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体的活性是通过磷酸化(非活性)和去磷酸化(活性)形式的相互转化来调节的。这种调节对于控制丙酮酸和碳水化合物燃料的代谢命运具有重要意义。通过去磷酸化激活该复合体增加了乙酰辅酶A在外周组织中的柠檬酸循环中完全氧化和在肝脏中合成脂肪酸的可用性。通过磷酸化使该复合体失活，限制了外周组织对葡萄糖的使用，并为肝脏和肾脏的葡萄糖合成保留了三种碳化合物。重要的是，糖尿病中也会发生丙酮酸脱氢酶复合体失活。面对充足的血糖水平，这会不适当地减少丙酮酸的摄入，从而加剧糖尿病状态。该项目的长期目标是了解在正常情况下以及在糖尿病中调节丙酮酸脱氢酶活性的分子机制。在这一调控中起关键作用的是丙酮酸脱氢酶激酶。该激酶是该复合体的一个组成部分，与转乙酰基酶成分密切相关。人们普遍认为，由于这种紧密的联系，激酶的功能在很大程度上是由位于复合体的转乙酰基酶成分上的激酶和硫辛基结构域之间的蛋白质-蛋白质相互作用决定的。在这一应用中，我们建议使用X射线结晶学和结构/功能分析相结合的方法来检验这一假设。该建议的具体目的是：1)确定含有内部含硫基结构域的复合体中激酶分子的三维结构；2)确定决定不同同工酶识别含硫脂基结构域的特异性的分子特征；3)建立负责含硫酰基结构域调节激酶活性的分子机制(S)；以及4)阐明被认为通过含硫酰基结构域结合的新型激酶抑制剂的作用机制(S)。在这项研究过程中产生的结构信息将是设计新一代特定于激酶的拖拉物不可或缺的。这些化合物可能被证明在缓解与糖尿病、代谢性酸中毒、缺血性心脏病和败血症相关的一些症状方面是有益的。

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.