The Structure-Function Study of An Immune Chaperone gp96 (HSP90b1, GRP94) And Its Implication in Cancer And Infectious Diseases

As the endoplasmic reticulum paralog of the cytosolic HSP90, gp96 (grp94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. Despite structural advancements and intense interests in drug development targeting the HSP90 family, little is known about the structural and functional regulation of gp96 or the mechanism of its action in client folding and maturation. Herein, we provide genetic and biochemical evidence to demonstrate definitively that N-linked glycosylation and disulfide-bond formation of gp96 do not affect its chaperone function. We have also proved that a C-terminal loop structure, formed by amino acid residues 652-678, is the critical region of the client binding domain (CBD) of gp96 for TLRs and integrins. Moreover, we have unveiled the role of gp96 in regulating other aspects of TLR9 biogenesis in addition to protein folding. During the course of this study, we also clarified that a peptide-based inhibitor that was previously reported to inhibit gp96 is in fact targeting cell surface HSP90 to block recognition of lipopolysaccharide (LPS). We generated a series of peptide derivatives (named as peptide inhibitors of endotoxin responsiveness, or PIERs) from the N-terminal helix structure of HSP90 and demonstrated that PIERs effectively inhibit NFκB responses to LPS but not to other TLR ligands. Our studies have not only advanced the understanding of the molecular basis of gp96-mediated substrate binding and regulation, but also paved the way for future applications and strategies in targeting HSP90 for therapy of a variety of diseases including cancer and infectious disease.