S-nitrosoglutathione reductase alleviates morphine analgesic tolerance by restricting PKCα S-nitrosation.

Morphine, a typical opiate, is widely used for controlling pain but can lead to various side effects with long-term use, including addiction, analgesic tolerance, and hyperalgesia. At present, however, the mechanisms underlying the development of morphine analgesic tolerance are not fully understood. This tolerance is influenced by various opioid receptor and kinase protein modifications, such as phosphorylation and ubiquitination. Here, we established a murine morphine tolerance model to investigate whether and how S-nitrosoglutathione reductase (GSNOR) is involved in morphine tolerance. Repeated administration of morphine resulted in the down-regulation of GSNOR, which increased excessive total protein S-nitrosation in the prefrontal cortex. Knockout or chemical inhibition of GSNOR promoted the development of morphine analgesic tolerance and neuron-specific overexpression of GSNOR alleviated morphine analgesic tolerance. Mechanistically, GSNOR deficiency enhanced S-nitrosation of cellular protein kinase alpha (PKCα) at the Cys78 and Cys132 sites, leading to inhibition of PKCα kinase activity, which ultimately promoted the development of morphine analgesic tolerance. Our study highlighted the significant role of GSNOR as a key regulator of PKCα S-nitrosation and its involvement in morphine analgesic tolerance, thus providing a potential therapeutic target for morphine tolerance.

De-nitrosylation Coordinates Appressorium Function for Infection of the Rice Blast Fungus.

As a signaling molecule, nitric oxide (NO) regulates the development and stress response in different organisms. The major biological activity of NO is protein S-nitrosylation, whose function in fungi remains largely unclear. Here, it is found in the rice blast fungus Magnaporthe oryzae, de-nitrosylation process is essential for functional appressorium formation during infection. Nitrosative stress caused by excessive accumulation of NO is harmful for fungal infection. While the S-nitrosoglutathione reductase GSNOR-mediated de-nitrosylation removes excess NO toxicity during appressorium formation to promote infection. Through an indoTMT switch labeling proteomics technique, 741 S-nitrosylation sites in 483 proteins are identified. Key appressorial proteins, such as Mgb1, MagB, Sps1, Cdc42, and septins, are activated by GSNOR through de-nitrosylation. Removing S-nitrosylation sites of above proteins is essential for proper protein structure and appressorial function. Therefore, GSNOR-mediated de-nitrosylation is an essential regulator for appressorium formation. It is also shown that breaking NO homeostasis by NO donors, NO scavengers, as well as chemical inhibitor of GSNOR, shall be effective methods for fungal disease control.

N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

GSNOR negatively regulates the NLRP3 inflammasome via S-nitrosation of MAPK14.

Hyperactivation of the NLRP3 inflammasome has been implicated in the pathogenesis of numerous diseases. However, the precise molecular mechanisms that modulate the transcriptional regulation of NLRP3 remain largely unknown. In this study, we demonstrated that S-nitrosoglutathione reductase (GSNOR) deficiency in macrophages leads to significant increases in the Nlrp3 and Il-1ß expression levels and interleukin-1ß (IL-1ß) secretion in response to NLRP3 inflammasome stimulation. Furthermore, in vivo experiments utilizing Gsnor-/- mice revealed increased disease severity in both lipopolysaccharide (LPS)-induced septic shock and dextran sodium sulfate (DSS)-induced colitis models. Additionally, we showed that both LPS-induced septic shock and DSS-induced colitis were ameliorated in Gsnor-/- Nlrp3-/- double-knockout (DKO) mice. Mechanistically, GSNOR deficiency increases the S-nitrosation of mitogen-activated protein kinase 14 (MAPK14) at the Cys211 residue and augments MAPK14 kinase activity, thereby promoting Nlrp3 and Il-1ß transcription and stimulating NLRP3 inflammasome activity. Our findings suggested that GSNOR is a regulator of the NLRP3 inflammasome and that reducing the level of S-nitrosylated MAPK14 may constitute an effective strategy for alleviating diseases associated with NLRP3-mediated inflammation.

GSNOR overexpression enhances CAR-T cell stemness and anti-tumor function by enforcing mitochondrial fitness.

Chimeric antigen receptor-T (CAR-T) cell has been developed as a promising agent for patients with refractory or relapsed lymphoma and leukemia, but not all the recipients could achieve a long-lasting remission. The limited capacity of in vivo expansion and memory differentiation post activation is one of the major reasons for suboptimal CAR-T therapeutic efficiency. Nitric oxide (NO) plays multifaceted roles in mitochondrial dynamics and T cell activation, but its function on CAR-T cell persistence and anti-tumor efficacy remains unknown. Herein, we found the continuous signaling from CAR not only promotes excessive NO production, but also suppressed S-nitrosoglutathione reductase (GSNOR) expression in T cells, which collectively led to increased protein S-nitrosylation, resulting in impaired mitochondrial fitness and deficiency of T cell stemness. Intriguingly, enforced expression of GSNOR promoted memory differentiation of CAR-T cell after immune activation, rendered CAR-T better resistance to mitochondrial dysfunction, further enhanced CAR-T cell expansion and anti-tumor capacity in vitro and in a mouse tumor model. Thus, we revealed a critical role of NO in restricting CAR-T cell persistence and functionality, and defined that GSNOR overexpression may provide a solution to combat NO stress and render patients with more durable protection from CAR-T therapy.

Differential S-nitrosylation and characterization of purified S-nitrosoglutathione reductase (GSNOR) from Brassica juncea shows multiple forms of the enzyme.

S-nitrosoglutathione reductase (GSNOR). a master regulator of NO homeostasis, is a single-copy gene in most plants. In Lotus japonicus, two GSNOR isoforms were identified exhibiting similar kinetic properties but differential tissue-specific expressions. Previously, a genome-wide identification in Brassica juncea revealed four copies of GSNOR, each encoding proteins that vary in subunit molecular weights and pI. Here, we report multiple forms of GSNOR using 2D immunoblot which showed 4 immunopositive spots of 41.5 kDa (pl 5.79 and 6.78) and 43 kDa (pl 6.16 and 6.23). To confirm, purification of GSNOR using anion-exchange chromatography yielded 2 distinct pools (GSNOR-A & GSNOR-B) with GSNOR activities. Subsequently, affinity-based purification resulted in 1 polypeptide from GSNOR-A and 2 polypeptides from GSNOR-B. Size exclusion-HPLC confirmed 3 GSNORs with molecular weight of 87.48 ± 2.74 KDa (GSNOR-A); 87.36 ± 3.25 and 82.74 ± 2.75 kDa (GSNOR-B). Kinetic analysis showed Km of 118 ± 11 µM and Vmax of 287 ± 22 nkat/mg for GSNOR-A, whereas Km of 96.4 ± 8 µM and Vmax of 349 ± 15 nkat/mg for GSNOR-B. S-nitrosylation and inhibition by NO showed redox regulation of all BjGSNORs. Both purified GSNORs exhibited variable denitrosylation efficiency as depicted by Biotin Switch assay. To the best of our knowledge, this is the first report confirming multiple isoforms of GSNOR in B. juncea.

S-nitrosoglutathione reductase-dependent p65 denitrosation promotes osteoclastogenesis by facilitating recruitment of p65 to NFATc1 promoter.

Osteoclasts, the exclusive bone resorptive cells, are indispensable for bone remodeling. Hence, understanding novel signaling modulators regulating osteoclastogenesis is clinically important. Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) is a master transcription factor in osteoclastogenesis, and binding of NF-κB p65 subunit to NFATc1 promoter is required for its expression. It is well-established that DNA binding activity of p65 can be regulated by various post-translational modifications, including S-nitrosation. Recent studies have demonstrated that S-nitrosoglutathione reductase (GSNOR)-mediated protein denitrosation participated in cell fate commitment by regulating gene transcription. However, the role of GSNOR in osteoclastogenesis remains unexplored and enigmatic. Here, we investigated the effect of GSNOR-mediated denitrosation of p65 on osteoclastogenesis. Our results revealed that GSNOR was up-regulated during osteoclastogenesis in vitro. Moreover, GSNOR inhibition with a chemical inhibitor impaired osteoclast differentiation, podosome belt formation, and bone resorption activity. Furthermore, GSNOR inhibition enhanced the S-nitrosation level of p65, precluded the binding of p65 to NFATc1 promoter, and suppressed NFATc1 expression. In addition, mouse model of lipopolysaccharides (LPS)-induced calvarial osteolysis was employed to evaluate the therapeutic effect of GSNOR inhibitor in vivo. Our results indicated that GSNOR inhibitor treatment alleviated the inflammatory bone loss by impairing osteoclast formation in mice. Taken together, these data have shown that GSNOR activity is required for osteoclastogenesis by facilitating binding of p65 to NFATc1 promoter via promoting p65 denitrosation, suggesting that GSNOR may be a potential therapeutic target in the treatment of osteolytic diseases.

Sterile inflammation induces vasculopathy and chronic lung injury in murine sickle cell disease.

Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by ∼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by ∼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.

Mechanistic study of the Aldo-keto reductase family 1 member A1 in regulating mesenchymal stem cell fate decision toward adipogenesis and osteogenesis.

AIMS: Akr1A1 is a glycolytic enzyme catalyzing the reduction of aldehyde to alcohol. This study aims to delineate the role of Akr1A1 in regulating the adipo-osteogenic lineage differentiation of mesenchymal stem cells (MSCs). MAIN METHODS: MSCs derived from human bone marrow and Wharton Jelly together with gain- and loss-of-function analysis as well as supplementation with the S-Nitrosoglutathione reductase (GSNOR) inhibitor N6022 were used to study the function of Akr1A1 in controlling MSC lineage differentiation into osteoblasts and adipocytes. KEY FINDINGS: Akr1A1 expression, PKM2 activity, and lactate production were found to be decreased in osteoblast-committed MSCs, but PGC-1α increased to induce mitochondrial oxidative phosphorylation. Increased Akr1A1 inhibited the SIRT1-dependent pathway for decreasing the expressions of PGC-1α and TAZ but increasing PPAR γ in adipocyte-committed MSCs, hence promoting glycolysis in adipogenesis. In contrast, Akr1A1 expression, PKM2 activity and lactate production were all increased in adipocyte-differentiated cells with decreased PGC-1α for switching energy utilization to glycolytic metabolism. Reduced Akr1A1 expression in osteoblast-committed cells relieves its inhibition of SIRT1-mediated activation of PGC-1α and TAZ for facilitating osteogenesis and mitochondrial metabolism. SIGNIFICANCE: Several metabolism-involved regulators including Akr1A1, SIRT1, PPARγ, PGC-1α and TAZ were differentially expressed in osteoblast- and adipocyte-committed MSCs. More importantly, Akr1A1 was identified as a new key regulator for controlling the MSC lineage commitment in favor of adipogenesis but detrimental to osteogenesis. Such information should be useful to develop perspective new therapeutic agents to reverse the adipo-osteogenic differentiation of BMSCs, in a way to increase in osteogenesis but decrease in adipogenesis.

Efficacies of S-nitrosoglutathione (GSNO) and GSNO reductase inhibitor in SARS-CoV-2 spike protein induced acute lung disease in mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which initially surfaced in late 2019, often triggers severe pulmonary complications, encompassing various disease mechanisms such as intense lung inflammation, vascular dysfunction, and pulmonary embolism. Currently, however, there's no drug addressing all these mechanisms simultaneously. This study explored the multi-targeting potential of S-nitrosoglutathione (GSNO) and N6022, an inhibitor of GSNO reductase (GSNOR) on markers of inflammatory, vascular, and thrombotic diseases related to COVID-19-induced acute lung disease. For this, acute lung disease was induced in C57BL/6 mice through intranasal administration of recombinant SARS-CoV-2 spike protein S1 domain (SP-S1). The mice exhibited fever, body weight loss, and increased blood levels and lung expression of proinflammatory cytokines (e.g., TNF-α and IL-6) as well as increased vascular inflammation mediated by ICAM-1 and VCAM-1 and lung infiltration by immune cells (e.g., neutrophils, monocytes, and activated cytotoxic and helper T cells). Further, the mice exhibited increased lung hyperpermeability (lung Evans blue extravasation) leading to lung edema development as well as elevated blood coagulation factors (e.g., fibrinogen, thrombin, activated platelets, and von Willebrand factor) and lung fibrin deposition. Similar to the patients with COVID-19, male mice showed more severe disease than female mice, along with higher GSNOR expression in the lungs. Optimization of GSNO by treatment with exogenous GSNO or inhibition of GSNOR by N6022 (or GSNO knockout) protects against SP-S1-induced lung diseases in both genders. These findings provide evidence for the potential efficacies of GSNO and GSNOR inhibitors in addressing the multi-mechanistic nature of SARS-CoV-2 SP-associated acute-lung disease.

Swamp eel aldehyde reductase is involved in response to nitrosative stress via regulating NO/GSH levels.

Aldehyde reductase (ALR) plays key roles in the detoxification of toxic aldehyde. In this study, the authors cloned the swamp eel ALR gene using rapid amplification of cDNA ends-PCR (RACE-PCR). The recombinant protein (rALR) was expressed in Escherichia coli and purified using a Ni2+ -NTA chelating column. The rALR protein exhibited efficient reductive activity towards several aldehydes, ketones and S-nitrosoglutathione (GSNO). A spot assay suggested that the recombinant E. coli strain expressing rALR showed better resistance to formaldehyde, sodium nitrite and GSNO stress, suggesting that swamp eel ALR is crucial for redox homeostasis in vivo. Consequently, the authors investigated the effect of rALR on the oxidative parameters of the liver in swamp eels challenged with Aeromonas hydrophila. The hepatic glutathione (GSH) content significantly increased, and the hepatic NO content and levels of reactive oxygen species and reactive nitrogen species significantly decreased when rALR was administered. In addition, the mRNA expression of hepatic Alr, HO1 and Nrf2 was significantly upregulated, whereas the expression levels of NF-κB, IL-1ß and NOS1 were significantly downregulated in the rALR-administered group. Collectively, these results suggest that ALR is involved in the response to nitrosative stress by regulating GSH/NO levels in the swamp eel.

Loss of S-nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

S-Nitrosoglutathione plays a central role in nitric oxide (NO) homeostasis, and S-nitrosoglutathione reductase (GSNOR) regulates the cellular levels of S-nitrosoglutathione across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. Silencing or knocking out of SlGSNOR intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin, and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set, and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

INTRODUCTION: Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. METHODS: In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 µg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. RESULTS: As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. CONCLUSIONS: Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Pharmacological Inhibition of Class III Alcohol Dehydrogenase 5: Turning Remote Ischemic Conditioning Effective in a Diabetic Stroke Model.

The restoration of cerebral blood flow (CBF) to achieve brain tissue oxygenation (PbtO2) is the primary treatment for ischemic stroke, a significant cause of adult mortality and disability worldwide. Nitric oxide (NO) and its bioactive s-nitrosylated (SNO) reservoirs, such as s-nitrosoglutathione (GSNO), induce hypoxic vasodilation to enhance CBF during ischemia. The endogenous pool of SNOs/GSNO is enhanced via the activation of endothelial NO synthase (eNOS/NOS3) and by the suppression of class III alcohol dehydrogenase 5 (ADH5), also known as GSNO reductase (GSNOR). Remote ischemic conditioning (RIC), which augments NOS3 activity and SNO, is an emerging therapy in acute stroke. However, RIC has so far shown neutral effects in stroke clinical trials. As the majority of stroke patients are presented with endothelial dysfunctions and comorbidities, we tested the hypothesis that NOS3 dysfunction and diabetes will abolish the protective effects of RIC therapy in stroke, and the prior inhibition of GSNOR will turn RIC protective. Our data demonstrate that RIC during thrombotic stroke failed to enhance the CBF and the benefits of thrombolysis in NOS3 mutant (NOS3+/-) mice, a genetic model of NOS3 dysfunction. Interestingly, thrombotic stroke in diabetic mice enhanced the activity of GSNOR as early as 3 h post-stroke without decreasing the plasma nitrite (NO2-). In thrombotic stroke, neither a pharmacological inhibitor of GSNOR (GRI) nor RIC therapy alone was protective in diabetic mice. However, prior treatment with GRI followed by RIC enhanced the CBF and improved recovery. In a reperfused stroke model, the GRI-RIC combination therapy in diabetic mice augmented PbtO2, a translatory signature of successful microvascular reflow. In addition, RIC therapy unexpectedly increased the inflammatory markers at 6 h post-stroke in diabetic stroke that were downregulated in combination with GRI while improving the outcomes. Thus, we conclude that preexisting NOS3 dysfunctions due to comorbidities may neutralize the benefits of RIC in stroke, which can be turned protective in combination with GRI. Our findings may support the future clinical trial of RIC in comorbid stroke. Further studies are warranted to test and develop SNO reservoirs as the blood-associated biomarker to monitor the response and efficacy of RIC therapy in stroke.

Roles and current applications of S-nitrosoglutathione in anti-infective biomaterials.

Bacterial infections can compromise the physical and biological functionalities of humans and pose a huge economical and psychological burden on infected patients. Nitric oxide (NO) is a broad-spectrum antimicrobial agent, whose mechanism of action is not affected by bacterial resistance. S-nitrosoglutathione (GSNO), an endogenous donor and carrier of NO, has gained increasing attention because of its potent antibacterial activity and efficient biocompatibility. Significant breakthroughs have been made in the application of GSNO in biomaterials. This review is based on the existing evidence that comprehensively summarizes the progress of antimicrobial GSNO applications focusing on their anti-infective performance, underlying antibacterial mechanisms, and application in anti-infective biomaterials. We provide an accurate overview of the roles and applications of GSNO in antibacterial biomaterials and shed new light on the avenues for future studies.

Bystander effect in photosensitized prostate cancer cells with a different grade of malignancy: The role of nitric oxide.

Photodynamic therapy (PDT) is a therapeutic modality based on the simultaneous action of three elements: photosensitizer, light and oxygen. This triad generates singlet oxygen and reactive oxygen species that can reduce the mass of a tumor. PDT is also able to stimulate iNOS, the enzyme that generates nitric oxide (NO). The role of NO in PDT-treated cancer cells has been investigated in several studies. They showed that low iNOS/NO levels stimulate signaling pathways that promote tumor survival, while high iNOS/NO levels arrest tumor growth. There is increasing evidence that ROS/RNS control both proliferation and migration of cells in the vicinity of PDT-treated tumor cells (so-called bystander cells). In this work, we addressed the question of how NO, which is generated by weak PDT, affects bystander cells. We used a conditioned medium: medium of PDT-treated tumor cells containing the stressors produced by the cells was added to untreated cells mimicking the neighboring bystander cells to investigate whether the conditioned medium affects cell proliferation. We found that low-level NO in prostate cancer cells affects the bystander tumor cells in a manner that depends on their malignancy grade.

Suppression of VEGFD expression by S-nitrosylation promotes the development of lung adenocarcinoma.

BACKGROUND: Vascular endothelial growth factor D (VEGFD), a member of the VEGF family, is implicated in angiogenesis and lymphangiogenesis, and is deemed to be expressed at a low level in cancers. S-nitrosylation, a NO (nitric oxide)-mediated post-translational modification has a critical role in angiogenesis. Here, we attempt to dissect the role and underlying mechanism of S-nitrosylation-mediated VEGFD suppression in lung adenocarcinoma (LUAD). METHODS: Messenger RNA and protein expression of VEGFD in LUAD were analyzed by TCGA and CPTAC database, respectively, and Assistant for Clinical Bioinformatics was performed for complex analysis. Mouse models with urethane (Ure)-induced LUAD or LUAD xenograft were established to investigate the role of S-nitrosylation in VEGFD expression and of VEGFD mutants in the oncogenesis of LUAD. Molecular, cellular, and biochemical approaches were applied to explore the underlying mechanism of S-nitrosylation-mediated VEGFD suppression. Tube formation and wound healing assays were used to examine the role of VEGFD on the angiogenesis and migration of LUAD cells, and the molecular modeling was applied to predict the protein stability of VEGFD mutant. RESULTS: VEGFD mRNA and protein levels were decreased to a different extent in multiple primary malignancies, especially in LUAD. Low VEGFD protein expression was closely related to the oncogenesis of LUAD and resultant from excessive NO-induced VEGFD S-nitrosylation at Cys277. Moreover, inhibition of S-nitrosoglutathione reductase consistently decreased the VEGFD denitrosylation at Cys277 and consequently promoted angiogenesis of LUAD. Finally, the VEGFDC277S mutant decreased the secretion of mature VEGFD by attenuating the PC7-dependent proteolysis and VEGFDC277S mutant thus reversed the effect of VEGFD on angiogenesis of LUAD. CONCLUSION: Low-expression of VEGFD positively correlates with LUAD development. Aberrant S-nitrosylation of VEGFD negates itself to induce the tumorigenesis of LUAD, whereas normal S-nitrosylation of VEGFD is indispensable for its secretion and repression of angiogenesis of LUAD.

GSNOR deficiency attenuates MPTP-induced neurotoxicity and autophagy by facilitating CDK5 S-nitrosation in a mouse model of Parkinson's disease.

The S-nitrosoglutathione reductase (GSNOR) is a key denitrosating enzyme that regulates protein S-nitrosation, a process which has been found to be involved in the pathogenesis of Parkinson's disease (PD). However, the physiological function of GSNOR in PD remains unknown. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that GSNOR expression was significantly increased and accompanied by autophagy mediated by MPTP-induced cyclin dependent kinase 5 (CDK5), behavioral dyskinesias and dopaminergic neuron loss. Whereas, knockout of GSNOR, or treatment with the GSNOR inhibitor N6022, alleviated MPTP-induced PD-like pathology and neurotoxicity. Mechanistically, deficiency of GSNOR inhibited MPTP-induced CDK5 kinase activity and CDK5-mediated autophagy by increasing S-nitrosation of CDK5 at Cys83. Our study indicated that GSNOR is a key regulator of CDK5 S-nitrosation and is actively involved in CDK5-mediated autophagy induced by MPTP.

Focus on Nitric Oxide Homeostasis: Direct and Indirect Enzymatic Regulation of Protein Denitrosation Reactions in Plants.

Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).