The Interferon-γ-inducible 11 S Regulator (PA28) and the LMP2/LMP7 Subunits Govern the Peptide Production by the 20 S Proteasome in Vitro

Abstract

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-␥, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

The Toll‐like receptor ligand MALP‐2 stimulates dendritic cell maturation and modulates proteasome composition and activity

“…This is in agreement with the observed expression of the catalytic subunits, since it has been demonstrated that the expression of LMP7 and MECL1 is associated with increased chymotryptic and tryptic activities [31][32][33][34]. It is known that the substitution of standard g -subunits with the IFN-+ -inducible subunits and the association of the proteasome-associated PA28 regulator alters the hydrolytic activity of proteasomes against tri-and tetrapeptides, as well as the quality of the resulting peptides [31][32][33][34][35]. Although it has been reported that some tumorderived peptide antigens are destroyed by immunoproteasomes [36], the general view is that CTL epitopes are more efficiently generated by immunoproteasomes.…”

No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes

“…The proteasome is constitutively expressed in most tissues. Through the treatment with the pro-inflammatory cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α, a replacement of the three constitutive catalytic subunits by their inducible homologues β1i (PSMB9), β2i (PSMB10), and β5i (PSMB8) is initiated (Groettrup et al 1996;Groettrup et al 1995). This results in the formation of a protease called the immunoproteasome the expression of which is typically restricted to immune cells and has altered proteolytic properties.…”

The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses

“…The purification and quantitation of the 20S proteasome from B8 cells as well as fluorogenic peptide assays were performed exactly as previously described (27). For the titration of lactacystin the inhibitor was added at the same time as the substrate, and fluorescence of the MCA and ␤-naphthylamide leaving groups was measured after 30, 60, and 90 min to ensure that the reaction proceeded in a linear fashion.…”

“…Clone B8 was derived from C4 cells by transfection of the immediate early gene 1 of MCMV encoding the pp89 protein (27). MC57 is a C57BL/ 6-derived methylcholanthrene-induced fibrosarcoma cell line (28).…”