Hsp90-mediated assembly of the 26 S proteasome is involved in major histocompatibility complex class I antigen processing.
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.
Yamano
Taketoshi
T
Laboratory for Immunochaperones, Research Center for Allergy and Immunology (RCAI), RIKEN Yokohama Inst., Yokohama 230-0045, Japan.
Mizukami
Shusaku
S
Murata
Shigeo
S
Chiba
Tomoki
T
Tanaka
Keiji
K
Udono
Heiichiro
H
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006868:Hydrolysis; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
D016227:Benzoquinones; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D018841:HSP90 Heat-Shock Proteins; D015395:Histocompatibility Antigens Class I; D047029:Lactams, Macrocyclic; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C001277:geldanamycin