Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics “biotin-switch” approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis

Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17//RAC1-mediated activation of NADPH oxidases

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2–mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans

A new role for RGS-1 in vascular function and blood pressure regulation

The Regulator of G-Protein Signalling-1 (RGS1) controls G protein coupled receptor signalling by acting as a GTPase-activating protein for heterotrimeric G proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. We have previously shown that Rgs1 regulates macrophage accumulation in Angiotensin II (Ang II)-induced aortic aneurysms and Rgs1-/-ApoE-/- mice are protected from Ang II-induced aortic aneurysm rupture compared to ApoE-/- mice. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1-/-ApoE-/- mice than ApoE-/- mice and this is mediated by non-haematopoietic cells as indicated by bone marrow chimeras. However the precise role of RGS1 in hypertension and vascular-derived cells is unknown

Vascular wall regulator of G-protein signalling-1 (RGS-1) is required for angiotensin II–mediated blood pressure control

G-Protein coupled receptors (GPCRs) activate intracellular signalling pathways by coupling to heterotrimeric G-proteins that control many physiological processes including blood pressure homeostasis. The Regulator of G-Protein Signalling-1 (RGS1) controls the magnitude and duration of downstream GPCR signalling by acting as a GTPase-activating protein for specific Gα-proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. Rgs1−/−ApoE−/− mice are protected from Angiotensin II (Ang II)-induced aortic aneurysm rupture. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1−/−ApoE−/− mice than ApoE−/− mice, independent of its role in myeloid cells. However the precise role of RGS1 in hypertension and vascular-derived cells remains unknown. We determined the effects of Rgs1 deletion on vascular function in ApoE−/− mice. Rgs1 deletion led to enhanced vasoconstriction in aortas and mesenteric arteries from ApoE−/− mice in response to phenylephrine (PE) and U46619 respectively. Rgs1 was shown to have a role in the vasculature, with endothelium-dependent vasodilation being impaired, and endothelium-independent dilatation to SNP being enhanced in Rgs1−/−ApoE−/− mesenteric arteries. To address the downstream signalling pathways in vascular smooth muscle cells (VSMCs) in response to Ang II-stimulation, we assessed pErk1/2, pJNK and pp38 MAPK activation in VSMCs transiently transfected with Rgs1. pErk1/2 signalling but not pJNK and pp38 signalling was impaired in the presence of Rgs1. Furthermore, we demonstrated that the enhanced contractile response to PE in Rgs1−/−ApoE−/− aortas was reduced by a MAPK/Erk (MEK) inhibitor and an L-type voltage gated calcium channel antagonist, suggesting that Erk1/2 signalling and calcium influx are major effectors of Rgs1-mediated vascular contractile responses, respectively. These findings indicate RGS1 is a novel regulator of blood pressure homeostasis and highlight RGS1-controlled signalling pathways in the vasculature that may be new drug development targets for hypertension

P683The role of nitric oxide synthase (NOS) and its essential cofactor tetrahydrobiopterin (BH4) in diabetic cardiomyopathy.

PURPOSE: Diabetes can impact on cardiovascular health by causing a distinct condition termed "diabetic cardiomyopathy". Its characteristic left ventricular (LV) diastolic dysfunction has been associated with interstitial fibrosis, reduced NO availability, and abnormal calcium handling. However, the early triggers and the underlying cellular mechanisms remain unknown. Here, we investigate changes in vascular and myocardial reactive oxygen species (ROS) and NO availability in a murine model of type 1 diabetes, and evaluate potential beneficial effects of inducing a myocardial-specific increase in the NOS cofactor tetrahydrobiopterin (BH4) on the development of LV dysfunction. Methods: Diabetes was induced in male mice by daily intraperitoneal streptozotocin (STZ) injection (43mg/kg, 5 consecutive days). To augment myocardial BH4 and increase NOS activity, transgenic mice were generated with myocardial-specific overexpression of the rate-limiting enzyme for BH4 synthesis, GTP cyclohydrolase 1 (mGCH1 Tg). Vascular function in isolated aortas was evaluated by isometric tension studies (myograph), NOS activity and biopterins by HPLC, and superoxide production by lucigenin-enhanced chemiluminescence. High-resolution echocardiography was used to assess LV function. Results: After 12 weeks of diabetes, WT and mGCH1 Tg mice showed impaired aortic endothelium-dependent vasodilatation, in association with increased superoxide production and reduced BH4 bioavailability (n=6-10 per group). By contrast, diabetic LV homogenates showed no increase in superoxide generation or reduced BH4:BH2 ratio and no reduction in NOS activity (n=9-12 per group). Nevertheless, in vivo echocardiography revealed significant LV diastolic dysfunction in WT diabetic mice, which was prevented in mGCH1 Tg mice (E'/A' diabetic vs control: 1.52±0.08 vs 1.53±0.08 in mGCH1 Tg; 0.89±0.07 vs 1.35±0.06 in WT, n=9 per group, P<0.01 for the interaction between genotype and diabetes). In line with these results, isolated LV myocytes from WT diabetic mice displayed prolonged relaxation, which was prevented in diabetic mGCH1 Tg (t50 relaxation, diabetic vs control: 105.3±2.8 vs 95.6±2.4 in WT and 77.3±3 vs 73.3±2 in mGCH1 Tg; n=25 cells from 2-5 hearts per group, P<0.05 for the interaction between genotype and diabetes). Conclusions: Impaired LV diastolic function in diabetic mice can be prevented by myocardial GCH1 overexpression in the absence of NOS dysfunction or increased oxidative stress, suggesting that GCH1/BH4 protect the diabetic myocardium by mechanisms other than redressing the local nitroso-redox balance

A key role for tetrahydrobiopterin-dependent endothelial NOS regulation in vascular resistance arteries: studies in endothelial cell tetrahydrobiopterin-deficient mice

Background and purpose: The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and reactive oxygen species (ROS) signalling in vascular physiology. To determine the physiological requirement for de-novo endothelial cell BH4 synthesis in vasomotor function in resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation. Experimental approach: Wire myography was used to assess the reactivity of mouse 2nd order mesenteric arteries. High-performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis. Key Results: Gch1fl/flTie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1fl/flTie2cre mice. Gch1fl/flTie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was equalised by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1fl/flTie2cre mice compared to wild-type littermates. The loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H2O2 and prostaglandin-derived vasodilator in Gch1fl/flTie2cre mesenteric arteries. Conclusions and implications: Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease