S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities

Abstract

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-L-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.atherosclerosis ͉ nitrosation ͉ vascular wall ͉ chaperone R ecent years have witnessed an increasing interest in the roles of nitric oxide (NO) in signal transduction pathways other than its activation of the cGMP pathway. Many of these roles rely on NO's ability to alter protein function through posttranslational modifications. Among these modifications, S-nitrosylation has emerged as a potential and fundamental regulator of protein function. S-nitrosylation is a covalent modification of thiol groups by formation of a thionitrite (-S-NϭO) group, facilitated by the formation of higher nitrogen oxides (1, 2). To date, several dozens of proteins have been shown to become S-nitrosylated and, in many cases, this modification was accompanied by altered function (see table S1 of ref. 1 for review).Nitric oxide, synthesized in the endothelium by endothelial nitric oxide synthase (eNOS), plays crucial roles in the vascular wall, including the maintenance of vascular tone. The possibility that NO might modify eNOS, or elements of the complex system involved in its activation, is an attractive hypothesis, suggesting a potential autoregulatory feedback mechanism. The eNOS enzyme is regulated by several posttranslational modifications including myristoylation, palmitoylation, and phosphorylation (3). This enzyme is also tightly regulated by specific interactions with inhibitory proteins such as caveolin-1 and by positive modulation by the scaffolding protein Hsp90. These interactions have been described in detail, and a relatively complete picture is beginning to emerge (4).We have previously used a proteomic approach to identify several proteins that were S-nitrosylated after exposure of vascular endothelial cells to the physiological nitrosothiol, Snitroso-L-cysteine (CSNO) (5). Further work led to the identification of Hsp90 as a protein susceptible to S-nitrosylation. This chaperone protein, known for its functions in protein folding, degradation, and scaffolding, has attracted renewed ...

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Similarly, mammalian Hsp25, 60, 70, and 90 also have redox active cysteine residues, and their oxidation has been linked to the modification of various chaperone functions in conditions of oxidative stress [150][151][152][153]. Various cysteine oxidations have been linked to Hsp activation that include nitrosylation, glutathionylation, and disulfide formation [151,152,154,155] (Figure 2, box 3). Hsp90 is, of note, regulated by S-nitrosylation [155].…”

“…Various cysteine oxidations have been linked to Hsp activation that include nitrosylation, glutathionylation, and disulfide formation [151,152,154,155] (Figure 2, box 3). Hsp90 is, of note, regulated by S-nitrosylation [155].…”

S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding

“…Further evidence suggests that PDI may transport NO to the extracellular space, where it could conceivably exert additional adverse effects. 51 In addition to PDI, S-nitrosylation is likely to affect critical thiol groups on other chaperones, such as heat-shock protein (HSP) 90 in the cytoplasm 92 and possibly GRP in the ER. Normally, HSP90 stabilizes misfolded proteins and modulates the activity of cell signaling proteins, including NOS and calreticulin.…”

“…59 In AD brains, levels of HSP90 are increased in both the cytosolic and membranous fractions, where HSP90 is thought to maintain tau and Ab in a soluble conformation, thereby averting their aggregation. 93,94 Martínez-Ruiz et al 92 recently demonstrated that S-nitrosylation of HSP90 can occur in endothelial cells, and this modification abolishes its ATPase activity, which is required for its function as a molecular chaperone. These studies suggest that S-nitrosylation of HSP90 in neurons of AD brains may possibly contribute to the accumulation of tau and Ab aggregates.…”

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium

“…Hsp90 plays a critical role in ILK stability and signaling, it protects ILK from protein degradation and promotes ILK complex with α-parvin and the cytoskeleton, 23 and we found that Hsp90-ILK interaction is necessary to prevent endothelial nitric oxide synthase enzymatic uncoupling that leads to endothelial dysfunction. It is accepted that S-nitrosylation of Hsp90 inhibits Hsp90 chaperone and ATPase activities 40 required to bind ILK to α-parvin and paxillin. So, S-nitrosylation of Hps90 could also support the loss of α-parvin and paxillin from the complex in NO-treated MAEC, which could explain how increasing amounts of NO contribute to vascular remodeling and also inhibit vascular relaxation.…”