This research includes two related projects: l. Studies of p67. A 67 KDa glycoprotein, p67 protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases (HRI and PKR) and is, therefore, necessary for protein synthesis. Our work has indicted that p67 activity is regulated at the mRNA level and also by protein glycosylation- deglycosylation. There are indications that p67 binds specifically to the eIF-2gamma subunit and uses its glycosyl residues to protect eIF-2 alpha- subunit.
Specific aims are: (A) Studies of regulation of p67 synthesis and degradation. A p67 deglycosylase activity will be purified from reticulocyte lysates and its roles in regulation of p67 activity will be studied. p67-regulation at the mRNA level will be studied using """"""""run-off"""""""" experiments. (B) Studies of the roles of p67 in regulation of protein synthesis in normal and viral infected cells. Effects of p67 deglycosylation and changing p67 levels on protein synthesis will be studied using animal cells under different physiological conditions such as serum starvation, mitogen stimulation and viral infection. p67 level will be changed by regulated expression of p67-cDNA or p67 antisense DNA in transfected cells. (C) Mapping the active sites on p67. Attempts will be made to map the glycosylation sites on p67 and also the sites on p67 responsible for p67 binding to eIF-2gamma subunit and protection of eIF-2 alpha-subunit from inhibitory phosphorylation. In vitro mutagenized p67 cDNA will be expressed in baculovirus system. The mutated proteins will then be isolated and analyzed. II. Mechanism of peptide chain initiation. Three protein factors eIF-2B, Co-eIF-2A and eIF-3 are required to promote ternary complex formation by eIF-2. The 180 kDa polypeptide in eIF-3 protein complex is possibly responsible for elF-3 activity to promote ternary complex formation.
Specific aims are: (A) Studies of the roles of p180, Co-eIF-2A and other protein factors in ternary and Met- tRNAf.40S.mRNA complex formation and also in over-all protein synthesis. Attempts will be made to extensively purify different protein factors and reconstitute efficient ternary and Met-tRNAf.40S.mRNA complex formation. (B) Cloning and characterization of a cDNA encoding Co-eIF-2A and (C) Preparation of anti sense Co-eIF-2A gene sequences and studies of the requirement of Co-eIF-2A in protein synthesis. Standard experimental procedures will be used for specific aims B and C. This research is expected to provide a better understanding of the molecular mechanism of gene expression at the translation level. An intriguing possibility is that we will be able to control viral infection by regulating p67 level in the cells. This will provide us with a tool to combat viral diseases.
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