? 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. ? ? ?
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