A major goal of this research is to increase our understanding of the 26S proteasome, the ATP-dependent complex, which preferentially degrades ubiquitin-conjugated proteins and catalyzes most of the protein degradation in animal cells. Unlike conventional proteases, this enzyme and its simpler homologs in prokaryotes are large ATP-hydrolyzing complexes that degrade polypeptides processively to small peptide. Recently, we discovered that the different active sites of the core 20S proteasome are allosterically regulated by peptide substrates and thus appear to function in a cyclical manner during protein breakdown (the """"""""bite-chew model). In coming years, we hope to elucidate the structural basis for these allosteric effects to determine whether the proteasome digests proteins in a preferred direction and to clarify how products are released by these particles. To understand the role of ATP hydrolysis, we are using as a model system the simpler proteasomes from archaea, where we recently discovered a proteasome ATPase complex, PAN, which homologous to the six ATPases in the 26S proteasome. We hope to clarify PAN's structure and how stimulates unfolding and translocation of substrates into the lumen of the 20S particle. Proteasomes degrade proteins to peptides that range from 3-22 residues. Most are quickly hydrolyzed in the cytosol to amino acids, but in mammals, some escape this fate and serve as precursors for the 8- to 9-residue peptides that are presented to the immune system on MHC-class I molecules. We have developed new methods to determine to what extent the 26S proteasomes and the alternative forms induced by gamma-interferon (immunoproteasomes and PA28-containing complexes) directly generate the antigenic peptides or yield larger precursors that are trimmed by aminopeptidases to the MHC-presented epitopes. We also hope to learn more about this trimming process and about the competing pathway by which most proteasome products are digested to amino acids.
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