Our work in the last funding period has focused on two enzyme systems first identified and isolated in our laboratory in the course of work funded by this grant. The first involves endopeptidase 24.15 (El) 24.15), a zinc-metalloenzyme, active in the metabolism of bioactive peptides. inhibitors of EP 24.15 synthesized in our laboratory have antinociceptive properties, increase the secretory response of the pituitary to LHRH, exert a profound hypotensive effect on blood pressure in rats, increase cardiac output renal blood flow, water and sodium excretion. The second involves the multicatalytic proteinase complex (MPC; proteasome), isolated from bovine pituitaries as an unusually high molecular mass (approximately 700 kDa), multisubunit complex, involved in such fundamental cell functions as antigen processing, mitosis, and degradation of regulatory proteins such as cyclins, oncogene products, and transcription factors, Progress in the last year on the identification of catalytic subunits of the MPC and the need to obtain information related to the unusual functional, structural and mechanistic aspects of the MPC induced us to concentrate our effort in the coming period on studies of the MPC. The MPC exhibits five distinct activities cleaving peptide bonds on the carboxyl side of basic, acidic, and neutral amino acids. Four out of the five catalytic components are sensitive to inactivation by 3,4- dichloroisocoumarin (DCI). The fifth DCI-resistant component cleaves peptide bonds on the carboxyl side of branched chain amino acids and is a major factor in the protein-degrading activity of the complex. Accumulating evidence indicates that the MPC constitutes the proteolytic core of the ubiquitin-dependent system of intracellular proteolysis and that cell viability and proliferation is dependent on its integrity and function. The primary structures of components of the MPC show no homology to any of the four traditional classes of proteolytic enzymes, and neither the mechanism of proteolysis nor the structures of the active centers of components of the MPC is known. The proteolytic activity of the MPC is mostly latent and expression of full activity requires the presence of activators.
The aims of this proposal are therefore: 1) To identify components of the MPC expressing specific proteolytic functions by selective labeling of their active sites with [14C]3,4-dichloroisocoumarin and other labeled isocoumarins, and by using specific substrates and inhibitors,for protection of defined components of the MPC from covalent modification; 2) To identify the amino acids involved in catalysis in the active centers of catalytically active subunits; and 3) to explore the mechanism involved in conversion of the latent form of the MPC to an active form. This work will identify subunits that express specific proteolytic functions, provide insight into the structure of the catalytic centers and the mechanistic aspects of proteolysis catalyzed by the MPC, and identify factors important for conversion of the latent form to an active form of the MPC.
Showing the most recent 10 out of 36 publications
