THE INTERFERON-GAMMA-INDUCIBLE 11S-REGULATOR (PA28) AND THE LMP2 LMP7SUBUNITS GOVERN THE PEPTIDE PRODUCTION BY THE 20S-PROTEASOME IN-VITRO/

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol b y the 20 S proteasome. Upon stimulation of antigen presenting cells wi th interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptid e (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 th e function of LMP2 and LMP7 in antigen presentation is still controver sial, we tested whether these subunits might operate by modifying prot easome activation through the 11 S regulator. We strongly overexpresse d the two LMP subunits separately or together by transfection in murin e fibroblasts. Isolated 20 S proteasomes from LMP transfectants were a pplied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analy sis 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. W hile the 11 S regulator did not preferentially activate LMP2 or 7 cont aining proteasomes, the binding of the 11 S regulator to any of the pr oteasome preparations markedly changed both the quality and quantity o f peptides produced. These results suggest that the 11 S regulator inc reases the spectrum of peptides which can be generated in antigen pres enting cells.