Hsp90-mediated Assembly of the 26 S Proteasome Is Involved in Major Histocompatibility Complex Class I Antigen Processing

Taketoshi Yamano,Tomoki Chiba,Heiichiro Udono,Shusaku Mizukami,Keiji Tanaka,Shigeo Murata

doi:10.1074/jbc.m803077200

Abstract

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.

Highlights

Which specific immunity is activated against infectious agents, tumors, and even self-antigens
Large proteolytic intermediates (Ͼ30 kDa) harboring major histocompatibility complex (MHC) class I epitope were found to associate with hsp90␣ in living cells [7], there is no direct evidence that the associated intermediates are physiologically relevant in terms of being T cell epitopes by proteasome-dependent processing
We observed that epoxomicin, a highly specific proteasome inhibitor, abrogated the production of Cytotoxic T lymphocytes (CTLs) epitopes from C-terminally extended peptides by the proteasome affinity-isolated by hsp90␣, indicating no association of hsp90␣ with cytosolic peptidases such as tripeptidyl protease, which acts downstream of the proteasome to process certain peptides independently [18]

Summary

Introduction

Which specific immunity is activated against infectious agents, tumors, and even self-antigens The majority of these 8 –10amino acid peptides are proteasome-degraded products of cellular proteins [1, 2]. We found that hsp stimulates MHC class I epitope production from C- but not N-terminally extended short synthetic peptides (13 ϳ 22-mer) in a proteasome-dependent manner in vivo [9]. Because ubiquitination of those short peptides is unnecessary to be processed by the proteasome, how hsp is involved in proteasome-dependent epitope production has still remained elusive. Our results showed that hsp90␣, by stimulating assembly of the 26 S proteasome, is directly involved in production of epitopes recognized by CD8ϩ T cells

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Conclusion