Disorders of protein metabolism in the liver are a principal feature of the pathogenesis of diabetes and other disease states that affect nutrient homeostasis. Development of strategies for the treatment of these disorders requires knowledge of the basic events involved in the regulation of hepatic protein metabolism. Therefore, the overall goal of this project is to provide a better understanding of the mechanisms by which hormones and nutrients regulate specific events in the path way of protein synthesis in the liver. The focus of the project continues to be on peptide-chain initiation, a process which is mediated by a series of proteins termed eukaryotic initiation-factors (abbreviated eIF) and which is a major control point for the regulation of mRNA translation in eukaryotic cells. Our previous work has established important roles for three initiation factors in the regulation of hepatic protein synthesis under conditions such as diabetes, deprivation of single essential amino acids, and depletion of calcium from the endoplasmic reticulum. The latter two conditions lead to increased phosphorylation of the alpha-subunit of eIF-2, a heterotrimeric protein which mediates the binding of initiator tRNA (Met-tRNAi) to the ribosome as a ternary complex (eIF-2.GTP.Met- tRNAi), and to decreased activity of eIF-2B, a heteropentameric protein which mediates guanine nucleotide exchange on eIF-2. The former condition leads to reduced function of eIF-4F, a heterotrimeric protein which mediates binding of mRNA to the 43S preinitiation complex. Presently, little is known about the regulation of these initiation factors. Therefore, the studies proposed in this application are designed to further elucidate the function and regulation of eIF-2, eIF-2B, and eIF- 4F, particularly their role in the control of hepatic protein synthesis under conditions of altered hormonal or nutrient status.
The specific aims of the studies are: (l) to clone, sequence, and express cDNAs encoding all five subunits of eIF-2B, investigate mechanisms involved in the regulation of eIF-2B activity including the roles of phosphorylation and allosteric factors, and examine the role of each subunit in terms of eIF-2B activity and regulation; (2) to clone,, sequence, and express cDNAs encoding elF- 2alpha: kinases, investigate the mode of regulation of each kinase, and examine the role of each kinase in mediating increased phosphorylation of eIF-2alpha under the physiological conditions described in the next two aims; (3) to investigate the involvement of a protein kinase cascade in the signal transduction path way by which depletion of calcium from the lumen of the endoplasmic reticulum results in increased phosphorylation of eIF-2alpha; (4) to identify and characterize the eIF-2alpha phosphatase and to investigate the possible role of an eIF-2alpha kinase in the mechanism by which deprivation of single essential amino acids results in increased phosphorylation of elF-2alpha; and (5) to test the hypothesis that insulin regulates peptide-chain initiation in liver by modulating the expression and/or state of phosphorylation of one or more of the subunits of eIF-4F. Overall, the studies described in this proposal should help identify mechanisms by which hormones and nutrients regulate protein synthesis in the liver. They should also provide new insights into the biochemical and molecular mechanisms involved in the initiation of protein synthesis.
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