DeepNitro: Prediction of Protein Nitration and Nitrosylation Sites by Deep Learning / 基因组蛋白质组与生物信息学报·英文版

Protein nitration and nitrosylation are essential post-translational modifications (PTMs) involved in many fundamental cellular processes. Recent studies have revealed that excessive levels of nitration and nitrosylation in some critical proteins are linked to numerous chronic diseases. Therefore, the identification of substrates that undergo such modifications in a site-specific manner is an important research topic in the community and will provide candidates for targeted therapy. In this study, we aimed to develop a computational tool for predicting nitration and nitrosylation sites in proteins. We first constructed four types of encoding features, including positional amino acid distributions, sequence contextual dependencies, physicochemical properties, and position-specific scoring features, to represent the modified residues. Based on these encoding features, we established a predictor called DeepNitro using deep learning methods for predicting protein nitration and nitrosylation. Using n-fold cross-validation, our evaluation shows great AUC values for DeepNitro, 0.65 for tyrosine nitration, 0.80 for tryptophan nitration, and 0.70 for cysteine nitrosylation, respectively, demonstrating the robustness and reliability of our tool. Also, when tested in the independent dataset, DeepNitro is substantially superior to other similar tools with a 7%-42% improvement in the prediction performance. Taken together, the application of deep learning method and novel encoding schemes, especially the position-specific scoring feature, greatly improves the accuracy of nitration and nitrosylation site prediction and may facilitate the prediction of other PTM sites. DeepNitro is implemented in JAVA and PHP and is freely available for academic research at http://deepnitro.renlab.org.

Chemical approaches for trapping protein thiols and their oxidative modification / 药学学报

Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.

SNObase, a database for S-nitrosation modification

S-Nitros(yl)ation is a ubiquitous redox-based post-translational modification of protein cysteine thiols by nitric oxide or its derivatives, which transduces the bioactivity of nitric oxide (NO) by regulation of protein conformation, activity, stability, localization and protein-protein interactions. These years, more and more S-nitrosated proteins were identified in physiological and pathological processes and the number is still growing. Here we developed a database named SNObase ( http://www.nitrosation.org ), which collected S-nitrosation targets extracted from literatures up to June 1st, 2012. SNObase contained 2561 instances, and provided information about S-nitrosation targets, sites, biological model, related diseases, trends of S-nitrosation level and effects of S-nitrosation on protein function. With SNObase, we did functional analysis for all the SNO targets: In the gene ontology (GO) biological process category, some processes were discovered to be related to S-nitrosation ("response to drug", "regulation of cell motion") besides the previously reported related processes. In the GO cellular component category, cytosol and mitochondrion were both enriched. From the KEGG pathway enrichment results, we found SNO targets were enriched in different diseases, which suggests possible significant roles of S-nitrosation in the progress of these diseases. This SNObase means to be a database with precise, comprehensive and easily accessible information, an environment to help researchers integrate data with comparison and relevancy analysis between different groups or works, and also an SNO knowledgebase offering feasibility for systemic and global analysis of S-nitrosation in interdisciplinary studies.

Quantitative proteomic analysis of S-nitrosated proteins in diabetic mouse liver with ICAT switch method

In this study we developed a quantitative proteomic method named ICAT switch by introducing isotope-coded affinity tag (ICAT) reagents into the biotin-switch method, and used it to investigate S-nitrosation in the liver of normal control C57BL/6J mice and type 2 diabetic KK-Ay mice. We got fifty-eight S-nitrosated peptides with quantitative information in our research, among which thirty-seven had changed S-nitrosation levels in diabetic mouse liver. The S-nitrosated peptides belonged to forty-eight proteins (twenty-eight were new S-nitrosated proteins), some of which were new targets of S-nitrosation and known to be related with diabetes. S-nitrosation patterns were different between diabetic and normal mice. Gene ontology enrichment results suggested that S-nitrosated proteins are more abundant in amino acid metabolic processes. The network constructed for S-nitrosated proteins by text-mining technology provided clues about the relationship between S-nitrosation and type 2 diabetes. Our work provides a new approach for quantifying S-nitrosated proteins and suggests that the integrative functions of S-nitrosation may take part in pathophysiological processes of type 2 diabetes.

Novos mecanismos pelos quais o exercício físico melhora a resistência à insulina no músculo esquelético: [revisão] / New mechanisms by which physical exercise improves insulin resistance in the skeletal muscle: [revision]

O prejuízo no transporte de glicose estimulada por insulina no músculo constitui um defeito crucial para o estabelecimento da intolerância à glicose e do diabetes tipo 2. Por outro lado, é notório o conhecimento de que tanto o exercício aeróbio agudo quanto o crônico podem ter efeitos benéficos na ação da insulina em estados de resistência à insulina. No entanto, pouco se sabe sobre os efeitos moleculares pós-exercício sobre a sinalização da insulina no músculo esquelético. Assim, esta revisãoapresenta novos entendimentos sobre os mecanismos por meio dos quais o exercício agudo restaura a sensibilidade à insulina, com destaque ao importante papel que proteínas inflamatórias e a S-nitrosação possuem sobre a regulação de proteínas da via de sinalização da insulina no músculo esquelético.

Insulin resistance of skeletal muscle glucose transport is a key-defect for the development of impaired glucose tolerance and type 2 diabetes. However, it is known that both an acute bout of exercise and chronic endurance exercise training can bring beneficial effects on insulin action in insulin-resistant states. However, little is currently known about the molecular effects of acute exercise on muscle insulin signaling in the post-exercise state in insulin-resistant organisms. This review provides new insight into the mechanism through which acute exercise restores insulin sensitivity, highlighting an important role for inflammatory proteins and S-nitrosation in the regulation of insulin signaling proteins in skeletal muscle.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Effects of Radix Ginseng and Radix Ophiopogonis extract (SMF) on protein S-nitrosylation in ischemic myocardial tissue / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Radix Ginseng and Radix Ophiopogonis extract (SMF) on protein S-nitrosylation in rats myocardial with ischemia/reperfusion injury (MI/RI).</p><p><b>METHOD</b>Myocardial ischemia/reperfusion in rats were produced by occlusion of the left anterior descending coronary artery. To study the cardioprotective effects of SMF on the acute MI/RI rats, the serum levels of creatine kinase (CK), lactate dehydrogenase (LDH), and nitric oxide (NO) were determined. The change of the expression of endothelial nitric oxide synthase (eNOS) was detected by Western blot. The content of related S-nitrosylation proteins in myocardial tissue was measured by Biotin-Switch method.</p><p><b>RESULT</b>SMF significantly decreased the serum levels of CK and LDH as well as increased the serum levels of NO and the expression of eNOS in myocardial tissue. The contents of S-nitrosylation proteins were significantly increased from (4.42 +/- 0.60) micromol x g(-1) to (8.78 +/- 1.37) micromol x g(-1). The molecular weight of the majority S-nitrosylation proteins were in the range of 90 x 10(3)-117 x 10(3).</p><p><b>CONCLUSION</b>Increased expression of eNOS and NO induced by SMF may activate S-nitrosylation of many proteins in rat hearts. The change of the activities or functions of those proteins by S-nitrosylation may be an important mechanism for myocardial protective effects of SMF.</p>

Validation of HPLC, DPPH• and nitrosation methods for mesalamine determination in pharmaceutical dosage forms

Mesalamine (5-aminosalicylic acid, 5-ASA) is used because of its local effects in the treatment of inflammatory bowel disease. Therefore, the aims of this work were to compare and validate three analytical methods for the quality control of commercial coated tablets containing 5-ASA: high performance liquid chromatography (HPLC), 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•) and nitrosation. The parameters linearity, precision and accuracy were studied in this work. HPLC with ultraviolet detection at 254 nm was carried out with a C18 column and a mobile phase constituted of 30 mmol/L monobasic phosphate buffer (pH 7.0) and methanol (70:30; v/v), with 25 percent tetrabutylammonium hydrogen sulphate. The DPPH• method was performed at 517 nm and using 100 mmol/L acetate buffer, pH 5.5, ethanol and 250 æmol/L ethanolic solution of DPPH•. The nitrosation method was accomplished by using a platinum electrode and standard 0.1 mol/L sodium nitrite as titrant solution. Repeatability (intra-day) and intermediate precision (inter-day), expressed as RSD, were lower than 3 percent. The experimental recoveries were between 72.5 and 99.9 percent. Statistical analysis by one-way ANOVA, followed by the multiple comparison test of Bonferroni showed no significant difference among the three methods. All proposed methods can be used for the reliable quantitation of 5-ASA in pharmaceutical dosage forms.

Mesalazina (ácido 5-aminosalicílico, 5-ASA) é utilizado devido seu efeito local no tratamento de doença inflamatória intestinal. Assim, o objetivo deste trabalho foi comparar e validar três métodos analíticos para o controle de qualidade de comprimidos comerciais revestidos contendo 5-ASA: cromatografia líquida de alta eficiência (CLAE), radical 1,1-difenil-2-picril-hidrazil (DPPH•) e nitrosação. Os parâmetros linearidade, precisão e exatidão foram estudados neste trabalho. CLAE com detecção ultravioleta em 254 nm foi realizada utilizando coluna C18 e a eluição em fase móvel constituída de tampão fosfato monobásico 30 mmol/L (pH 7,0) e metanol (70:30; v/v), com 25 por cento de sulfato hidrogênio de tetrabutilamônio. Para o método de DPPH• utilizou-se tampão acetato 100 mmol/L, pH 5,5, álcool etílico e 250 æmol/L solução etanólica de DPPH• a 517 nm. Para o método de nitrosação utilizou-se um eletrodo de platina e um padrão de nitrito de sódio 0.1 mol/L como solução titulante. Repetibilidade (intra-dia) e precisão intermediária (inter-dia), expressado como DPR, foi menor que 3 por cento. A recuperação experimental foi entre 72,5 e 99,9 por cento. Análise estatística por "one-way" ANOVA, seguida de comparação múltipla do teste de Bonferroni, não mostrou significância entre os três métodos. Os métodos propostos podem ser usados para análise quantitativa do5-ASA em formas farmacêuticas.

Endothelin-1 induced alterations in oxidative-nitrosative stress markers

The aim of this preliminary study is to investigate the effects of exogenous Endothelin-1 [ET-1] on systolic blood pressure and heart rate as well as on plasma nitric oxide metabolites, malondialdehyde, copper and zinc concentrations and red cell superoxide dismutase and catalase activities. Thirty Wistar-Albino male rats, 8-10 weeks old, with a mean body weight of 285 gm were used in the study. Daily systolic blood pressures were measured by tail plethysmography. Following exogenous administration of ET-1 [1 nmol/kg] systolic arterial blood pressures were recorded and blood samples of control and experimental groups were drawn. Nitric oxide metabolites [nitrite, nitrate], malondialdehyde, copper, zinc concentrations in plasma, superoxide dismutase and catalase activities and copper, zinc concentrations in red cell were determined both in control and experimental groups. All laboratory procedures were performed at the Department of Pathophysiology, School of Medicine, Ankara University, Ankara, Turkey in 2003. There were statistically significant increases in plasma nitrate, red cell superoxide dismutase activity, systolic arterial blood pressure and statistically significant decreases in red cell catalase activity, plasma copper, red cell zinc concentrations in experimental group due to exogenous ET-1 administration compared to controls. There appears an important interaction between exogenous ET-1 and oxidative-nitrosative stress markers which may affect the progression of hypertension

Effect of Dietary Factors in the Etiology of Stomach Cancer / 한국역학회지

BACKGROUND: Stomach cancer is the most malignant neoplasm among Koreans. There ane a number of epidemiological studies on dietary factors of stomach cancer in many countries. However, analytical studies on Korean dietary factors are very scarce. SUBJECTS AND METHODS: A case-control study was conducted at the Korea Cancer Center Hospital in Seoul between April and September in 1996. One hundred twenty-six stomach cancer patients confirmed by the histological diagnosis were compared with 234 control subjects matched by age, sex, and admission date. A food frequency questionnaire asking the consumption frequency of 85 selected food items was used to gather the information from all subjects via a face-to-face interview. Multiple logistic regression models were used to estimate relative risks when controlling simultaneously for covariates. RESULTS: An increased risk of stomach cancer was noted among those with low economic status, fast eating rate, high eating out, hot-temperature soup preference, salt preference, cucumber Kimchi intake, use of pickled fish in Kimchi. Intake of garlic, green onion, tofu, mung bean pancake, acorn-starch paste, starch vermicelli with mixed vegetables, total fruits, citrus fruits, cabbage, green peppers, spinach, mushrooms and total meat appeared to be protective. Stomach cancer risk was not associated with intake of rice, dairy product, fishes, condiments, coffee, tea, and the cooking methods. These data suggested that the high intake of salt and smoked or pickled food may be associated with a hig risk of stomach cancer, and this association could be due to a intragastric formation of nitrosamines. The negative association with fruits and some vegetable consumption may be due to the inhibition of nitrosation process. CONCLUSION: Our findings indicated that dietary factors contributed to stomach cancer occurrence in Korea, and this may offer clues for further ethnical and prevention research.

Presencia de actividad mutagénica en frijoles (Phaseolus vulgaris L) sin nitrosar / Mutagenic activity of beans (Phaseolus vulgaris) without nitrosation

Extractos acuosos de trece muestras de frijoles (Phaseolus vulgaris L) adquiridos en el mercado local fueron analizados por el método de Ames utilizando la cepa Salmonella typhimurium LT2 TA102. Cinco de las muestras mostraron actividad mutagénica (tasa de reversión mayor a 1,5). A diferencia de estudios anteriores, no se observó efecto sinérgico entre la sal y la nitrosación, individual o conjuntamente, sobre la actividad mutagénica detectada