Biotin Switch Assay Research Articles

Abstract 13341: Homoarginine Supplementation Improves Cardiac Function in a Murine Model of Ischaemic Heart Failure

Introduction: Homoarginine (HA) is an emerging biomarker with low levels associated with increased risk of stroke, myocardial infarction and heart failure (HF) in epidemiological studies. Recently, we identified HA as an independent predictor of mortality in patients with HF. We therefore hypothesized that HA supplementation might be beneficial in a murine model of chronic heart failure. Methods: C57BL/6J mice were treated with 14 mg/L HA in drinking water for 4 weeks prior to coronary artery occlusion to induce infarction. Infarct size and HF severity were assessed 6 weeks later by cine-MRI and LV haemodynamics under isoflurane anaesthesia. HA and biopterins were measured using LC-MS/MS and HPLC. Protein S-nitrosylation (SNO) was quantified using biotin switch assay. Results: We first established that HA supplementation significantly increased both plasma and myocardial HA levels in normal C57BL/6J mice (mean±SEM 0.29±0.03 vs. 0.89±0.07 μmol/L and 8.8±1.4 vs. 24.4±3.4 nmol/g protein; n=5-10; P<0.01), without affecting mean arterial pressure (76±1 vs. 73±2 mmHg; n=5-6; P=0.23). Infarct sizes were retrospectively matched between control and HA groups (mean 39±2%) and both developed profound LV remodelling (i.e. hypertrophy and dilatation), but to a similar extent. HA-treated mice had improved LV systolic pressure (83.4±1.3 vs. 87.6±1.3 mmHg), contractility (dP/dtmax 4636±238 vs. 5515±270 mmHg/s), relaxation (dP/dtmin -3183±225 vs. -4029±302 mmHg/s) and β-adrenergic responsiveness (+928±184 vs. +1906±327 mmHg/s; n=21-23; all P<0.05). HA-treated hearts showed an increase in tetrahydrobiopterin levels (1.24±0.34 vs. 3.15±0.41 pmol/mg protein; n=4-6, P<0.01) and elevated global SNO (0.98±0.04 vs. 1.76±0.25; n=7/7; P<0.05), but with no change in nitric oxide synthase protein or activity. Conclusions: HA supplementation improves LV haemodynamic function and contractile reserve in mice with chronic HF, potentially via altered redox regulation or SNO status. Our data suggest HA is more than a simple biomarker, since higher levels directly influence HF pathophysiology. Further studies aimed at clinical translation are merited.

Protein cysteine S-nitrosylation inhibits vesicular uptake of neurotransmitters

Previous studies have shown that nitric oxide can induce cysteine S-nitrosylation of total protein in synaptosomes, suggesting that nitric oxide may contribute to the regulation of synaptic protein function. Vesicular neurotransmitter transporters pack neurotransmitters into synaptic vesicles and play an important role in neurotransmission. In the central nervous system, vesicular monoamine transporter 2 (VMAT2) is responsible for the uptake of monoamines, vesicular acetylcholine transporter (VAChT) is responsible for the uptake of acetylcholine, while vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) are responsible for the uptake of glutamate. The purpose of this study was to investigate the role of cysteine S-nitrosylation in the regulation of these vesicular neurotransmitter transporters. Using the biotin switch assay followed by avidin precipitation and immunoblotting we found that the nitric oxide donor nitrosoglutathione (GSNO) not only increased total cysteine S-nitrosylation, but also increased cysteine S-nitrosylation of VMAT2, VAChT, VGLUT1 and VGLUT2 in the mouse brain. Further, GSNO also decreased the vesicular uptake of [3H]dopamine, [3H]acetylcholine and [3H]glutamate. Our studies suggest that the cysteine S-nitrosylation may play an important role in the regulation of vesicular neurotransmitter transport.

H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells

BackgroundCystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H2S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H2S S-sulfhydration is now proposed as a major mechanism for H2S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H2S and its implication in gluconeogenesis in the liver have been unknown. MethodsGene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. ResultsExogenously applied H2S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H2S-induced gluconeogenesis. H2S had no effect on the expressions of PC mRNA and protein, while H2S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H2S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H2S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. ConclusionsCSE/H2S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. General significanceTissue-specific regulation of CSE/H2S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.

Nitric oxide modulates Lycopersicon esculentum C-repeat binding factor 1 (LeCBF1) transcriptionally as well as post-translationally by nitrosylation

Nitric oxide (NO) production increases in the cold stress. This cold enhanced NO manifests its effect either by regulating the gene expression or by modulating proteins by NO based post-translational modifications (PTMs) including S-nitrosylation. CBF (C-repeat binding factor) dependent cold stress signaling is most studied cold stress-signaling pathway in plants. SNP (sodium nitroprusside, a NO donor) treatment to tomato seedlings showed four fold induction of LeCBF1 (a cold inducible CBF) transcript in cold stress. S-nitrosylation as PTM of CBF has not been analyzed till date. In silico analysis using GPS-SNO 1.0 software predicted Cys 68 as the probable site for nitrosylation in LeCBF1. The 3D structure and motif prediction showed it to be present in the beta hairpin loop and hence available for S-nitrosylation. LeCBF1 was cloned and expressed in Escherichia coli. LeCBF1 accumulated in the inclusion bodies, which were solubilized under denaturing conditions and purified after on column refolding by Ni-NTA His tag affinity chromatography. Purified LeCBF1 resolved as a 34 kDa spot with a slightly basic pI (8.3) on a 2-D gel. MALDI-TOF mass spectrometry identified it as LeCBF1 and western blotting using anti-LeCBF1 antibodies confirmed its purification. Biotin switch assay and neutravidin affinity chromatography showed LeCBF1 to be S-nitrosylated in presence of GSNO (NO donor) as well as endogenously (without donor) in cold stress treated tomato seedlings. Dual regulation of LeCBF1 by NO at both transcriptional as well as post-translational level (by S-nitrosylation) is shown for the first time.

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation—the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus.

S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen–glucose deprivation

Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca2+ dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO–PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen–glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.

Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

Modes of Physiologic H2S Signaling in the Brain and Peripheral Tissues

Hydrogen sulfide (H2S), once associated with rotten eggs and sewers, is now recognized as a gasotransmitter that is synthesized in vivo in a regulated fashion. This ancient gaseous molecule has been retained throughout evolution to perform various roles in different life forms. H2S modulates important signaling functions in diverse cellular processes ranging from regulation of blood pressure to redox homeostasis. One of the modes by which H2S signals is by post-translational modification of reactive cysteine residues in a process designated as sulfhydration, resulting in conversion of the -SH groups of target cysteine residues to -SSH. Using the modified biotin-switch assay and a fluorescent maleimide-based analysis, sulfhydration of several proteins has been detected in various cell types. Aberrant sulfhydration patterns occur in neurodegenerative conditions such as Parkinson's disease. The exact concentration, source of H2S, and conditions under which various stores of H2S are utilized have not been fully elucidated. Currently, available inhibitors of the biosynthetic enzymes of H2S lack sufficient specificity to shed light on detailed mechanisms of H2S action. Probes with a higher sensitivity that can reliably detect cellular and tissue H2S levels are yet to be developed. Availability of advanced probes and biosynthesis inhibitors would help in the measurement of real-time changes of endogenous H2S levels in an in vivo context. The study of the dynamics of sulfhydration and nitrosylation of critical cysteine residues of regulatory proteins involved in physiology and pathophysiology is an area of interest for the future.

Strategies and Tools for Detection of Protein S-Nitrosylation and S-Sulfhydration

H2S interacts with the free thiol group in the active cysteine residues from target proteins and forms a hydropersulfide moiety (-SSH), termed as S-sulfhydration, which is similar to nitric oxide (NO) regulation of protein by S-nitrosylation. Protein S-sulfhydration now is proposed to mediate most of H2S bioactivities in various cellular functions. A number of methods have been developed for detection of S-nitrosylation and S-sulfhydration. Selective recognition of hydropersulfide (SSH) is a main target for the detection of S-sulfhydration. Protein Ssulfhydration can be detected by biotin switch assay, which is developed and further modified based on the detection approaches for protein S-nitrosylation. Here we highlight the different tools for detection of protein S-sulfhydration and S-nitrosylation, and provide strategies for developing new methods to locate the modified cysteine residues.

Protein sulfhydration.

Hydrogen sulfide (H2S) is one of the gasotransmitters that modulates various biological processes and participates in multiple signaling pathways. H2S signals by a process termed sulfhydration. Sulfhydration has recently been recognized as a posttranslational modification similar to nitrosylation. Sulfhydration occurs at reactive cysteine residues in proteins and results in the conversion of an -SH group of cysteine to an -SSH or a persulfide group. Sulfhydration is highly prevalent in vivo, and aberrant sulfhydration patterns have been observed under several pathological conditions ranging from heart disease to neurodegenerative diseases such as Parkinson's disease. The biotin switch assay, originally developed to detect nitrosylation, has been modified to detect sulfhydration. In this chapter, we discuss the physiological roles of sulfhydration and the methodologies used to detect this modification.

Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation.

Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex

Hydrogen sulfide (H₂S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H₂S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H₂S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H₂S in inflammation.

Abstract 20500: Trx1 Promotes Trans-Nitrosylation of Cellular Proteins to Stimulate Autophagy and Cell Survival

Thioredoxin-1 (Trx1) is cardioprotective during oxidative stress, mainly through its antioxidant activity. Trx1 is also S-nitrosylated and, in turn, trans-nitrosylates other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is unknown. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival during stress in vitro. Using biotin-switch assays, we found that wild-type Trx1 (Trx1WT) is S-nitrosylated at baseline, but the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Trx1WT and Trx1C73S do not differ in their redox activity, as determined by Amplex Red assays. Cellular protein S-nitrosylation levels were increased after 4 hours of glucose deprivation (GD), an energy stress condition (1.64±0.27 fold, p<0.05), as determined by biotin switch assays. Overexpression of Trx1WT increased (3.94-fold), whereas knockdown of Trx1 (0.66±0.01 fold, p<0.01) or overexpression of Trx1C73S (0.77±0.02 fold, p<0.01) decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1C73 regulates protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hours of GD (1.42±0.08 fold vs LacZ, p<0.05), as evaluated with propidium iodide assays. Conversely, shTrx1 (2.13±0.05 fold vs control, p<0.01) or Trx1C73S (1.73±0.034 fold vs LacZ, p<0.01) increased cell death during GD. Either knockdown of Trx1 (LC3-II/Tubulin: 0.55 fold vs control) or overexpression of Trx1C73S (vs LacZ: LC3-II/Tubulin, 0.60 fold; autophagosomes, 0.83±0.16-fold, p<0.005; autolysosomes, 0.62±0.13-fold, p<0.005) significantly decreased autophagy during GD. Mechanistically, Trx1 co-immunoprecipitates with Atg7, an E1-like protein which plays a critical role in mediating autophagy. Using mass spectroscopy analyses, we found that SNO-Trx1 can trans-nitrosylate Atg7 in vitro. These results suggest that Trx1 trans-nitrosylates Atg7 during GD. Taken all together, our results indicate that Trx1 promotes trans-nitrosylation of cellular proteins, including Atg7, and autophagy, thereby promoting cell survival during energy stress in CMs.

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

Exogenous H2 S enhances mice gastric smooth muscle tension through S-sulfhydration of KV 4.3, mediating the inhibition of the voltage-dependent potassium current.

Hydrogen sulfide (H2 S) has been shown to have an excitatory effect on gastric motility, but the underlying molecular mechanism is unclear. In this study, we aimed to investigate the possible targets of H2 S and determine how H2 S affects its target proteins during H2 S-induced contraction. Patch-clamp and potentiometric fluorescence dye were utilized to measure the electrophysiological changes. The Biotin-switch assay was utilized to detect the protein S-sulfhydration. The isometric tension measurement was conducted too. Exogenous H2 S enhanced the tonic contraction of gastric antral smooth muscle, and voltage-dependent potassium channel (KV ) blocker and Dithiothreitol (DTT, a reducing agent) abolished the excitatory effect of NaHS. Exogenous H2 S inhibited the fast inactivation component of the voltage-dependent potassium channel current (IKVfast ) in isolated gastric antral smooth muscle cells. H2 S inhibited the KV 4.3 current in H293 cells with heterologous expression of KV 4.3, but did not inhibit the KV 4.1 and KV 4.2 currents, which together contribute greatly to IKVfast . NaHS significantly decreased the membrane potential in cultured gastric smooth muscle cells, but the NaHS-induced depolarization was suppressed by knockdown of KV 4.3 and N-ethylamaleimide (NEM), a free thiol group blocker. In addition, NaHS sulfhydrated KV 4.3 in H293 cells and in gastric smooth muscle tissue. However, this S-sulfhydration was inhibited by NEM and DTT. Meanwhile the NaHS-induced inhibition of IKVfast and KV 4.3 was also blocked by NEM and DTT. These results suggest that exogenous H2 S sulfhydrates KV 4.3 to decrease the membrane potential, thereby enhancing the basal tension of gastric antral smooth muscle.

ENOS-JNK1-AR signaling pathway mediates deltamethrin-induced germ cells apoptosis in testes of adult rats

The purpose of this study is to examine germ cells apoptosis and reduction of spermatogenesis which might be induced by deltamethrin (DM). Furthermore, the study is performed to determine if the apoptosis is mediated by the signaling proteins: eNOS, JNK1 and androgen receptor (AR). Fifty-four male SD rats were divided into nine groups (six rats each): blank control group; corn oil treated group; DM treated group; saline treated group; DM+saline treated group; DM+histamine (eNOS specific agonist) treated group; 50% ethanol treated group; DM+50% ethanol group and DM+quercetagetin (JNK1 specific inhibitor) treated group. The experiment was conducted for 15 days. Apoptosis was evaluated by TUNEL; S-nitrosylation of JNK1 was examined by the biotin switch assay; eNOS expression and Ser650 phosphorylation of AR were assessed by immunoblotting and immunohistochemical analysis, respectively. DM treated group showed notable apoptotic cells and reduced production of sperm, while DM plus histamine group and DM plus quercetagetin group showed remarkably decreased apoptosis and improved production of sperm. Administration of DM inhibited spermatogenesis, the activity of eNOS and S-nitrosylation of JNK1. Meanwhile, phosphorylation of AR was shown to be elevated. Histamine and quercetagetin were also examined to have a further confirmation. It is suggested DM-induced germ cells apoptosis and reduction of sperm production were mediated by eNOS-JNK1-AR signaling pathway.

S-nitroso-proteome in poplar leaves in response to acute ozone stress.

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = −3.6) and caffeic acid O-methyltransferase (−3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.

High fat, high sucrose diet causes cardiac mitochondrial dysfunction due in part to oxidative post-translational modification of mitochondrial complex II

Background: Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. Methods and results: Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. Conclusions: MHD due to consumption of a HFHS “Western” diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

The role of regulatory proteins and S-nitrosylation of endothelial nitric oxide synthase in the human clitoris: implications for female sexual function.

During female sexual arousal, clitoral blood flow is controlled by endothelial nitric oxide synthase (eNOS) and its product, nitric oxide (NO). The mechanisms regulating eNOS activity and NO bioavailability in the clitoris are largely unknown. To identify proteins involved in regulation of eNOS activity within the clitoris and to evaluate the effects of S-nitrosoglutathione reductase (GSNO-R) and eNOS nitrosylation/denitrosylation on clitoral blood flow. Immunohistochemistry for eNOS, caveolin-1 (Cav1), heat shock protein-90 (Hsp90), phosphodiesterase type 5 (PDE5), GSNO-R, and soluble guanylate cyclase (sGC) was performed on human and murine clitoral tissue. Western blot analysis was performed for eNOS, phosphorylated eNOS (phospho-eNOS, Ser1177), Cav1, Hsp90, sGC, PDE5, phosphoinositide 3-kinase (PI3K), Akt (protein kinase B), and GSNO-R on protein from human clitoral tissue. A biotin switch assay was used to analyze the S-nitrosylation of eNOS, nNOS, and GSNO-R. Clitoral blood flow was measured in wild-type and GSNO-R(-/-) mice at baseline and during cavernous nerve electrical stimulation (CNES). Localization of eNOS regulatory proteins and clitoral blood flow. eNOS and GSNO-R co-localized to the vascular endothelium and sinusoids of human clitoral tissue. Immunohistochemistry also localized Cav1 and Hsp90 to the endothelium and PDE5 and sGC to the trabecular smooth muscle. Expression of S-nitrosylated (SNO)-eNOS and SNO-GSNO-R was detected by biotin switch assays. Wild-type control mice exhibited increased clitoral blood flow with CNES whereas GSNO-R(-/-) animals failed to show an increase in blood flow. Several key eNOS regulatory proteins are present in the clitoral tissue in a cellular specific pattern. S-nitrosylation of eNOS may also represent a key regulatory mechanism governing eNOS activation/deactivation since mice deficient in GSNO-R failed to increase clitoral blood flow. Additional studies are necessary to define the role of S-nitrosylation in the genital vascular response and its subsequent impact on female sexual function.

A GAPDH-Mediated Trans-Nitrosylation Pathway Is Required for Feedback Inhibition of Bile Salt Synthesis in Rat Liver

Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in thecontrol of CYP7A1 expression and the involvement in thisprocess of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals. We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers. Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHPup-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2(HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions. In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.