Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Bender, Andrew T.; Demady, Damon R.; Osawa, Yoichi

doi:10.1074/jbc.m000155200

Cited by 87 publications

(112 citation statements)

References 35 publications

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“…It has been demonstrated that CHIP ubiquitinates nNOS and that the quantity of nNOS-ubiquitin conjugates is greatly enhanced upon the interaction of CHIP with nNOS-bound Hsp70 [23]. In contrast, binding of Hsp90 to nNOS has been reported to enhance the enzyme's catalytic activity [41], together with calmodulin-dependent inhibition of its ubiquitination [29]. We showed that CHIP and Hsp70 are both present in skeletal muscle.…”

Section: Discussionmentioning

confidence: 74%

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Mezghenna¹,

Leroy²,

Azay-Milhau³

et al. 2013

Diabetologia

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Aims/hypothesis Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect. Methods Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays. Results nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulininduced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes. Conclusions/interpretation Recovery of nNOS in insulinresistant muscles should be considered a potential new approach to address insulin resistance.

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Section: Discussionmentioning

confidence: 74%

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Mezghenna¹,

Leroy²,

Azay-Milhau³

et al. 2013

Diabetologia

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“…Indeed, techniques to directly study eNOS dimerisation in living cells and tissues are currently lacking. Some recent studies suggest that the neuronal NOS homodimer is more stable than free monomers, as monomers are more readily degraded through the ubiquitin-proteasome pathway [39,40]. However, the finding that total eNOS protein is increased in diabetes suggests that unstable eNOS homodimerisation, leading to eNOS degradation, is unlikely to be the major mechanism mediating the effects of BH4 on endothelial function in diabetes.…”

Section: Discussionmentioning

confidence: 99%

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

et al. 2005

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Aims/hypothesis: Impaired nitric oxide (NO) bioactivity and increased superoxide (SO) production are characteristics of vascular endothelial dysfunction in diabetes. The underlying mechanisms remain unknown. In this regard, we investigated the role of tetrahydrobiopterin (BH4) bioavailability in regulating endothelial nitric oxide synthase (eNOS) activity, dimerisation and SO production in streptozotocin-induced diabetic mice. Methods: Mouse aortas were used for assays of the following: (1) aortic function by isometric tension; (2) NO by electronic paramagnetic resonance; (3) SO by lucigenin-enhanced chemiluminescence and dihydroethidine fluorescence; (4) total biopterin and BH4 by high-performance liquid chromatography; and (5) eNOS protein expression and dimerisation by immunoblotting. Results: In diabetic mouse aortas, relaxations to acetylcholine and NO levels were significantly decreased, but SO production was increased, in association with reductions in total biopterins and BH4. Although total eNOS levels were increased in diabetes, the protein mainly existed in monomeric form. Conversely, specifically augmented BH4 in diabetic endothelium preserved eNOS dimerisation, but the expression remained unchanged. Conclusions/interpretation: Our results demonstrate that BH4 plays an important role in regulating eNOS activity and its functional protein structure, suggesting that increasing endothelial BH4 and/or protecting it from oxidation may be a rational therapeutic strategy to restore eNOS function in diabetes.

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“…nNOS exists in native complexes with Hsp90, and Hsp90 inhibitors enhance the ubiquitination and turnover of nNOS (3,6,27). Hsp90 regulates signaling proteins by modulating ligand-binding clefts (43), and we have suggested that Hsp90 stabilizes a more open state of the nNOS heme/substrate-binding cleft to enhance enzyme activity (36).…”

Section: Discussionmentioning

confidence: 99%

“…In this system, the predominant ubiquitin conjugate detected is the mono-ubiquitinated form, although polyubiquitin conjugates also form. The monoubiquitin conjugate is the predominant species in human embryonic kidney cells and rat brain cytosol (6). We used GST-tagged ubiquitin so that the conjugates could be more easily detected by Western blotting with anti-nNOS IgG.…”

Section: C331a Nnos Is Preferentially Ubiquitinated By Fraction Ii-mentioning

confidence: 99%

“…Another mechanism of regulation is the ubiquitination and proteasomal degradation of NOS (3). Of particular pharmacological interest is the finding that certain drugs cause the suicide inactivation, covalent alteration, ubiquitination, and proteasomal degradation of nNOS (3)(4)(5)(6)(7)(8). This phenomenon is not unique to nNOS as it is well documented that the suicide inactivation of other P450 cytochromes leads to covalent alteration, enhanced ubiquitination, and proteasomal turnover of the enzymes (9).…”

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confidence: 99%

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C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

Clapp

Peng

Morishima

et al. 2010

Journal of Biological Chemistry

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It is established that suicide inactivation of neuronal nitricoxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N G -nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.Nitric-oxide synthases (NOS) are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to nitric oxide and citrulline by a process that requires NADPH and molecular oxygen (1). There are three major mammalian isoforms as follows: neuronal NOS (nNOS), 2 endothelial NOS, and inducible NOS. NOS is bidomain in structure with an oxygenase domain, which contains the binding site for the heme, L-arginine, and tetrahydrobiopterin, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH (2). NOS is a highly regulated enzyme requiring homodimerization and bound calmodulin for efficient electron transfer from the flavins to the heme moiety to enable synthesis of NO. Another mechanism of regulation is the ubiquitination and proteasomal degradation of NOS (3). Of particular pharmacological interest is the finding that certain drugs cause the suicide inactivation, covalent alteration, ubiquitination, and proteasomal degradation of nNOS (3-8). This phenomenon is not unique to nNOS as it is well documented that the suicide inactivation of other P450 cytochromes leads to covalent alteration, enhanced ubiquitination, and proteasomal turnover of the enzymes (9). The C terminus of Hsc70-interacting protein (CHIP) has been shown to be an E3 ligase that ubiquitinates cytochromes P450 3A4 and 2E1 as well as nNOS (10 -12). The...

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Ubiquitination of Neuronal Nitric-oxide Synthase in Vitro and in Vivo

Cited by 87 publications

References 35 publications

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Endothelial nitric oxide synthase dysfunction in diabetic mice: importance of tetrahydrobiopterin in eNOS dimerisation

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

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