Role of cytosolic chaperone HSP90 (HSP90AA1/AB1) in antigen presentation by MHC I molecules

Antigen presentation by MHC I molecules involves generation of peptides, loading of MHC I with peptides, and transport of MHC I-peptide complexes to the cell surface. The peptides are derived from the degradation of endogenous proteins in the cytosol. In this thesis, the chicken ovalbumin epitope SIINFEKL has been used as the model antigen and mechanism of its generation has been investigated. My results show that complete inhibition of proteasome activity results in only a modest inhibition in generation of new Kb-SIINFEHL complexes. Only a combined inhibition of proteasomes and other cytosolic proteases leads to a complete inhibition of generation of such complexes. Subsequent to their generation, peptides are chaperoned by heat shock proteins (HSPs). My studies provide direct evidence for the involvement of cytosolic hsp90 in chaperoning of peptides that are destined for loading onto MHC I. My studies define the nature of the peptides chaperoned by hsp90, and the precise step at which hsp90 associates with peptides. The role of hsp90 in presentation of an ovalbumin epitope is found to be at a post-proteasomal step: hsp90 associates with N-terminally extended precursors of the SIINFEHL epitope, and such peptides are depleted from hsp90 preparations in inhibitor-treated cells. My results indicate that treatment of cells with hsp90 inhibitors leads to generation of "empty" MHC I due to inhibited loading of MHC I with peptides. Hsp90-inhibited cells are poor stimulators of T lymphocytes. Altogether, these results define the mechanisms of generation of SIINFEHL or its precursors from intact ovalbumin, and the chaperoning of such precursors by hsp90. In addition, the role of hsp90 in processing of a variety of antigens, such as epitope I of larger T antigen is shown. I have also examined the form of the antigen, which is transferred from an antigen donor cell to an antigen presenting cell during cross-priming. My results are consistent with the idea that peptides, rather than the intact protein constitute the transferred form of antigen. ^