Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase.
Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays crucial roles in inflammation and host defense. With an intrinsically bound calmodulin, iNOS is fully active once expressed in cells. Thus, regulation of NO production from iNOS was thought to primarily occur at the enzyme transcriptional level. Here we show that NO synthesis from iNOS can be profoundly modulated by heat shock protein 90 (hsp90) through protein-protein interaction. To study whether hsp90 affects iNOS function, recombinant murine iNOS was purified from an Escherichia coli expression system by affinity chromatography. Hsp90, at physiological concentrations (10-500 nm), dose-dependently increased iNOS activity. This was a specific effect because neither denatured hsp90 nor irrelevant bovine serum albumin affected iNOS function. Overexpression of hsp90 enhanced NO production in iNOS-transfected cells. On the contrary, hsp90 inhibition dramatically decreased NO formation from iNOS in macrophages. Co-immunoprecipitation studies showed that hsp90 and iNOS associated with each other in cells. Overexpression of iNOS resulted in NO-mediated cellular injury. Hsp90 inhibition markedly attenuated NO formation and prevented cellular injury. These results demonstrated that hsp90 is an allosteric enhancer of iNOS. iNOS is coupled with hsp90 in cells, and this coupling facilitates NO synthesis. In light of the critical role of hsp90 in iNOS-mediated cytotoxic action, modulating the interaction between hsp90 and iNOS may be a new approach to intervene inflammation and immune response.
Yoshida
Masako
M
Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Xia
Yong
Y
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002956:Citrulline / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019097:Spin Trapping; D014158:Transcription, Genetic; D014162:Transfection
D018841:HSP90 Heat-Shock Proteins; D011994:Recombinant Proteins; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse