Two Distinct Pathways Mediated by PA28 and hsp90 in Major Histocompatibility Complex Class I Antigen Processing

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28α−/−/β−/− lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-γ, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-γ enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-γ did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-γ–stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type

cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region

AbstractThe nucleotide sequence of a cDNA that encodes a new subunit, named RC1, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23,130, which is consistent with the size obtained by electrophoretic analysis of purified RC1. The partial amino acid sequences of several fragments of RC1, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RC1 was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RC1 is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway

Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-γ for acquirement of the functional diversity responsible for antigen processing

AbstractProteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-γ) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-γ appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens

NEDD8 recruits E2-ubiquitin to SCF E3 ligase

NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2–E3 complex, which stimulates protein polyubiquitylation