains by different kinase isozymes; 3) to establish the molecular mechanism(s) responsible for the lipoyl-bearing domain-mediated regulation of kinase activity; and 4) to elucidate the mechanism(s) of action of novel kinase-specific inhibitors that are thought to be acting through the lipoyl-bearing domain binding site of kinase molecule. The structural information generated in the course of this study will be indispensable for the design of a new generation of kinase-specific drags. These compounds may prove to be beneficial in alleviating some of the symptoms associated with diabetes, as well as metabolic acidosis, ischemic heart disease, and sepsis.