The long-range goals of this research are to elucidate mechanisms of intracellular protein catabolism and the role of cellular proteinases in mediation of cellular events in health and diseases such as diabetes mellitus. My approach has been to identify and characterize cellular proteinases and mechanisms used by living systems to degrade cellular proteins, and to determine factors that control the rate of degradation of specific enzymes according to the metabolic needs of the organism. The work has provided fundamental information about structure and activity of meprins, and a-new family of metalloendopeptidases, and has expanded to include studies of the biosynthesis, processing, and activation of cell-surface mammalian proteinases, and to areas that may apply to renal failure and disease. In the next period, it is proposed to investigate: (A) factors and motifs that drive the covalent and non-covalent association of meprin subunits to form active and latent homo- and heterooligomers, and the association of meprins with other membrane, endoplasmic reticulum, and cytosolic proteins. Methods to be used include mutational analyses of recombinant meprin subunits expressed in human 293 cells, measurements of activity, stability, and oligomerization of the enzymes, and assessment of interacting units using the yeast two-hybrid system. (B) the function of meprins by disrupting the structural genes for the subunits, examining the embryonic expression of the subunits, and by determining whether adult mice with high and low meprin A phenotypes react differently to stresses on the kidney. Urinary forms of human meprin will also be characterized, and the proposition that adults are polymorphic for expression of the meprin alpha-subunit, as are mice, will be examined. Meprins have provided an elegant model to study the regulation of cell surface proteinases. These studies will provide fundamental information about the biosynthesis, interactions, regulation, and functions <)f these membrane-bound and secreted proteinases, as well as information about isoforms of the enzymes in developmental, mature, and diseased states.
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