Regulators of Osteoblast Protein Synthesis Initiation

Yale University, New Haven CT

Investigators

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Abstract

DESCRIPTION (provided by applicant): Eukaryotic protein synthesis initiation is an intricate, highly stress-sensitive event that requires formation of a ternary complex composed of initiation factor 2 (eIF2), GTP, and methionine-charged initiator tRNA (met-tRNAi). During studies to reconstruct this process, we produced unique evidence for a potent ternary complex inhibitor (TCI) in the low molecular mass pool of eukaryotic RNA (sRNA). TCI readily isolates from the bulk of sRNA and tRNAs by ion exchange chromatography. In cell lysates, TCI suppresses the rate of protein synthesis, and disrupts assembly of heavy polymeric ribosomes. Moreover, another component in ribosome depleted cytoplasmic lysates significantly antagonizes TCI (¿TCI) activity. In this proposal we intend three focused goals. First, we will purify and determin the primary nucleotide sequence of TCI, using standard extraction, co-immunoprecipitation, chromatography, and sequencing techniques. Second, we will measure TCI and ¿TCI activity levels in primary proliferating, differentiating, and mineralizing osteoblast cultures. We will quantify TCI and ¿TCI activity by rate and dosage assays, by relation to positive cell and tissue sources and to co-isolated primary fibroblasts using highly purified assay substrates and reagents. Third, we will examine the effect of two well known physiological stressors, amino acid deprivation and hypoxia, which each control protein synthesis through the eIF2 pathway, on TCI and ¿TCI activity in osteoblasts, using the methods and results developed in the previous goals. Results from the first goal will permit quantitative assessment of TCI and secondary structure predictions. Results from goal two will provide evidence for TCI and ¿TCI in cells within the physiological context with application to bone growth. Results from goal three will provide support for the importance of these factors by relation to stress induced bone pathology. Since assembly of the met- tRNAi/eIF2/GTP ternary complex is integral to all aspects of protein synthesis, and by extension to cellular physiology, our results will establish proof of principal fr future studies to purify ¿TCI to homogeneity, to expand stress studies in normal and transgenic animals, and develop a better definition of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.