Interferon-γ inducible exchanges of 20S proteasome active site subunits: Why?

Abstract

When cells are stimulated with the cytokines IFN-γ or TNF-α, the synthesis of three proteasome subunits LMP2 (β1i), LMP7 (β5i), and MECL-1 (β2i) is induced. These subunits replace the three subunits delta (β1), MB1 (β5), and Z (β2), which bear the catalytically active sites of the proteasome, during proteasome neosynthesis. The cytokine-induced exchanges of three active site subunits of a complex protease is unprecedented in biology and one may expect a strong functional driving force for this system to evolve. These cytokine-induced replacements of proteasome subunits are believed to favour the production of peptide ligands of major histocompatibility complex (MHC) class I molecules for the stimulation of cytotoxic T cells. Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be pivotal for the generation or destruction of several unique epitopes. In this review we discuss the recent data on LMP2/LMP7/MECL-1-dependent epitope generation and the functions of each of these subunit exchanges. We propose that these subunit exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not proteolytically generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues.

Introduction

The proteasome is by far the most important provider of peptide ligands for the presentation on major histocompatibility complex (MHC) class I molecules 〚1〛, 〚2〛, 〚3〛. However, the proteasome executes a host of important cellular functions other than antigen processing and evolved much earlier than the specific immune system 〚4〛, 〚5〛. It is therefore assumed that both the peptide binding groove of MHC class I molecules as well as the transporter associated with antigen processing (TAP) 〚6〛 adapted to the range of peptide products which can be produced by the proteasome 〚7〛. In vitro, the 20S and 26S proteasomes produce peptides of 3–25 amino acids in length of which about 15% meet the strict length requirement of eight or nine amino acids for MHC class I ligands 〚8〛, 〚9〛, 〚10〛, 〚11〛. In order to bind with reasonable affinity to the cleft of class I proteins, the peptide ligands need to contain anchor residues which are situated at the C terminus and at some other position within the peptide sequence which varies among different MHC molecules and alleles 〚12〛. The proteasome is responsible for generating the C-termini of class I ligands but the binding pockets for P1 residues in proteasomes do not perfectly match the C-terminal anchor residues of MHC class I peptide ligands which are hydrophobic in mice and either hydrophobic or basic in humans. The proteasome contains three different active site subunits named β5, β2, and β1 which according to mutagenesis experiments in yeast preferentially cleave C-terminal of hydrophobic, basic, and acidic residues 〚10〛, 〚13〛. These three activities have been classified with fluorogenic peptides according to their P1 specificity as chymotrypsin-like, trypsin-like, and caspase-like, respectively. Since no class I molecules with acidic anchor residues have been described in mice or humans in spite of extensive investigations it appears that the caspase-like activity would not be able to generate class I ligands in mice and humans and may even destroy them by internally cleaving potential class I ligands.

It appears that the immune systems of mice and humans have invented a mechanism to adjust the specificity of the proteasome to the needs of MHC class I restricted antigen presentation. When the antiviral cytokines IFN-γ or TNF-α are produced by T cells during an acute immune response, the cytokine-stimulated cells in the inflammatory site will transcriptionally induce the synthesis of three extra proteasome subunits called LMP2 (β1i), LMP7 (β5i), and MECL-1 (β2i). They are homologous to the three active site bearing subunits delta (β1), MB1 (β5), and Z (β2) of the proteasome and due to a strong overproduction, the inducible subunits are incorporated into newly assembled proteasomes instead of the constitutively expressed subunits. The inducible subunits also possess the residues which are required for the peptidolytic activity like ¹Thr or ³³Lys and can thus be expected to be catalytically active. Altough these subunit exchanges have been intensively investigated for the past 10 years, their precise function is still a matter of debate and largely unknown.