Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones

Feng Chen, Steven Haigh, Yanfang Yu, Tyler Benson, Yusi Wang, Xueyi Li, Huijuan Dou, Zsolt Bagi, Alexander Dmitriyevich Verin, David W Stepp, Gabor Csanyi, Ahmed Chadli, Neal Lee Weintraub, Susan M.E. Smith, David J Fulton

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)793-805
Number of pages13
JournalFree Radical Biology and Medicine
Volume89
DOIs
StatePublished - Dec 1 2015

Fingerprint

HSP90 Heat-Shock Proteins
Carbon Monoxide
Superoxides
Enzyme activity
Protein Binding
Assays
Hydrogen Peroxide
Cell signaling
Oligomerization
Molecular Chaperones

Keywords

  • Co-chaperones
  • Hsp70
  • Hsp90
  • NADPH oxidase
  • Nox
  • Vascular biology

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

Cite this

Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones. / Chen, Feng; Haigh, Steven; Yu, Yanfang; Benson, Tyler; Wang, Yusi; Li, Xueyi; Dou, Huijuan; Bagi, Zsolt; Verin, Alexander Dmitriyevich; Stepp, David W; Csanyi, Gabor; Chadli, Ahmed; Weintraub, Neal Lee; Smith, Susan M.E.; Fulton, David J.

In: Free Radical Biology and Medicine, Vol. 89, 01.12.2015, p. 793-805.

Research output: Contribution to journalArticle

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AU - Chen, Feng

AU - Haigh, Steven

AU - Yu, Yanfang

AU - Benson, Tyler

AU - Wang, Yusi

AU - Li, Xueyi

AU - Dou, Huijuan

AU - Bagi, Zsolt

AU - Verin, Alexander Dmitriyevich

AU - Stepp, David W

AU - Csanyi, Gabor

AU - Chadli, Ahmed

AU - Weintraub, Neal Lee

AU - Smith, Susan M.E.

AU - Fulton, David J

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N2 - 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.

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KW - Hsp70

KW - Hsp90

KW - NADPH oxidase

KW - Nox

KW - Vascular biology

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