Work on pituitary proteases funded by this grant led in 1979-1981 to the discovery of the multi catalytic proteinase complex (MPC; proteasome), a cytoplasmic and nuclear macromolecule essential for fundamental cell functions and cell survival. Two of the five catalytic components of the MPC, the branched chain amino acid preferring (BrAAP) and chymotrypsin-like (ChT-L) components constitute major factors in the protein degrading activity of the MPC. The importance of these two components is indicated by the finding that the majority of cleavage sites found in natural peptides, proteins, and class I antigenic peptides contain a branched chain or aromatic amino acid at the carboxyl terminus. Our recent work has shown that the properties of these components in two classes of MPCs, one containing the X, Y, and Z, the other containing the LMP2, LMP7 and LMP10 beta-subunits differ with respect to specificity and susceptibility to inhibitors. However, their role in defined intracellular functions and the regulation of their activity remains to be determined. Furthermore, nothing is know about the functional role of the BrAAP component, one of the catalytically most active components of the MPC.
The aim of this proposal is: 1. To examine the specificity of the two components in two classes of 20S MPCs after incorporation into the large 26S complex involved in ubiquitin- dependent and ubiquitin independent proteolysis; 2. To examine activity changes in the two MPC classes induced by PA28, and endogenous protein activator, and histone H3, an activator of the BrAAP component, using synthetic and natural peptides, including those containing class I epitope, and to identify the structural elements of histone H3 responsible for activation; 3. To determine the effect of inhibitors synthesized in this laboratory, specific for the BrAAP and ChT-L activities and the two classes of MPCs, on the activity of the native and activated forms of the MPC in the isolated 20S, 26S and 20S-PA28 complexes; 4. To use specific inhibitors for examination of the involvement of the BrAAP and ChT-L components in the degradation of membrane bound, cytoplasmic and nuclear proteins in two cell types, one containing the X, Y and Z subunits, the other containing the LMP7, LMP2 and LMP10 subunits. This work will gain insight into the role of two major catalytic components in intracellular function of two classes of proteasomes and provide a basis for modulation of their functions by the use of selective inhibitors having pharmacological potential. The work should also contribute to the understanding of the mechanisms involved in the regulation of MPC activity.
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