title: Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones.
abstract: Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398-719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1-550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490-550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310-529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher molecular weight complexes of Nox5 and decreased interaction between monomers. Collectively these results show that the C-terminal region of Nox5 binds to the M domain of Hsp90 and that the binding of Hsp90 and select co-chaperones facilitate oligomerization and the efficient production of superoxide.
authors.affiliation: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, 210029 China; Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA. Electronic address: fchen@gru.edu.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Haigh
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Benson
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Yusi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Xueyi
authors.initials: X
authors.lastname: Li
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Dou
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bagi
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Stepp
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Gabor
authors.initials: G
authors.lastname: Csanyi
authors.affiliation: Cancer Research Center, Molecular Chaperones Program, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Chadli
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA.
authors.forename: Neal L
authors.initials: NL
authors.lastname: Weintraub
authors.affiliation: Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw GA 30152, USA.
authors.forename: Susan M E
authors.initials: SM
authors.lastname: Smith
authors.affiliation: Vascular Biology Center, Georgia Regents University, Augusta, Georgia 30912, USA. Electronic address: dfulton@gru.edu.
authors.forename: David J R
authors.initials: DJ
authors.lastname: Fulton
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000378:metabolism*; D011485:Protein Binding; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection
chemical_list: D018841:HSP90 Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013481:Superoxides; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human
keywords: Co-chaperones; Hsp70; Hsp90; NADPH oxidase; Nox; Vascular biology