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.

RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G + -bacterial toxins

“…COS-7 and HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 U/ml penicillin, 100 mg/ ml streptomycin, and 10% FBS [28,29]. Human Lung Microvascular Endothelial Cells (HLMVECs) were isolated and grown in house as previously described [8,23] or purchased from Lonza and grown in Endothelial Growth Medium-2-Microvessel (EGM-2MV) containing the requisite growth factors and 5%FBS (Lonza, Allendale, NJ) and used below passage 8.…”

NOX5: Molecular biology and pathophysiology

“…NOX5 is activated when the [Ca 2+ ] i increases and in response to various activators, including regulatory proteins (calmodulin), kinases (PKCα, ERK1/2, CAM kinase II and c‐Abl) and through post‐translational modifications (phosphorylation). NOX5 is inactivated by regulatory proteins [caveolin‐1 (Cav‐1)] and chaperone molecules [heat shock protein 70 (Hsp70)] and through post‐translational modifications (oxidation, nitrosylation and SUMOylation) (Chen, Haigh et al., ; Chen, Wang et al., ; Chen, Yin, Dimitropoulou, & Fulton, ; Fulton, )…”

“…Although NOX5 does not require NADPH oxidase subunits for its activation, it is influenced by various regulatory proteins, some of which interact directly with NOX5, including protein kinase C (PKC), calmodulin, caveolin‐1, c‐Abl1 and chaperone molecules (Hsp90 and Hsp70) (Chen, Yu, et al., ; Chen et al., ). These interactions influence NOX5 activity differentially and might also stabilize the enzyme.…”

Targeting the Diabetic Chaperome to Improve Peripheral Neuropathy

“…Mechanistically, recovery from the sensory neuropathy correlated with an Hsp70-dependent improvement in mitochondrial bioenergetics of the diabetic sensory neurons [59]. Though the underlying basis of how the chaperone improves mitochondrial function remains unclear, it may relate to a decrease in glucose-induced superoxide production in mitochondria [61•, 62], inhibition of various NADPH oxidase isoforms [85], or an Hsp70-dependent clearance of damaged organelles [72]. Similar to DMAG, KU-596 therapy also blocked a diabetes-induced increase in genes associated with inflammation [61•].…”