The major objective of this proposal will be to gain greater insight into the molecular and cell biological mechanisms underlying the production of insulin and other peptide hormones of the islet of Langerhans, including their genetic bases, evolutionary origins and disorders which may contribute to the pathophysiology of diabetes and/or other diseases in man. Extensions of previous work and new projects are proposed in 8 areas: (1) Studies on the proteolytic mechanisms involved in the processing of prohormones, e.g. proinsulin, including the initiation of studies on the biosynthesis and transport of cathespin B precursors in islet cells, the mechanisms of targeting and activation of cathespin B precursors in secretion granules versus lysosomes, and investigation via molecular cloning of the complexity and tissue distribution of the multigene family of tissue proteinases containing cysteine in their active centers (cathespins B, H, L and papain). (2) Regulation of insulin biosynthesis in cells via translation and transcriptional control mechanisms, including the identification of regions in the insulin gene that may play a role in regulation of insulin production. (3) Studies on defects in insulin biosynthesis and structure due to mutations in the insulin gene, or others, and through development of improved genetic screening methods, evaluation of their contribution to the incidence of various forms of diabetes. (4) Studies on the evolution of insulin and related gene products, with particular emphasis on extending present studies to invertebrates and other more primitive species. (5) Studies on islet-central nervous system interrelationships, including studies via molecular cloning on the precursors of pancreatic polypeptide and TRH in islets and their counterparts in brain, as well as on the extra-pancreatic expression of insulin genes. (6) Development of new approaches to the molecular cloning and primary structural analysis of insulin receptors and their precursor forms. (7) New approaches to the study of prohormone structure and function via use of monoclonal antibodies or via expression, by means of DNA-mediated gene transfer methods, of altered precursor molecules having site-specific mutations that may affect their synthesis, folding, intracellular transport and routing, conversion to active forms or ultimate secretion and function as active hormones. (8) New studies on tumorigenesis in islet cells directed towards elucidating the possible roles of various cellular oncogenes in the genesis of islet cell tumors in man and animals and exploring the possible use of modified cellular oncogenes or their viral counterparts to generate new islet cell lines for investigational use.
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