Protein Nitration Research Articles

Molecular and biochemical biomarkers in the American oyster Crassostrea virginica exposed to herbicide Roundup® at high temperature.

Aquatic organisms are frequently exposed to various environmental stressors. Thus, the effects of high temperatures and herbicides on aquatic organisms are a major subject of interest. In this study, we studied the effects of short-term exposure (1week) to Roundup®, a glyphosate-based herbicide (concentrations: 0.5 and 5µg/L), on the morphology of gills, digestive glands, and connective tissues, and the expression of heat shock protein-70 (HSP70, a chaperone protein), cytochrome P450 (CYP450, a biomarker of environmental contaminants), dinitrophenyl protein (DNP, a biomarker of protein oxidation), nitrotyrosine protein (NTP, a biomarker of protein nitration), antioxidant enzymes such as superoxidase dismutase (SOD) and catalase (CAT) in tissues of American oyster, Crassostrea virginica (Gmelin, 1791) maintained at high temperature (30°C). Histological analyses showed an increase in mucous production in the gills and digestive glands, and in hemocyte aggregation in the connective tissues as well as a structural change of lumen in the digestive glands of oysters exposed to Roundup. Immunohistochemical and quantitative RT-PCR analyses showed significant (P < 0.05) increases in HSP70, CYP450, DNP, NTP, CAT, and SOD mRNA and protein expressions in the tissues of oysters exposed to Roundup. Taken together, these results suggest that exposure to Roundup at high temperature induces overproduction of reactive oxygen species/reactive nitrogen species which in turn leads to altered prooxidant-antioxidant activity in oyster tissues. Moreover, our results provide new information on protein oxidation/nitration and antioxidant-dependent mechanisms for HSP70 and CYP450 regulations in oysters exposed to Roundup at high temperature.

Roundup® disrupts tissue architecture, attenuates Na+/K+-ATPase expression, and induces protein oxidation/nitration, cellular apoptosis, and antioxidant enzyme expressions in the gills of goldfish, Carassius auratus

Extensive agricultural activities to feed the growing population are one major driving force behind aquatic pollution. Different types of pesticides are used in farmlands to increase crop production and wash up into water bodies. Glyphosate-based herbicide Roundup® is one of the most used pesticides in the United States; however, its effects on teleost species are still poorly understood. This study focused on the effects of environmentally relevant concentrations of Roundup exposure (low- and high-dose: 0.5 and 5 μg/L for 2-week) on Na+/K+-ATPase (NKA, a biomarker for sodium‑potassium ion pump efficacy), cytochrome P450-1A (CYP1A, a monooxygenase enzyme), 2,4-dinitrophenyl protein (DNP, a biomarker for protein oxidation), 3-nitrotyrosine protein (NTP, a biomarker for protein nitration), superoxidase dismutase (SOD, an antioxidant enzyme), catalase (CAT, an antioxidant enzyme) expressions, and cellular apoptosis in the gills of goldfish. Histopathological and in situ TUNEL analyses showed widespread tissue damage, including lamellar fusion, loss of gill architecture, club shape of primary lamellae, mucous formation, and distortion in the epithelium layer, as well as apoptotic nuclei in gills. Immunohistochemical and qRT-PCR analyses provided insights into the expressions of molecular indicators in gills. Fish exposed to Roundup exhibited a significant (P < 0.05) downregulation of NKA expression in gills. Additionally, we observed upregulation of CYP1A, DNP, NTP, SOD, and CAT expressions in the gills of goldfish. Overall, our results suggest that exposure to Roundup causes disruption of gill architecture, induces protein oxidation/nitration and cellular apoptosis, and alters prooxidant-antioxidant homeostasis in tissues, which may lead to reduced fitness and survivability of teleost species.

Pepper Fruit Extracts Show Anti-Proliferative Activity against Tumor Cells Altering Their NADPH-Generating Dehydrogenase and Catalase Profiles.

Cancer is considered one of the main causes of human death worldwide, being characterized by an alteration of the oxidative metabolism. Many natural compounds from plant origin with anti-tumor attributes have been described. Among them, capsaicin, which is the molecule responsible for the pungency in hot pepper fruits, has been reported to show antioxidant, anti-inflammatory, and analgesic activities, as well as anti-proliferative properties against cancer. Thus, in this work, the potential anti-proliferative activity of pepper (Capsicum annuum L.) fruits from diverse varieties with different capsaicin contents (California < Piquillo < Padrón < Alegría riojana) against several tumor cell lines (lung, melanoma, hepatoma, colon, breast, pancreas, and prostate) has been investigated. The results showed that the capsaicin content in pepper fruits did not correspond with their anti-proliferative activity against tumor cell lines. By contrast, the greatest activity was promoted by the pepper tissues which contained the lowest capsaicin amount. This indicates that other compounds different from capsaicin have this anti-tumor potentiality in pepper fruits. Based on this, green fruits from the Alegría riojana variety, which has negligible capsaicin levels, was used to study the effect on the oxidative and redox metabolism of tumor cell lines from liver (Hep-G2) and pancreas (MIA PaCa-2). Different parameters from both lines treated with crude pepper fruit extracts were determined including protein nitration and protein S-nitrosation (two post-translational modifications (PTMs) promoted by nitric oxide), the antioxidant capacity, as well as the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), among others. In addition, the activity of the NADPH-generating enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and NADP-isocitrate dehydrogenase (NADP-ICDH) was followed. Our data revealed that the treatment of both cell lines with pepper fruit extracts altered their antioxidant capacity, enhanced their catalase activity, and considerably reduced the activity of the NADPH-generating enzymes. As a consequence, less H2O2 and NADPH seem to be available to cells, thus avoiding cell proliferation and possibly triggering cell death in both cell lines.

Nitric Oxide in Plant Functioning: Metabolism, Signaling, and Responses to Infestation with Ecdysozoa Parasites.

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological processes in plants, including responses to biotic and abiotic stresses. Changes in endogenous NO concentration lead to activation/deactivation of NO signaling and NO-related processes. This paper presents the current state of knowledge on NO biosynthesis and scavenging pathways in plant cells and highlights the role of NO in post-translational modifications of proteins (S-nitrosylation, nitration, and phosphorylation) in plants under optimal and stressful environmental conditions. Particular attention was paid to the interactions of NO with other signaling molecules: reactive oxygen species, abscisic acid, auxins (e.g., indole-3-acetic acid), salicylic acid, and jasmonic acid. In addition, potential common patterns of NO-dependent defense responses against attack and feeding by parasitic and molting Ecdysozoa species such as nematodes, insects, and arachnids were characterized. Our review definitely highlights the need for further research on the involvement of NO in interactions between host plants and Ecdysozoa parasites, especially arachnids.

Identification and quantification of protein nitration sites in human coronary artery smooth muscle cells in the absence and presence of peroxynitrous acid/peroxynitrite

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO−) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO−, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO−, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO− (50 μM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO− concentrations (500 μM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.

Participation of Protein Nitration in Keratotic Plug Formation

毛孔に生じる角栓は，タンパクを主成分とする固形物で，その形成予防は難しかった。角栓は，ケラチン17（K17）を特徴的に含有し，K17の生成に関わるインターフェロンγ（IFN-γ）の角栓形成への関与が窺われた。一方，IFN-γは一酸化窒素（NO）の生成に関与すること，またNOを介するチロシンのニトロ化はタンパクの凝集を引き起こす例があることから，タンパクが固形物となる機構にこれらが寄与している可能性が考えられた。そこで本研究では，角栓形成に対する IFN-γやタンパクニトロ化の関与の有無を明らかにするため，IFN-γ量の角栓数への関与，角栓タンパク成分の解析，ニトロ化がタンパクの分子サイズに与える影響について調査した。その結果，IFN-γ が多い人は角栓数が多く，角栓中にはニトロチロシンが存在することが明らかとなった。また，ニトロ化によりタンパクの分子サイズは増大し，培養角化細胞においては，IFN-γ が NO ドナーと同様にニトロチロシン生成を促進することがわかった。本研究により，毛孔内に固形物を生じる原因として IFN-γ による NO 生成を介するタンパクのニトロ化が示唆され，タンパクニトロ化やそれによる凝集の阻害が新たな角栓形成予防策として提案された。

Probing Tyrosine Nitration with a Small TbIII -Metallopeptide.

Tyrosine nitration, a post-translational modification (PTM) that takes place under nitrosative stress conditions, occurs through a non-enzymatic peroxynitrite-mediated reaction. Although protein nitration has long been considered an irreversible PTM involved in nitrosative stress-associated diseases, it has also been suggested to be a regulatory mechanism of signal transduction. Therefore, the development of tools that help to understand this protein modification is of great interest. Herein, we explore a TbIII -chelating metallopeptide to monitor tyrosine nitration. The luminescence of this probe decreases significantly between its non-nitrated and nitrated states, and this reduction in the luminescence intensity is directly related to the degree of tyrosine nitration after treatment with peroxynitrite. Remarkably, the luminescence intensity changes after nitration are not affected in the presence of complex biological media, which makes it a promising tool for understanding this protein modification.

Cavin-2 loss exacerbates hypoxia-induced pulmonary hypertension with excessive eNOS phosphorylation and protein nitration

Pulmonary hypertension (PH) is associated with a poor prognosis even in recent years. Caveolin-1 (CAV1), a caveolae-associated protein, is a causal gene in PH. Cavin-2, one of the other caveolae-associated proteins, forms protein complexes with CAV1 and influences each other's functions. However, the role of Cavin-2 in PH has not been thoroughly investigated. To clarify the role of Cavin-2 in PH, we exposed Cavin-2-deficient (Cavin-2 KO) mice to hypoxia. A part of the analyses was confirmed in human pulmonary endothelial cells (HPAECs). After 4-week 10% O2 hypoxic exposure, we performed physiological, histological, and immunoblotting analyses. Right ventricular (RV) systolic pressure elevation and RV hypertrophy were exacerbated in Cavin-2 KO mice with hypoxia-induced PH (Cavin-2 KO PH mice). The vascular wall thickness of pulmonary arterioles was aggravated in Cavin-2 KO PH mice. Cavin-2 loss reduced CAV1 and induced sustained endothelial nitric oxide synthase (eNOS) hyperphosphorylation in the Cavin-2 KO PH lungs and HPAECs. NOx production associated with eNOS phosphorylation was also increased in the Cavin-2 KO PH lung and HPAECs. Furthermore, the nitration of proteins, including protein kinase G (PKG), was raised in the Cavin-2 KO PH lungs. In conclusion, we revealed that Cavin-2 loss exacerbated hypoxia-induced PH. Our results suggest that Cavin-2 loss leads to sustained eNOS hyperphosphorylation in pulmonary artery endothelial cells via CAV1 reduction, resulting in Nox overproduction-mediated nitration of proteins, including PKG, in smooth muscle cells.

A platform for distributed production of synthetic nitrated proteins in live bacteria.

The incorporation of the nonstandard amino acid para-nitro-L-phenylalanine (pN-Phe) within proteins has been used for diverse applications, including the termination of immune self-tolerance. However, the requirement for the provision of chemically synthesized pN-Phe to cells limits the contexts where this technology can be harnessed. Here we report the construction of a live bacterial producer of synthetic nitrated proteins by coupling metabolic engineering and genetic code expansion. We achieved the biosynthesis of pN-Phe in Escherichia coli by creating a pathway that features a previously uncharacterized nonheme diiron N-monooxygenase, which resulted in pN-Phe titers of 820 ± 130 µM after optimization. After we identified an orthogonal translation system that exhibited selectivity toward pN-Phe rather than a precursor metabolite, we constructed a single strain that incorporated biosynthesized pN-Phe within a specific site of a reporter protein. Overall, our study has created a foundational technology platform for distributed and autonomous production of nitrated proteins.

Mitochondrial H2O2 metabolism as central event of heart complex I syndrome in early diabetes.

Hydrogen peroxide is the main metabolite effective in redox regulation and it is considered an insulinomimetic agent, with insulin signalling being essential for normal mitochondrial function in cardiomyocytes. Therefore, the aim of this work was to deeply analyse the heart mitochondrial H2O2 metabolism, in the early stage of type 1 diabetes. Diabetes was induced by Streptozotocin (STZ, single dose, 60mg×kg-1, ip.) in male Wistar rats and the animals were sacrificed 10 days after injection. Mitochondrial membrane potential and ATP production, using malate-glutamate as substrates, in the heart of diabetic animals were like the ones observed in control group. Mn-SOD activity was lower (15%) in the heart of diabetic rats even though its expression was increased (29%). The increment in heart mitochondrial H2O2 production (117%) in diabetic animals was accompanied by an enhancement in the activities and expressions of glutathione peroxidase (26% and 42%) and of catalase (200% and 133%), with no changes in the peroxiredoxin activity, leading to [H2O2]ss ∼40nM. Heart mitochondrial lipid peroxidation and protein nitration were higher in STZ-injected animals (45% and 42%) than in control group. The mitochondrial membrane potential and ATP production preservation suggest the absence of irreversible damage at this early stage of diabetes 1. The increase in mitochondrial [H2O2]ss above the physiological range, but still below supraphysiological concentration (∼100nM) seems to be part of the adaptive response triggered in cardiomyocytes due to the absence of insulin. The signs of mitochondrial dysfunction observed in this very early stage of diabetes are consistent with the mitochondrial entity called ″complex I syndrome″.

Alleviation of Meloidogyne incognita induced biotic stress in Daucus carota by nitrogen: Insights into cellular viability and reactive oxygen species of the host

Developing innovative nematode management solutions is becoming more important since ecologically hazardous nematicides are being phased out of the global markets. As a result, alternative measures that are less harmful to the environment are preferred for dealing with the root-knot nematodes which cause huge economic losses to farmers. This investigation was therefore, entailed to find the optimum nitrogen fertilizer rate to maximize plant growth promotion and upsurge nematicidal activity, while minimizing the harmful effects on environment. Among the various nitrogen fertilizer treatments from 50 kg/ha to 200 kg/ha, the most effective concentration was found to be 150 kg/ha N (U150Mi) at which significant (P ≤ 0.05) improvements in growth, yield, photosynthetic pigments, nitrate reductase activity (NRA), protein, and carbohydrate content of carrot were observed as compared to the nematode inoculated untreated plants. Scanning electron microscopy showed that compared to the untreated inoculated control, the stomatal characteristics of carrot improved significantly at U150Mi (150 kg/ha N). Laser confocal microscopy illustrated that nitrogen fertilizer markedly improved the cellular viability of carrot at U150Mi (150 kg/ha N) in comparison to nematode infected plants. Histochemical analysis revealed that the reactive oxygen species (ROS) marker, superoxide anion radicals were significantly scavenged by the enhanced antioxidant defense system of carrot at U150Mi (150 kg/ha N). In vitro studies demonstrate that nitrogen fertilizer has nematicidal properties that were dose dependent. Gall/egg mass indices and reproduction factor were dramatically reduced when U150Mi (150 kg/ha N) was applied to carrot plants inoculated with Meloidogyne incognita.

Mitoquinone protects against acetaminophen-induced liver injury in an FSP1-dependent and GPX4-independent manner

Mitochondrial oxidative stress has been a crucial mediator in acetaminophen (APAP)-induced hepatotoxicity. MitoQ, an analog of coenzyme Q10, is targeted towards mitochondria and acts as a potent antioxidant. This study aimed to explore the effect of MitoQ on APAP-induced liver injury and its possible mechanisms. To investigate this, CD-1 mice and AML-12 cells were treated with APAP. Hepatic MDA and 4-HNE, two markers of lipid peroxidation (LPO), were elevated as early as 2 h after APAP. Oxidized lipids were rapidly upregulated in APAP-exposed AML-12 cells. Hepatocyte death and mitochondrial ultrastructure alterations were observed in APAP-induced acute liver injury. The in vitro experiments showed that mitochondrial membrane potentials and OXPHOS subunits were downregulated in APAP-exposed hepatocytes. MtROS and oxidized lipids were elevated in APAP-exposed hepatocytes. We discovered that APAP-induced hepatocyte death and liver injury were ameliorated by attenuation of protein nitration and LPO in MitoQ-pretreated mice. Mechanistically, knockdown of GPX4, a key enzyme for LPO defense systems, exacerbated APAP-induced oxidized lipids, but did not influence the protective effect of MitoQ on APAP-induced LPO and hepatocyte death. Whereas knockdown of FSP1, another key enzyme for LPO defense systems, had little effect on APAP-induced lipid oxidation but partially weakened the protection of MitoQ on APAP-induced LPO and hepatocyte death. These results suggest that MitoQ may alleviate APAP-evoked hepatotoxicity by eliminating protein nitration and suppressing hepatic LPO. MitoQ prevents APAP-induced liver injury partially dependent of FSP1 and independent of GPX4.

Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5.

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO-) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO- and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO--induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.

Combined chronic copper exposure and aging lead to neurotoxicity in vivo

The environment, containing pollutants, toxins, and transition metals (copper, iron, manganese, and zinc), plays a critical role in neurodegenerative disease development. Copper occupational exposure increases Parkinson’s disease (PD) risk. Previously, we determined the mechanisms by which copper induces dopaminergic cell death in vitro. The copper transporter protein 1 (Ctr1) overexpression led to intracellular glutathione depletion potentiating caspase-3 mediated cell death; oxidative stress was primarily cytosolic, and Nrf2 was upregulated mediating an antioxidant response; and protein ubiquitination, AMPK-Ulk1 signaling, p62, and Atg5-dependent autophagy were increased as a protective mechanism. However, the effect of chronic copper exposure on the neurodegenerative process has not been explored in vivo. We aimed to elucidate whether prolonged copper treatment reproduces PD features and mechanisms during aging. Throughout 40 weeks, C57BL/6J male mice were treated with copper at 0, 100, 250, and 500 ppm in the drinking water. Chronic copper exposure altered motor function and induced dopaminergic neuronal loss, astrocytosis, and microgliosis in a dose-dependent manner. α-Synuclein accumulation and aggregation were increased in response to copper, and the proteasome and autophagy alterations, previously observed in vitro, were confirmed in vivo, where protein ubiquitination, AMPK phosphorylation, and the autophagy marker LC3-II were also increased by copper exposure. Finally, nitrosative stress was induced by copper in a concentration-dependent fashion, as evidenced by increased protein nitration. To our knowledge, this is the first study combining chronic copper exposure and aging, which may represent an in vivo model of non-genetic PD and help to assess potential prophylactic and therapeutic approaches. Data AvailabilityThe data underlying this article are available in the article.

The Combined Treatment of Curcumin with Verapamil Ameliorates the Cardiovascular Pathology in a Williams–Beuren Syndrome Mouse Model

Williams-Beuren syndrome (WBS) is a rare disorder caused by a recurrent microdeletion with hallmarks of cardiovascular manifestations, mainly supra-valvular aortic stenosis (SVAS). Unfortunately, there is currently no efficient treatment. We investigated the effect of chronic oral treatment with curcumin and verapamil on the cardiovascular phenotype of a murine model of WBS harbouring a similar deletion, CD (complete deletion) mice. We analysed systolic blood pressure in vivo and the histopathology of the ascending aorta and the left ventricular myocardium to determine the effects of treatments and their underlying mechanism. Molecular analysis showed significantly upregulated xanthine oxidoreductase (XOR) expression in the aorta and left ventricular myocardium of CD mice. This overexpression is concomitant with increased levels of nitrated proteins as a result of byproduct-mediated oxidative stress damage, indicating that XOR-generated oxidative stress impacts the pathophysiology of cardiovascular manifestations in WBS. Only the combined therapy of curcumin and verapamil resulted in a significant improvement of cardiovascular parameters via activation of the nuclear factor erythroid 2 (NRF2) and reduction of XOR and nitrated protein levels. Our data suggested that the inhibition of XOR and oxidative stress damage could help prevent the severe cardiovascular injuries of this disorder.

Nitration of cAMP-Response Element Binding Protein Participates in Myocardial Infarction-Induced Myocardial Fibrosis via Accelerating Transcription of Col1a2 and Cxcl12.

Aims: Myocardial fibrosis after myocardial infarction (MI) leads to heart failure. Nitration of protein can alter its function. cAMP-response element binding protein (CREB) is a key transcription factor involved in fibrosis. However, little is known about the role of nitrated CREB in MI-induced myocardial fibrosis. Meanwhile, downstream genes of transcription factor CREB in myocardial fibrosis have not been identified. This study aims to verify the hypothesis that nitrated CREB promotes MI-induced myocardial fibrosis via regulating the transcription of Col1a2 and Cxcl12. Results: Our study showed that (1) the level of nitrative stress was elevated and nitrated CREB was higher in the myocardium after MI. Tyr182, 307, and 336 were the nitration sites of CREB; (2) with the administration of peroxynitrite (ONOO-) scavengers, CREB phosphorylation, nuclear translocation, and binding activity to TORC2 (transducers of regulated CREB-2) were attenuated; (3) the expressions of extracellular matrix (ECM) proteins were upregulated and downregulated in accordance with the expression alteration of CREB both in vitro and in vivo; (4) CREB accelerated transcription of Col1a2 and Cxcl12 after MI directly. With the administration of ONOO- scavengers, ECM protein expressions were attenuated; meanwhile, the messenger RNA (mRNA) levels of Col1a2 and Cxcl12 were alleviated as well. Innovation and Conclusion: Nitration of transcription factor CREB participates in MI-induced myocardial fibrosis through enhancing its phosphorylation, nuclear translocation, and binding activity to TORCs, among which CREB transcripts Col1a2 and Cxcl12 directly. These data indicated that nitrated CREB might be a potential therapeutic target against MI-induced myocardial fibrosis. Antioxid. Redox Signal. 38, 709-730.

Episodic Binge-like Ethanol Reduces Skeletal Muscle Strength Associated with Atrophy, Fibrosis, and Inflammation in Young Rats.

Binge Drinking (BD) corresponds to episodes of ingestion of large amounts of ethanol in a short time, typically ≤2 h. BD occurs across all populations, but young and sports-related people are especially vulnerable. However, the short- and long-term effects of episodic BD on skeletal muscle function have been poorly explored. Young rats were randomized into two groups: control and episodic Binge-Like ethanol protocol (BEP) (ethanol 3 g/kg IP, 4 episodes of 2-days ON-2-days OFF paradigm). Muscle function was evaluated two weeks after the last BEP episode. We found that rats exposed to BEP presented decreased muscle strength and increased fatigability, compared with control animals. Furthermore, we observed that skeletal muscle from rats exposed to BEP presented muscle atrophy, evidenced by reduced fiber size and increased expression of atrophic genes. We also observed that BEP induced fibrotic and inflammation markers, accompanied by mislocalization of nNOSµ and high levels of protein nitration. Our findings suggest that episodic binge-like ethanol exposure alters contractile capacity and increases fatigue by mechanisms involving atrophy, fibrosis, and inflammation, which remain for at least two weeks after ethanol clearance. These pathological features are common to several neuromuscular diseases and might affect muscle performance and health in the long term.

Decreased methylglyoxal-mediated protein glycation in the healthy aging mouse model of ectopic expression of UCP1 in skeletal muscle

Mice with ectopic expression of uncoupling protein-1 (UCP1) in skeletal muscle exhibit a healthy aging phenotype with increased longevity and resistance to impaired metabolic health. This may be achieved by decreasing protein glycation by the reactive metabolite, methylglyoxal (MG). We investigated protein glycation and oxidative damage in skeletal muscle of mice with UCP1 expression under control of the human skeletal actin promoter (HSA-mUCP1) at age 12 weeks (young) and 70 weeks (aged). We found both young and aged HSA-mUCP1 mice had decreased advanced glycation endproducts (AGEs) formed from MG, lysine-derived Nε(1-carboxyethyl)lysine (CEL) and arginine-derived hydroimidazolone, MG-H1, whereas protein glycation by glucose forming Nε-fructosyl-lysine (FL) was increased ca. 2-fold, compared to wildtype controls. There were related increases in FL-linked AGEs, Nε-carboxymethyl-lysine (CML) and 3-deoxylglucosone-derived hydroimidazolone 3DG-H, and minor changes in protein oxidative and nitration adducts. In aged HSA-mUCP1 mice, urinary MG-derived AGEs/FL ratio was decreased ca. 60% whereas there was no change in CML/FL ratio – a marker of oxidative damage. This suggests that, normalized for glycemic status, aged HSA-mUCP1 mice had a lower flux of whole body MG-derived AGE exposure compared to wildtype controls. Proteomics analysis of skeletal muscle revealed a shift to increased heat shock proteins and mechanoprotection and repair in HSA-mUCP1 mice. Decreased MG-derived AGE protein content in skeletal muscle of aged HSA-mUCP1 mice is therefore likely produced by increased proteolysis of MG-modified proteins and increased proteostasis surveillance of the skeletal muscle proteome. From this and previous transcriptomic studies, signaling involved in enhanced removal of MG-modified protein is likely increased HSPB1-directed HUWE1 ubiquitination through eIF2α-mediated, ATF5-induced increased expression of HSPB1. Decreased whole body exposure to MG-derived AGEs may be linked to increased weight specific physical activity of HSA-mUCP1 mice. Decreased formation and increased clearance of MG-derived AGEs may be associated with healthy aging in the HSA-mUCP1 mouse.

Effects of Nitrogen Application Strategy on Nitrogen Enzyme Activities and Protein Content in Spring Wheat Grain

The aim of this study was to determine the regulatory effect of different nitrogen (N) fertilizer application rates on the grain N metabolism enzymes and protein content of drip-irrigated spring wheat under the climatic conditions in Xinjiang, China. A split plot experiment was conducted with strong gluten wheat Xinchun 38 (XC 38) and medium gluten wheat Xinchun 49 (XC 49) as experimental materials. We set up seven nitrogen treatments, in amounts of 300 (Nck), 285 (N5), 270 (N10), 255 (N15), 240 (N20), 225 (N25) and 0 (N0) kg hm−2. The effects of N application rate on nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate aminotransferase (GPT), protein content, protein composition, and yield of wheat grain were studied. The results showed that NR, GS, GPT, protein content, albumin, globulin, glutenin, gliadin, and yield first increased and then decreased with the decrease in N application. Furthermore, different responses to different applications between different wheat varieties was also observed. The indexes of XC 38 reached the maximum in the N15 treatment, and the yield increased 2.99~81.45%. XC 49 showed the best indicators under the N25 treatment and the yield increased 0.37~71.29%. Under the same N level, all indicators of XC 38 were better than XC 49. Correlation analysis showed that the yield and protein yield were significantly positively correlated with NR, GS, and GPT. The interaction of N fertilizer and variety had significant effects on NR, GS, GPT, protein content, components, and yield. These results show that the protein content and yield of wheat grain can be improved by reasonably adjusting the N fertilizer application strategy.

Abstract 13989: Glutaredoxin-1: A Redox Regulator of Metalloproteases in Vascular Endothelial Cells

Background: Endothelial dysfunction is a common mediator of aneurysmal remodeling regardless of etiology and the vascular bed that occur (e.g., genetic mutations, hypertension, aging, smoking, abdominal or thoracic artery or aortic root), but the underlying mechanisms remain unknown. This work aims to test the hypothesis that perturbing thiol redox signaling in endothelial cells triggers aberrant activation of matrix metalloproteinase-2 and -9, thereby precipitating aneurysmal remodeling. Methods and Results: In human aortic aneurysm (AA) samples from individuals with Marfan syndrome, levels of MMP-2/-9 were increased drastically that coincided with elevated oxidative stress markers: tyrosine nitrated proteins and lipid peroxidation product 4-hydroxynonenal . In mouse model of AA: fibrillin-1 mutant( Fbn1 mgR/mgR ) mice, reversible oxidation of cysteine residues—S-glutathionylation (PrS-SG) that mediate thiol redox signaling by modulating target proteins’ functions—were markedly elevated as an early redox event. Endothelial specific deletion of glutaredoxin-1(Grx1), a specific de-glutathionylation enzyme was found to massively increase gelatinase activity of MMP-2/-9 in whole aortas, and worsen vessel wall integrity. Using primary human aortic ECs, we showed that Grx1 can directly prevent pro-MMP-2/-9 from glutathionylation of their cysteine switch and consequent activation. Furthermore, the medium collected from HAECs that lack functional Grx1 stimulated MMP-2 activity in smooth muscle cells, thus setting up an amplifying mechanism of MMP activation. Conclusions: this study is the first to provide experiment evidence for a pathogenic role of dysregulated thiol redox signaling in vascular endothelial cells in aneurysmal remodeling.