Genetic Variability Impacts Genotoxic and Transcriptome Responses in the Human Colon after the Consumption of Processed Red Meat Products and Those with Added Phytochemical Extracts.

The PHYTOME study investigated the effect of consuming processed meat products on outcomes related to colorectal cancer risk without testing the impact of genetic variability on these responses. This research aims to elucidate the genetic impact on apparent total N-nitroso compound (ATNC) excretion, colonic DNA adduct formation, ex vivo-induced DNA damage, and gene expression changes in colon biopsies of healthy participants. Through a systematic literature review, candidate polymorphisms were selected and then detected using TaqMan and PCR analysis. The effect of genotype on study outcomes was determined via a linear mixed model and analysis of variance. Machine learning was used to evaluate relative allele importance concerning genotoxic responses, which established a ranking of the most protective alleles and a combination of genotypes (gene scores). Participants were grouped by GSTM1 genotype and differentially expressed genes (DEGs), and overrepresented biological pathways were compared between groups. Stratifying participants by ten relevant genes revealed significant variations in outcome responses. After consumption of processed red meat, variations in NQO1 and COMT impacted responses in ATNC levels (µmol/L) (+9.56 for wildtype vs. heterozygous) and DNA adduct levels (pg/µg DNA) (+1.26 for variant vs. wildtype and +0.43 for variant vs. heterozygous), respectively. After phytochemicals were added to the meat, GSTM1 variation impacted changes in DNA adduct levels (-6.12 for deletion vs. wildtype). The gene scores correlated with these responses and DEGs were identified by GSTM1 genotype. The altered pathways specific to the GSTM1 wildtype group included 'metabolism', 'cell cycle', 'vitamin D receptor', and 'metabolism of water-soluble vitamins and co-factors'. Genotype impacted both the potential genotoxicity of processed red meat and the efficacy of protective phytochemical extracts.

DNA Damage and Oxidative Stress of Tobacco Smoke Condensate in Human Bladder Epithelial Cells.

Smoking is a major risk factor for bladder cancer (BC), with up to 50% of BC cases being attributed to smoking. There are 70 known carcinogens in tobacco smoke; however, the principal chemicals responsible for BC remain uncertain. The aromatic amines 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are implicated in BC pathogenesis of smokers on the basis of the elevated BC risk in factory workers exposed to these chemicals. However, 4-ABP and 2-NA only occur at several nanograms per cigarette and may be insufficient to induce BC. In contrast, other genotoxicants, including acrolein, occur at 1000-fold or higher levels in tobacco smoke. There is limited data on the toxicological effects of tobacco smoke in human bladder cells. We have assessed the cytotoxicity, oxidative stress, and DNA damage of tobacco smoke condensate (TSC) in human RT4 bladder cells. TSC was fractionated by liquid-liquid extraction into an acid-neutral fraction (NF), containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, phenols, and aldehydes, and a basic fraction (BF) containing aromatic amines, heterocyclic aromatic amines, and N-nitroso compounds. The TSC and NF induced a time- and concentration-dependent cytotoxicity associated with oxidative stress, lipid peroxide formation, glutathione (GSH) depletion, and apurinic/apyrimidinic (AP) site formation, while the BF showed weak effects. LC/MS-based metabolomic approaches showed that TSC and NF altered GSH biosynthesis pathways and induced more than 40 GSH conjugates. GSH conjugates of several hydroquinones were among the most abundant conjugates. RT4 cell treatment with synthetic hydroquinones and cresol mixtures at levels present in tobacco smoke accounted for most of the TSC-induced cytotoxicity and the AP sites formed. GSH conjugates of acrolein, methyl vinyl ketone, and crotonaldehyde levels also increased owing to TSC-induced oxidative stress. Thus, TSC is a potent toxicant and DNA-damaging agent, inducing deleterious effects in human bladder cells at concentrations of <1% of a cigarette in cell culture media.

Interaction between Melatonin and NO: Action Mechanisms, Main Targets, and Putative Roles of the Emerging Molecule NOmela.

Melatonin (MEL), a ubiquitous indolamine molecule, has gained interest in the last few decades due to its regulatory role in plant metabolism. Likewise, nitric oxide (NO), a gasotransmitter, can also affect plant molecular pathways due to its function as a signaling molecule. Both MEL and NO can interact at multiple levels under abiotic stress, starting with their own biosynthetic pathways and inducing a particular signaling response in plants. Moreover, their interaction can result in the formation of NOmela, a very recently discovered nitrosated form of MEL with promising roles in plant physiology. This review summarizes the role of NO and MEL molecules during plant development and fruit ripening, as well as their interactions. Due to the impact of climate-change-related abiotic stresses on agriculture, this review also focuses on the role of these molecules in mediating abiotic stress tolerance and the main mechanisms by which they operate, from the upregulation of the entire antioxidant defense system to the post-translational modifications (PTMs) of important molecules. Their individual interaction and crosstalk with phytohormones and H2S are also discussed. Finally, we introduce and summarize the little information available about NOmela, an emerging and still very unknown molecule, but that seems to have a stronger potential than MEL and NO separately in mediating plant stress response.

Salivary nitrate/nitrite and acetaldehyde in humans: potential combination effects in the upper gastrointestinal tract and possible consequences for the in vivo formation of N-nitroso compounds-a hypothesis.

Subsequent to the dietary uptake of nitrate/nitrite in combination with acetaldehyde/ethanol, combination effects resulting from the sustained endogenous exposure to nitrite and acetaldehyde may be expected. This may imply locoregional effects in the upper gastrointestinal tract as well as systemic effects, such as a potential influence on endogenous formation of N-nitroso compounds (NOC). Salivary concentrations of the individual components nitrate and nitrite and acetaldehyde are known to rise after ingestion, absorption and systemic distribution, thereby reflecting their respective plasma kinetics and parallel secretion through the salivary glands as well as the microbial/enzymatic metabolism in the oral cavity. Salivary excretion may also occur with certain drug molecules and food constituents and their metabolites. Therefore, putative combination effects in the oral cavity and the upper digestive tract may occur, but this has remained largely unexplored up to now. In this Guest Editorial, published evidence on exposure levels and biokinetics of nitrate/nitrite/NOx, NOC and acetaldehyde in the organism is reviewed and knowledge gaps concerning combination effects are identified. Research is suggested to be initiated to study the related unresolved issues.

Risk assessment for nitrosated pharmaceuticals: A future perspective in drug development.

Since June 2018, thousands of drug products from around the world had to be recalled due to the unexpected presence of nitrosamines (NAs). Starting with the pharmaceutical group of sartans, antidiabetic drugs, antihistamines, and antibiotics also became the subject of investigation. The occurrence of NAs has shown that pharmaceutical companies and regulatory agencies did not focus on these substances in the past during drug development. In this study, we incorporated a nitrosation assay procedure into high-resolution supercritical fluid chromatography (SFC)-mass spectrometry screening to test the potential of direct nitrosation of active pharmaceutical ingredients (APIs). The forced degradation study was performed with a four-fold molar excess of sodium nitrite, relative to the drug substance, at pH 3-4 for 4 h at 37°C. Chromatographic separation was performed on a porous graphitic carbon column by SFC. The mass analysis then focused on direct N-nitrosation or N-nitroso compounds (NOCs) formed after dealkylation. Substances (n = 67) from various pharmaceutical classes were evaluated and 49.3% of them formed NOCs, of which 21.2% have not yet been reported in the literature. In addition, for two APIs, which are known to form an unidentified NOC, the structure could be identified. A few substances also showed multiple NOCs and even N,N'-dinitroso-species. As NAs are carcinogens, they have to be eliminated or at least limited to prevent cancer in patients, who rely on these drugs. This study contributes a procedure that can be implemented in preapproval drug development and postapproval risk assessment to prevent unexpected findings in the future.

Atrial nitroso-redox balance and refractoriness following on-pump cardiac surgery: a randomized trial of atorvastatin.

AIMS: Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial. METHODS AND RESULTS: We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71-76%, n = 46 paired samples, P < 0.0001) and a reduction in atrial tetrahydrobiopterin (BH4) (34% CI 33-35%, n = 36 paired samples, P < 0.01), and in GCH-1 (56% CI 55-58%, n = 26 paired samples, P < 0.001) and NOS activity (58% CI 52-67%, n = 20 paired samples, P < 0.001). Perioperative atorvastatin treatment prevented the effect of CPB and reperfusion on all parameters but had no significant effect on the postoperative right AERP, troponin release, or NT-proBNP after cardiac surgery. CONCLUSION: Perioperative statin therapy prevents post-reperfusion atrial nitroso-redox imbalance in patients undergoing on-pump cardiac surgery but has no significant impact on postoperative atrial refractoriness, perioperative myocardial injury, or markers of postoperative LV function. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT01780740.

Expressed Soybean Leghemoglobin: Effect on Escherichia coli at Oxidative and Nitrosative Stress.

Leghemoglobin (Lb) is an oxygen-binding plant hemoglobin of legume nodules, which participates in the symbiotic nitrogen fixation process. Another way to obtain Lb is its expression in bacteria, yeasts, or other organisms. This is promising for both obtaining Lb in the necessary quantity and scrutinizing it in model systems, e.g., its interaction with reactive oxygen (ROS) and nitrogen (RNS) species. The main goal of the work was to study how Lb expression affected the ability of Escherichia coli cells to tolerate oxidative and nitrosative stress. The bacterium E. coli with the embedded gene of soybean leghemoglobin a contains this protein in an active oxygenated state. The interaction of the expressed Lb with oxidative and nitrosative stress inducers (nitrosoglutathione, tert-butyl hydroperoxide, and benzylviologen) was studied by enzymatic methods and spectrophotometry. Lb formed NO complexes with heme-nitrosylLb or nonheme iron-dinitrosyl iron complexes (DNICs). The formation of Lb-bound DNICs was also detected by low-temperature electron paramagnetic resonance spectroscopy. Lb displayed peroxidase activity and catalyzed the reduction of organic peroxides. Despite this, E. coli-synthesized Lb were more sensitive to stress inducers. This might be due to the energy demand required by the Lb synthesis, as an alien protein consumes bacterial resources and thereby decreases adaptive potential of E. coli.

Replacement of Nitrite in Meat Products by Natural Bioactive Compounds Results in Reduced Exposure to N-Nitroso Compounds: The PHYTOME Project.

SCOPE: It has been proposed that endogenously form N-nitroso compounds (NOCs) are partly responsible for the link between red meat consumption and colorectal cancer (CRC) risk. As nitrite has been indicated as critical factor in the formation of NOCs, the impact of replacing the additive sodium nitrite (E250) by botanical extracts in the PHYTOME project is evaluated. METHOD AND RESULTS: A human dietary intervention study is conducted in which healthy subjects consume 300 g of meat for 2 weeks, in subsequent order: conventional processed red meat, white meat, and processed red meat with standard or reduced levels of nitrite and added phytochemicals. Consumption of red meat products enriched with phytochemicals leads to a significant reduction in the faecal excretion of NOCs, as compared to traditionally processed red meat products. Gene expression changes identify cell proliferation as main affects molecular mechanism. High nitrate levels in drinking water in combination with processed red meat intake further stimulates NOC formation, an effect that could be mitigated by replacement of E250 by natural plant extracts. CONCLUSION: These findings suggest that addition of natural extracts to conventionally processed red meat products may help to reduce CRC risk, which is mechanistically support by gene expression analyses.

S-nitrosylation-mediated coupling of G-protein alpha-2 with CXCR5 induces Hippo/YAP-dependent diabetes-accelerated atherosclerosis.

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis.

Determination of the N-Nitroso Compounds in Mouse Following RDX Exposure.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, commonly called RDX, is an important explosive, which is widely used in military and civic activities. As it is used, RDX is widely found in many locations and caused soil and water contamination. Many studies show that RDX is toxic to many organisms, including plants, animals, and microbes. RDX causes genetic toxicity and neurotoxicity as well as potential carcinogenesis. Even it is worse that RDX can be biotransformed into other N-nitroso derivatives, such as MNX, DNX, and TNX; these derivatives can be found in both naturally in RDX-contaminated soil and also in the animal GI tracks. To study the potential effect of RDX and its N-nitroso derivatives, this chapter presents a step-by-step method for detect RDX and its N-nitroso derivatives in animal stomach and GI tracts followed RDX exposure by gas chromatography with electron capture detector (GC/ECD). This method can also be used to detect RDX and its N-nitroso derivatives in other tissues and in other animals and plants.

Nitric oxide down-regulates voltage-gated Na+ channel in cardiomyocytes possibly through S-nitrosylation-mediated signaling.

Nitric oxide (NO) is produced from endothelial cells and cardiomyocytes composing the myocardium and benefits cardiac function through both vascular-dependent and-independent effects. This study was purposed to investigate the possible adverse effect of NO focusing on the voltage-gated Na+ channel in cardiomyocytes. We carried out patch-clamp experiments on rat neonatal cardiomyocytes demonstrating that NOC-18, an NO donor, significantly reduced Na+ channel current in a dose-dependent manner by a long-term application for 24 h, accompanied by a reduction of Nav1.5-mRNA and the protein, and an increase of a transcription factor forkhead box protein O1 (FOXO1) in the nucleus. The effect of NOC-18 on the Na+ channel was blocked by an inhibitor of thiol oxidation N-ethylmaleimide, a disulfide reducing agent disulfide 1,4-Dithioerythritol, or a FOXO1 activator paclitaxel, suggesting that NO is a negative regulator of the voltage-gated Na+ channel through thiols in regulatory protein(s) for the channel transcription.

Red light stimulates vasodilation through extracellular vesicle trafficking.

Red light (670 nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670 nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670 nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.

N-Nitrosomelatonin, an efficient nitric oxide donor and transporter in Arabidopsis seedlings.

Nitric oxide (NO) produced in plant cells has the unique ability to interact with various other biomolecules, thereby facilitating its own as well as their signaling and associated actions at their sites of biosynthesis and at other sites via transcellular long distance transport of the molecular complexes. Melatonin (Mel) is one such biomolecule produced in plant cells which has fascinated plant biologists with regard to its molecular crosstalk with other molecules to serve its roles as a growth regulator. Present work reports the synthesis of N-nitrosomelatonin (NOMela) and its preferential uptake by Arabidopsis seedlings roots and long distance transport to the leaves through vascular strands. Equimolar (250 µM) concentrations of NOMela and S-nitrosoglutathione (GSNO) in aqueous solutions bring about 52.8% more release of NO from NOMela than from GSNO. Following confocal laser scanning microscopic (CLSM) imaging, Pearson's correlation coefficient analysis of the Scatter gram of endogenously taken up NOMela demonstrates significant NO signal in roots emanating from mitochondria. NOMela (250 µM) taken up by Arabidopsis seedling roots also proved more efficient as a NO transporter from primary root to leaves than 250 µM of GSNO. These novel observations on NOMela thus hold promise to decipher its crucial role as a NO carrier and reservoir in plant cells, and also as a facilitator of melatonin action in plant development.

DeepCys: Structure-based multiple cysteine function prediction method trained on deep neural network: Case study on domains of unknown functions belonging to COX2 domains.

Cysteine (Cys) is the most reactive amino acid participating in a wide range of biological functions. In-silico predictions complement the experiments to meet the need of functional characterization. Multiple Cys function prediction algorithm is scarce, in contrast to specific function prediction algorithms. Here we present a deep neural network-based multiple Cys function prediction, available on web-server (DeepCys) (https://deepcys.herokuapp.com/). DeepCys model was trained and tested on two independent datasets curated from protein crystal structures. This prediction method requires three inputs, namely, PDB identifier (ID), chain ID and residue ID for a given Cys and outputs the probabilities of four cysteine functions, namely, disulphide, metal-binding, thioether and sulphenylation and predicts the most probable Cys function. The algorithm exploits the local and global protein properties, like, sequence and secondary structure motifs, buried fractions, microenvironments and protein/enzyme class. DeepCys outperformed most of the multiple and specific Cys function algorithms. This method can predict maximum number of cysteine functions. Moreover, for the first time, explicitly predicts thioether function. This tool was used to elucidate the cysteine functions on domains of unknown functions belonging to cytochrome C oxidase subunit-II like transmembrane domains. Apart from the web-server, a standalone program is also available on GitHub (https://github.com/vam-sin/deepcys).

Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe.

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.

iCysMod: an integrative database for protein cysteine modifications in eukaryotes.

As important post-translational modifications, protein cysteine modifications (PCMs) occurring at cysteine thiol group play critical roles in the regulation of various biological processes in eukaryotes. Due to the rapid advancement of high-throughput proteomics technologies, a large number of PCM events have been identified but remain to be curated. Thus, an integrated resource of eukaryotic PCMs will be useful for the research community. In this work, we developed an integrative database for protein cysteine modifications in eukaryotes (iCysMod), which curated and hosted 108 030 PCM events for 85 747 experimentally identified sites on 31 483 proteins from 48 eukaryotes for 8 types of PCMs, including oxidation, S-nitrosylation (-SNO), S-glutathionylation (-SSG), disulfide formation (-SSR), S-sulfhydration (-SSH), S-sulfenylation (-SOH), S-sulfinylation (-SO2H) and S-palmitoylation (-S-palm). Then, browse and search options were provided for accessing the dataset, while various detailed information about the PCM events was well organized for visualization. With human dataset in iCysMod, the sequence features around the cysteine modification sites for each PCM type were analyzed, and the results indicated that various types of PCMs presented distinct sequence recognition preferences. Moreover, different PCMs can crosstalk with each other to synergistically orchestrate specific biological processes, and 37 841 PCM events involved in 119 types of PCM co-occurrences at the same cysteine residues were finally obtained. Taken together, we anticipate that the database of iCysMod would provide a useful resource for eukaryotic PCMs to facilitate related researches, while the online service is freely available at http://icysmod.omicsbio.info.

Mind the GAP: Purification and characterization of urea resistant GAPDH during extreme dehydration.

The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a Km value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I50 of 7.3 M, while the I50 from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.

Nitrite Reductase 1 Is a Target of Nitric Oxide-Mediated Post-Translational Modifications and Controls Nitrogen Flux and Growth in Arabidopsis.

Plant growth is the result of the coordinated photosynthesis-mediated assimilation of oxidized forms of C, N and S. Nitrate is the predominant N source in soils and its reductive assimilation requires the successive activities of soluble cytosolic NADH-nitrate reductases (NR) and plastid stroma ferredoxin-nitrite reductases (NiR) allowing the conversion of nitrate to nitrite and then to ammonium. However, nitrite, instead of being reduced to ammonium in plastids, can be reduced to nitric oxide (NO) in mitochondria, through a process that is relevant under hypoxic conditions, or in the cytoplasm, through a side-reaction catalyzed by NRs. We use a loss-of-function approach, based on CRISPR/Cas9-mediated genetic edition, and gain-of-function, using transgenic overexpressing HA-tagged Arabidopsis NiR1 to characterize the role of this enzyme in controlling plant growth, and to propose that the NO-related post-translational modifications, by S-nitrosylation of key C residues, might inactivate NiR1 under stress conditions. NiR1 seems to be a key target in regulating nitrogen assimilation and NO homeostasis, being relevant to the control of both plant growth and performance under stress conditions. Because most higher plants including crops have a single NiR, the modulation of its function might represent a relevant target for agrobiotechnological purposes.

The nitrosoamphetamine metabolite is accommodated in the active site of human hemoglobin: Spectroscopy and crystal structure.

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. We report the 2.15 Å resolution X-ray crystal structure of the AmphNO adduct of adult human hemoglobin as the Hb [α-FeIII(H2O)][ß-FeII(AmphNO)] derivative. We show that the binding of AmphNO to the ß subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV-vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs.

S-Nitrosylation at the active site decreases the ubiquitin-conjugating activity of ubiquitin-conjugating enzyme E2 D1 (UBE2D1), an ERAD-associated protein.

S-Nitrosylation of protein cysteine thiol is a post-translational modification mediated by nitric oxide (NO). The overproduction of NO causes nitrosative stress, which is known to induce endoplasmic reticulum (ER) stress. We previously reported that S-nitrosylation of protein disulfide isomerase (PDI) and the ER stress sensor inositol-requiring enzyme 1 (IRE1) decreases their enzymatic activities. However, it remains unclear whether nitrosative stress affects ER-associated degradation (ERAD), a separate ER stress regulatory system responsible for the degradation of substrates via the ubiquitin-proteasomal pathway. In the present study, we found that the ubiquitination of a known ERAD substrate, serine/threonine-protein kinase 1 (SGK1), is attenuated by nitrosative stress. C-terminus of Hsc70-interacting protein (CHIP) together with ubiquitin-conjugating enzyme E2 D1 (UBE2D1) are involved in this modification. We detected that UBE2D1 is S-nitrosylated at its active site, Cys85 by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, in vitro and cell-based experiments revealed that S-nitrosylated UBE2D1 has decreased ubiquitin-conjugating activity. Our results suggested that nitrosative stress interferes with ERAD, leading to prolongation of ER stress by co-disruption of various pathways, including the molecular chaperone and ER stress sensor pathways. Given that nitrosative stress and ER stress are upregulated in the brains of patient with Parkinson's disease (PD) and of those with Alzheimer's disease (AD), our findings may provide further insights into the pathogenesis of these neurodegenerative disorders.