The long-term objectives are the elucidation of the regulation of hemoglobin synthesis and erythropoiesis in normal and abnormal hematologic conditions. The focus of this research is to study the mechanisms of regulation of heme-regulated EIF-2a kinase (HRI) at the molecular structural level. HRI is a potent inhibitor of protein synthesis in immature erythroid cells. Protein synthesis in intact reticulocytes and their lysates is dependent on the availability of heme. In heme-deficiency, protein synthesis is inhibited at the level of initiation due to the activation of HRI. HRI is a heme-regulated protein kinase that phosphorylates the a-subunit of EIF-2 impairs its recycling in translational initiation and results in the cessation of protein synthesis. HRI is mainly expressed in erythroid cells and the physiological role of HRI is to maintain the balanced synthesis of heme and globin chains.
The specific aims of this proposed study are (1) Regulation of HRI by phosphorylation; (2) Molecular mechanisms in the regulation of HRI by heme, the prosthetic group of hemoglobin which is the principal proteins in reticulocytes; ( 3) Determination of HRI structure by X-ray crystallography. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, synthesis of oligodeoxynucleotides, high pressure liquid chromatography, amino acid microsequencing, in vitro transcription and translation, protein kinase assays, gel electrophoresis, deoxynucleotide sequencing, expression of HRI and other proteins using recombinant baculovirus, immunoaffinity column chromatography, immunoprecipitation, Western blot analysis, and X-ray crystallography. HRI is a key regulator of protein synthesis, a process which is essential for the regulation of cell growth, differentiation, antiviral response and hormonal responses. Structure-function analysis of HRI will advance our knowledge not only on the regulation of protein kinases which are vital in the regulation of various cellular processes, but also on the regulation of heme proteins.
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