RESUMEN
The specific roles that immunoproteasome variants play in MHC class I antigen presentation are unknown at present. To investigate the biochemical properties of different immunoproteasome forms and unveil the molecular mechanisms of PA28 activity, we performed in vitro degradation of full-length proteins by 20S, 26S, and PA28αß-20S immunoproteasomes and analyzed the spectrum of peptides released. Notably, PA28αß-20S immunoproteasomes hydrolyze proteins at the same low rates as 20S alone, which is in line with PA28, neither stimulating nor preventing entry of unfolded polypeptides into the core particle. Most importantly, binding of PA28αß to 20S greatly reduces the size of proteasomal products and favors the release of specific, more hydrophilic, longer peptides. Hence, PA28αß may either allosterically modify proteasome active sites or act as a selective "smart" sieve that controls the efflux of products from the 20S proteolytic chamber.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación Alostérica , Dominio Catalítico , Interacciones Hidrofóbicas e Hidrofílicas , Tamaño de la Partícula , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co- and post-translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N-terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N-terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho-amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with lambda phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N- and O-linked oligosaccharides nor O-linked beta-N-acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co- and post-translational modifications, including N(alpha)-acetylation, N(alpha)-myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.