The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro.

Marcus Groettrup,Sybille Standera,Lothar Kuehn,Michael Seeger,Peter M Kloetzel,Thomas Ruppert,Ullrich Koszinowski

doi:10.1074/jbc.270.40.23808

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-gamma, 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.

Highlights

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
We have shown that binding of the 11 S regulator markedly alters the quality and quantity of peptide products generated by the 20 S proteasome
In the same in vitro digestion assay using a 25-mer peptide as a substrate, we demonstrate that single or joint incorporation of LMP2 and LMP7 subunits into the 20 S proteasome likewise changes the quantity of different petide products generated

Summary

Introduction

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. 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 20 S proteasome is the major cytosolic endoprotease in eukaryotes (6 – 8) These 700-kDa protease complexes, which constitute 0.5–1% of total cell protein, consist of 14 different subunits ranging in molecular mass from 21 to 32 kDa and with isoelectric points from 3 to 10, as evidenced by two-dimensional analysis on NEPHGE-PAGE gels [9]. At least four of the 13–15 subunits of the 19 S regulator belong to a novel family of ATPases, and one subunit has been shown to be the receptor of ubiquitin, crucial for the function of the 26 S protease in degrading ubiquitinated proteins [29]

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Conclusion