Renal dysfunction in adults following cardiopulmonary bypass is linked to declines in S-nitroso hemoglobin: a case series.

Background: Impaired kidney function is frequently observed in patients following cardiopulmonary bypass (CPB). Our group has previously linked blood transfusion to acute declines in S-nitroso haemoglobin (SNO-Hb; the main regulator of tissue oxygen delivery), reductions in intraoperative renal blood flow, and postoperative kidney dysfunction. While not all CPB patients receive blood, kidney injury is still common. We hypothesized that the CPB procedure itself may negatively impact SNO-Hb levels leading to renal dysfunction. Materials and methods: After obtaining written informed consent, blood samples were procured immediately before and after CPB, and on postoperative day (POD) 1. SNO-Hb levels, renal function (estimated glomerular filtration rate; eGFR), and plasma erythropoietin (EPO) concentrations were quantified. Additional outcome data were extracted from the patients' medical records. Results: Twenty-seven patients were enroled, three withdrew consent, and one was excluded after developing bacteremia. SNO-Hb levels declined after surgery and were directly correlated with declines in eGFR (R=0.48). Conversely, plasma EPO concentrations were elevated and inversely correlated with SNO-Hb (R=-0.53) and eGFR (R=-0.55). Finally, ICU stay negatively correlated with SNO-Hb concentration (R=-0.32). Conclusion: SNO-Hb levels are reduced following CPB in the absence of allogenic blood transfusion and are predictive of decreased renal function and prolonged ICU stay. Thus, therapies directed at maintaining or increasing SNO-Hb levels may improve outcomes in adult patients undergoing cardiac surgery.

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. This results in marked reduction in the amount of S-nitrosylation of liver proteins including notably acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in DNL. We further show that ACC S-nitrosylation markedly increases enzymatic activity. Diminished endothelial nitric oxide synthase expression and ACC S-nitrosylation may thus represent a physiological adaptation to caloric excess by constraining lipogenesis. Our findings demonstrate that S-nitrosylation of liver proteins is subject to dietary control and suggest that DNL is coupled to dietary and metabolic conditions through ACC S-nitrosylation.

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). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

Protocol for preparing Thiopropyl Sepharose resin used for capturing S-nitrosylated proteins.

S-nitrosothiol (SNO)-Resin Assisted Capture (SNO-RAC) relies on a Thiopropyl Sepharose resin to identify S-nitrosylated proteins (SNO-proteins) and sites of S-nitrosylation. Here, we present a protocol for preparing Thiopropyl Sepharose resin with efficiency of SNO-protein capture comparable to the discontinued commercial version. We describe steps for amine coupling, disulfide reduction, and generation of thiol reactive resin. We then detail quality control procedures. This resin is also suitable for Acyl-RAC assays to capture palmitoylated proteins. For complete details on the use and execution of the SNO-RAC protocol, please refer to Forrester et al.,1 Fonseca et al.,2 and Seth et al.3.

Identification of a Selective SCoR2 Inhibitor That Protects Against Acute Kidney Injury.

Acute kidney injury (AKI) is associated with high morbidity and mortality, and no drugs are available clinically. Metabolic reprogramming resulting from the deletion of S-nitroso-coenzyme A reductase 2 (SCoR2; AKR1A1) protects mice against AKI, identifying SCoR2 as a potential drug target. Of the few known inhibitors of SCoR2, none are selective versus the related oxidoreductase AKR1B1, limiting therapeutic utility. To identify SCoR2 (AKR1A1) inhibitors with selectivity versus AKR1B1, analogs of the nonselective (dual 1A1/1B1) inhibitor imirestat were designed, synthesized, and evaluated. Among 57 compounds, JSD26 has 10-fold selectivity for SCoR2 versus AKR1B1 and inhibits SCoR2 potently through an uncompetitive mechanism. When dosed orally to mice, JSD26 inhibited SNO-CoA metabolic activity in multiple organs. Notably, intraperitoneal injection of JSD26 in mice protected against AKI through S-nitrosylation of pyruvate kinase M2 (PKM2), whereas imirestat was not protective. Thus, selective inhibition of SCoR2 has therapeutic potential to treat acute kidney injury.

Comparison of the Nitric Oxide Synthase Interactomes and S-Nitroso-Proteomes: Furthering the Case for Enzymatic S-Nitrosylation.

Aims: S-nitrosylation of proteins is the main mechanism through which nitric oxide (NO) regulates cellular function and likely represents the archetype redox-based signaling system across aerobic and anaerobic organisms. How NO generated by different nitric oxide synthase (NOS) isoforms leads to specificity of S-nitrosylation remains incompletely understood. This study aimed to identify proteins interacting with, and whose S-nitrosylation is mediated by, human NOS isoforms in the same cellular system, thereby illuminating the contribution of individual NOSs to specificity. Results: Of the hundreds of proteins interacting with each NOS, many were also S-nitrosylated. However, a large proportion of S-nitrosylated proteins (SNO-proteins) did not associate with NOS. Moreover, most NOS interactors and SNO-proteins were unique to each isoform. The amount of NO produced by each NOS isoform was unrelated to the numbers of SNO-proteins. Thus, NOSs promoted S-nitrosylation of largely distinct sets of target proteins. Different signaling pathways were enriched downstream of each NOS. Innovation and Conclusion: The interactomes and SNOomes of individual NOS isoforms were largely distinct. Only a small fraction of SNO-proteins interacted with their respective NOS. Amounts of S-nitrosylation were unrelated to the amount of NO generated by NOSs. These data argue against free diffusion of NO or NOS interactions as being necessary or sufficient for S-nitrosylation and favor roles for additional enzymes and/or regulatory elements in imparting SNO-protein specificity. Antioxid. Redox Signal. 39, 621-634.

Kinase Activity Is Not Required for G Protein-Coupled Receptor Kinase 4 Restraining mTOR Signaling during Cilia and Kidney Development.

SIGNIFICANCE STATEMENT: G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause. BACKGROUND: G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development. METHODS: To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model. RESULTS: Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause. CONCLUSIONS: These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.

Control of tissue oxygenation by S-nitrosohemoglobin in human subjects.

S-Nitrosohemoglobin (SNO-Hb) is unique among vasodilators in coupling blood flow to tissue oxygen requirements, thus fulfilling an essential function of the microcirculation. However, this essential physiology has not been tested clinically. Reactive hyperemia following limb ischemia/occlusion is a standard clinical test of microcirculatory function, which has been ascribed to endothelial nitric oxide (NO). However, endothelial NO does not control blood flow governing tissue oxygenation, presenting a major quandary. Here we show in mice and humans that reactive hyperemic responses (i.e., reoxygenation rates following brief ischemia/occlusion) are in fact dependent on SNO-Hb. First, mice deficient in SNO-Hb (i.e., carrying C93A mutant Hb refractory to S-nitrosylation) showed blunted muscle reoxygenation rates and persistent limb ischemia during reactive hyperemia testing. Second, in a diverse group of humans-including healthy subjects and patients with various microcirculatory disorders-strong correlations were found between limb reoxygenation rates following occlusion and both arterial SNO-Hb levels (n = 25; P = 0.042) and SNO-Hb/total HbNO ratios (n = 25; P = 0.009). Secondary analyses showed that patients with peripheral artery disease had significantly reduced SNO-Hb levels and blunted limb reoxygenation rates compared with healthy controls (n = 8 to 11/group; P < 0.05). Low SNO-Hb levels were also observed in sickle cell disease, where occlusive hyperemic testing was deemed contraindicated. Altogether, our findings provide both genetic and clinical support for the role of red blood cells in a standard test of microvascular function. Our results also suggest that SNO-Hb is a biomarker and mediator of blood flow governing tissue oxygenation. Thus, increases in SNO-Hb may improve tissue oxygenation in patients with microcirculatory disorders.

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. SAR1 acts as a SURF4 nitrosylase and SURF4 as a PCSK9 nitrosylase to inhibit PCSK9 secretion, while SCoR2 counteracts nitrosylase activity by promoting PCSK9 denitrosylation. Inhibition of PCSK9 by an NO-based drug requires nitrosylase activity, and small-molecule inhibition of SCoR2 phenocopies the PCSK9-mediated reductions in cholesterol observed in SCoR2-deficient mice. Our results reveal enzymatic machinery controlling cholesterol levels through S-nitrosylation and suggest a distinct treatment paradigm for cardiovascular disease.

ß-PIX cooperates with GIT1 to regulate endothelial nitric oxide synthase in sinusoidal endothelial cells.

Previous studies have demonstrated that G protein-coupled receptor kinase interacting-1 protein (GIT1) associates with endothelial nitric oxide synthase (eNOS) to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly associated binding partner, ß-PIX (p21-activated kinase-interacting exchange factor ß, ARHGEF7) is specifically important in the regulation of eNOS activity. We examined ß-PIX expression in normal rat liver by immunohistochemistry and explored ß-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of ß-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and ß-PIX required to regulate eNOS activity were mapped. ß-PIX was expressed primarily in SECs in normal liver and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, and hepatocytes). ß-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs and was important in stimulating eNOS activity. Of note, GIT1-ß-PIX interaction led to synergistic enhancement of eNOS activity, and ß-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of ß-PIX or GIT1 in normal SECs using ß-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains [Spa2 homology domain (SHD) and synaptic localization domain (SLD), aa 331-596] and the ß-PIX COOH terminal (aa 496-555) appeared to be critical in the regulation eNOS activity. The data indicate that ß-PIX regulates eNOS phosphorylation and function in normal SECs and highlight the importance of the GIT1/ß-PIX/eNOS trimolecular complex in normal liver SEC function.NEW & NOTEWORTHY ß-PIX is a multidomain protein known to be a GIT1 binding partner. We report here that in the normal liver, the distribution and cellular localization of ß-PIX are restricted largely to sinusoidal endothelial cells. Furthermore, ß-PIX interacts with eNOS and GIT1 promotes eNOS activity and NO production and therefore exerts a novel posttranslational regulatory function on eNOS activity in sinusoidal endothelial cells. We also have identified specific molecular domains important in GIT1 and ß-PIX's interaction with eNOS, which may represent novel therapeutic targets in the control of sinusoidal blood flow and intrahepatic resistance.

S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Optimized S-nitrosohemoglobin Synthesis in Red Blood Cells to Preserve Hypoxic Vasodilation Via ßCys93.

Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. ß-globin Cys93Ala (ßCys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions. However, the means to replete SNO in mouse red blood cells (RBCs) to restore RBC function is not known. In particular, although methods have been developed to selectively S-nitrosylate ßCys93 in human Hb and intact human RBCs, conditions have not been optimized for mouse RBCs that are used experimentally. Here we show that loading SNO onto Hb in mouse RBC lysates can be achieved with high stoichiometry and ß-globin selectivity. However, S-nitrosylation of Hb within intact mouse RBCs is ineffective under conditions that work well with human RBCs, and levels of metHb are prohibitively high. We developed an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and shows that loss of SNO loading in ßCys93Ala mutant RBCs results in reduced vasodilation. We also demonstrate that differences in SNO/met/nitrosyl Hb stoichiometry can account for differences in RBC function among studies. RBCs loaded with quasi-physiologic amounts of SNO-Hb will produce vasodilation proportionate to hypoxia, whereas RBCs loaded with higher amounts lose allosteric regulation, thus inducing vasodilation at both high and low oxygen level. SIGNIFICANCE STATEMENT: Red blood cells from mice exhibit poor hemoglobin S-nitrosylation under conditions used for human RBCs, frustrating tests of vasodilatory activity. Using an optimized S-nitrosylation protocol, mouse RBCs exhibit hypoxic vasodilation that is significantly reduced in hemoglobin ßCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of ßCys93 in oxygen delivery.

Brain-specific deletion of GIT1 impairs cognition and alters phosphorylation of synaptic protein networks implicated in schizophrenia susceptibility.

Despite tremendous effort, the molecular and cellular basis of cognitive deficits in schizophrenia remain poorly understood. Recent progress in elucidating the genetic architecture of schizophrenia has highlighted the association of multiple loci and rare variants that may impact susceptibility. One key example, given their potential etiopathogenic and therapeutic relevance, is a set of genes that encode proteins that regulate excitatory glutamatergic synapses in brain. A critical next step is to delineate specifically how such genetic variation impacts synaptic plasticity and to determine if and how the encoded proteins interact biochemically with one another to control cognitive function in a convergent manner. Towards this goal, here we study the roles of GPCR-kinase interacting protein 1 (GIT1), a synaptic scaffolding and signaling protein with damaging coding variants found in schizophrenia patients, as well as copy number variants found in patients with neurodevelopmental disorders. We generated conditional neural-selective GIT1 knockout mice and found that these mice have deficits in fear conditioning memory recall and spatial memory, as well as reduced cortical neuron dendritic spine density. Using global quantitative phospho-proteomics, we revealed that GIT1 deletion in brain perturbs specific networks of GIT1-interacting synaptic proteins. Importantly, several schizophrenia and neurodevelopmental disorder risk genes are present within these networks. We propose that GIT1 regulates the phosphorylation of a network of synaptic proteins and other critical regulators of neuroplasticity, and that perturbation of these networks may contribute specifically to cognitive deficits observed in schizophrenia and neurodevelopmental disorders.

The manifold roles of protein S-nitrosylation in the life of insulin.

Insulin, which is released by pancreatic islet ß-cells in response to elevated levels of glucose in the blood, is a critical regulator of metabolism. Insulin triggers the uptake of glucose and fatty acids into the liver, adipose tissue and muscle, and promotes the storage of these nutrients in the form of glycogen and lipids. Dysregulation of insulin synthesis, secretion, transport, degradation or signal transduction all cause failure to take up and store nutrients, resulting in type 1 diabetes mellitus, type 2 diabetes mellitus and metabolic dysfunction. In this Review, we make the case that insulin signalling is intimately coupled to protein S-nitrosylation, in which nitric oxide groups are conjugated to cysteine thiols to form S-nitrosothiols, within effectors of insulin action. We discuss the role of S-nitrosylation in the life cycle of insulin, from its synthesis and secretion in pancreatic ß-cells, to its signalling and degradation in target tissues. Finally, we consider how aberrant S-nitrosylation contributes to metabolic diseases, including the roles of human genetic mutations and cellular events that alter S-nitrosylation of insulin-regulating proteins. Given the growing influence of S-nitrosylation in cellular metabolism, the field of metabolic signalling could benefit from renewed focus on S-nitrosylation in type 2 diabetes mellitus and insulin-related disorders.

Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin ß-cysteine93 S-nitrosylation.

Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian Hbs, ß-globin cysteine93 (ßCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during O2 delivery. ßCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting O2 normally. Here, we show that ßCys93Ala mutant mice had reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion and that hypoxic vasodilation by RBCs was impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, ßCys93Ala mutant mice showed marked impairment of hypoxic peripheral vasodilation and developed signs of pulmonary hypertension with age. Mutant mice also died prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low O2. Altogether, we identify a major role for RBC SNO in clinically relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.

The enzymatic function of the honorary enzyme: S-nitrosylation of hemoglobin in physiology and medicine.

The allosteric transition within tetrameric hemoglobin (Hb) that allows both full binding to four oxygen molecules in the lung and full release of four oxygens in hypoxic tissues would earn Hb the moniker of 'honorary enzyme'. However, the allosteric model for oxygen binding in hemoglobin overlooked the essential role of blood flow in tissue oxygenation that is essential for life (aka autoregulation of blood flow). That is, blood flow, not oxygen content of blood, is the principal determinant of oxygen delivery under most conditions. With the discovery that hemoglobin carries a third biologic gas, nitric oxide (NO) in the form of S-nitrosothiol (SNO) at ß-globin Cys93 (ßCys93), and that formation and export of SNO to dilate blood vessels are linked to hemoglobin allostery through enzymatic activity, this title is honorary no more. This chapter reviews evidence that hemoglobin formation and release of SNO is a critical mediator of hypoxic autoregulation of blood flow in tissues leading to oxygen delivery, considers the physiological implications of a 3-gas respiratory cycle (O2/NO/CO2) and the pathophysiological consequences of its dysfunction. Opportunities for therapeutic intervention to optimize oxygen delivery at the level of tissue blood flow are highlighted.

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 et al. [1] 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. These findings are consistent with emerging roles for protein S-nitrosylation in developmental biology (including cardiomyocyte development), aging/longevity, and cancer.

An optimized protocol for isolation of S-nitrosylated proteins from C. elegans.

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).

Red Blood Cell-Mediated S-Nitrosohemoglobin-Dependent Vasodilation: Lessons Learned from a ß-Globin Cys93 Knock-In Mouse.

Significance: Red blood cell (RBC)-mediated vasodilation plays an important role in oxygen delivery. This occurs through hemoglobin actions, at least in significant part, to convert heme-bound nitric oxide (NO) (in tense [T]/deoxygenated-state hemoglobin) into vasodilator S-nitrosothiol (SNO) (in relaxed [R]/oxygenated-state hemoglobin), convey SNO through the bloodstream, and release it into tissues to increase blood flow. The coupling of hemoglobin R/T state allostery, both to NO conversion into SNO and to SNO release (along with oxygen), under hypoxia supports the model of a three-gas respiratory cycle (O2/NO/CO2). Recent Advances: Oxygenation of tissues is dependent on a single, strictly conserved Cys residue in hemoglobin (ßCys93). Hemoglobin couples SNO formation/release at ßCys93 to O2 binding/release at hemes ("thermodynamic linkage"). Mice bearing ßCys93Ala hemoglobin that is unable to generate SNO-ßCys93 establish that SNO-hemoglobin is important for R/T allostery-regulated vasodilation by RBCs that couple blood flow to tissue oxygenation. Critical Issues: The model for RBC-mediated vasodilation originally proposed by Stamler et al. in 1996 has been largely validated: SNO-ßCys93 forms in vivo, dilates blood vessels, and is hypoxia-regulated, and RBCs actuate vasodilation proportionate to hypoxia. Numerous compensations in ßCys93Ala animals to alleviate tissue hypoxia (discussed herein) are predicted to preserve vasodilatory responses of RBCs but impair linkage to R/T transition in hemoglobin. This is borne out by loss of responsivity of mutant RBCs to oxygen, impaired blood flow responses to hypoxia, and tissue ischemia in ßCys93-mutant animals. Future Directions: SNO-hemoglobin mediates hypoxic vasodilation in the respiratory cycle. This fundamental physiology promises new insights in vascular diseases and blood disorders.

Microcephaly with altered cortical layering in GIT1 deficiency revealed by quantitative neuroimaging.

G Protein-Coupled Receptor Kinase-Interacting Protein-1 (GIT1) regulates neuronal functions, including cell and axon migration and synapse formation and maintenance, and GIT1 knockout (KO) mice exhibit learning and memory deficits. We noted that male and female GIT1-KO mice exhibit neuroimaging phenotypes including microcephaly, and altered cortical layering, with a decrease in neuron density in cortical layer V. Micro-CT and magnetic resonance microscopy (MRM) were used to identify morphometric phenotypes for the skulls and throughout the GIT1-KO brains. High field MRM of actively-stained mouse brains from GIT1-KO and wild type (WT) controls (n = 6 per group) allowed segmenting 37 regions, based on co-registration to the Waxholm Space atlas. Overall brain size in GIT1-KO mice was ~32% smaller compared to WT controls. After correcting for brain size, several regions were significantly different in GIT1-KO mice relative to WT, including the gray matter of the ventral thalamic nuclei and the rest of the thalamus, the inferior colliculus, and pontine nuclei. GIT1-KO mice had reduced volume of white matter tracts, most notably in the anterior commissure (~26% smaller), but also in the cerebral peduncle, fornix, and spinal trigeminal tract. On the other hand, the basal ganglia appeared enlarged in GIT1-KO mice, including the globus pallidus, caudate putamen, and particularly the accumbens - supporting a possible vulnerability to addiction. Volume based morphometry based on high-resolution MRM (21.5 µm isotropic voxels) was effective in detecting overall, and local differences in brain volumes in GIT1-KO mice, including in white matter tracts. The reduced relative volume of specific brain regions suggests a critical, but not uniform, role for GIT1 in brain development, conducive to brain microcephaly, and aberrant connectivity.