Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development.

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca release from the sarcoplasmic reticulum through the ryanodine receptor/Ca-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood.

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet.

Nitric oxide (NO), produced primarily by nitric oxide synthase enzymes, is known to influence energy metabolism by stimulating fat uptake and oxidation. The effects of NO on de novo lipogenesis (DNL), however, are less clear. Here we demonstrate that hepatic expression of endothelial nitric oxide synthase is reduced following prolonged administration of a hypercaloric high-fat diet.

An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling.

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1).

A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis.

Accumulating evidence suggests that protein S-nitrosylation is enzymatically regulated and that specificity in S-nitrosylation derives from dedicated S-nitrosylases and denitrosylases that conjugate and remove S-nitrosothiols, respectively. Here, we report that mice deficient in the protein denitrosylase SCoR2 (S-nitroso-Coenzyme A Reductase 2; AKR1A1) exhibit marked reductions in serum cholesterol due to reduced secretion of the cholesterol-regulating protein PCSK9. SCoR2 associates with endoplasmic reticulum (ER) secretory machinery to control an S-nitrosylation cascade involving ER cargo-selection proteins SAR1 and SURF4, which moonlight as S-nitrosylases.

GSNOR regulates cardiomyocyte differentiation and maturation through protein S-nitrosylation.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme responsible for reverting protein S-nitrosylation (SNO). In this issue, Salerno provide evidence that GSNOR deficiency - and thus elevated protein S-nitrosylation - accelerates cardiomyocyte differentiation and maturation of induced pluripotent stem cells (iPSCs). GSNOR inhibition (GSNOR iPSCs) expedites the epithelial-mesenchymal transition (EMT) and promotes cardiomyocyte progenitor cell proliferation, differentiation, and migration.

AKR1A1 is a novel mammalian -nitroso-glutathione reductase.

Oxidative modification of Cys residues by NO results in -nitrosylation, a ubiquitous post-translational modification and a primary mediator of redox-based cellular signaling. Steady-state levels of -nitrosylated proteins are largely determined by denitrosylase enzymes that couple NAD(P)H oxidation with reduction of -nitrosothiols, including protein and low-molecular-weight (LMW) -nitrosothiols (-nitroso-GSH (GSNO) and -nitroso-CoA (SNO-CoA)). SNO-CoA reductases require NADPH, whereas enzymatic reduction of GSNO can involve either NADH or NADPH.

Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage.

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI).