Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

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Antonella Pantaleo

2016-01-01

Full Text Available In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.

BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells

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Lagadinou, Eleni D.; Sach, Alexander; Callahan, Kevin; Rossi, Randall M.; Neering, Sarah J.; Minhajuddin, Mohammad; Ashton, John M.; Pei, Shanshan; Grose, Valerie; O’Dwyer, Kristen M.; Liesveld, Jane L.; Brookes, Paul S.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

Summary Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally-defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly over-express BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation. PMID:23333149

Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

OpenAIRE

Korzeniewski, Bernard; Zoladz, Jerzy A

2003-01-01

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial protein...

Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

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Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-01-01

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways

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Wen Xiong

2017-09-01

Full Text Available The duck virus hepatitis (DVH caused by the duck hepatitis virus A (DHAV has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA and its phosphorylated derivative (pICA possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2 as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD and glutathione peroxidase (GSH-Px of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2 and c-Jun N-terminal kinase (JNK, which were related to mitogen-activated protein kinases (MAPKs signaling pathways. Ultimately, compared to ICA, pICA exhibited more

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

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Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

OpenAIRE

Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

1990-01-01

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

Xenobiotics that affect oxidative phosphorylation alter differentiation of human adipose-derived stem cells at concentrations that are found in human blood

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Laura Llobet

2015-11-01

Full Text Available Adipogenesis is accompanied by differentiation of adipose tissue-derived stem cells to adipocytes. As part of this differentiation, biogenesis of the oxidative phosphorylation system occurs. Many chemical compounds used in medicine, agriculture or other human activities affect oxidative phosphorylation function. Therefore, these xenobiotics could alter adipogenesis. We have analyzed the effects on adipocyte differentiation of some xenobiotics that act on the oxidative phosphorylation system. The tested concentrations have been previously reported in human blood. Our results show that pharmaceutical drugs that decrease mitochondrial DNA replication, such as nucleoside reverse transcriptase inhibitors, or inhibitors of mitochondrial protein synthesis, such as ribosomal antibiotics, diminish adipocyte differentiation and leptin secretion. By contrast, the environmental chemical pollutant tributyltin chloride, which inhibits the ATP synthase of the oxidative phosphorylation system, can promote adipocyte differentiation and leptin secretion, leading to obesity and metabolic syndrome as postulated by the obesogen hypothesis.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

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Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-09-19

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic inhibition of PKA phosphorylation of RyR2 prevents dystrophic cardiomyopathy

NARCIS (Netherlands)

Sarma, Satyam; Li, Na; van Oort, Ralph J.; Reynolds, Corey; Skapura, Darlene G.; Wehrens, Xander H. T.

2010-01-01

Aberrant intracellular Ca(2+) regulation is believed to contribute to the development of cardiomyopathy in Duchenne muscular dystrophy. Here, we tested whether inhibition of protein kinase A (PKA) phosphorylation of ryanodine receptor type 2 (RyR2) prevents dystrophic cardiomyopathy by reducing SR

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

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Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Science.gov (United States)

Sun, Jiying; Shi, Lin; Kinomura, Aiko; Fukuto, Atsuhiko; Horikoshi, Yasunori; Oma, Yukako; Harata, Masahiko; Ikura, Masae; Ikura, Tsuyoshi; Kanaar, Roland; Tashiro, Satoshi

2018-05-08

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities. © 2018, Sun et al.

Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation

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Zhou, Yan [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); College of Food Safety, Guizhou Medical University, Guiyang 550025 (China); Ruan, Zheng, E-mail: ruanzheng@ncu.edu.cn [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Zhou, Lili; Shu, Xugang [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Sun, Xiaohong [College of Food Safety, Guizhou Medical University, Guiyang 550025 (China); Mi, Shumei; Yang, Yuhui [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Yin, Yulong, E-mail: yinyulong@isa.ac.cn [State Key Laboratory of Food Science and Technology and School of Food Science, Nanchang University, Nanchang 330047 (China); Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125 (China)

2016-01-22

Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. - Highlights: • Dietary supplementation with chlorogenic acid (CGA) improved endotoxin-induced liver injury. • Chlorogenic acid enhances ATP increase and shifts energy metabolism, which is correlated with up-regulation AMPK and PGC-1α. • The possible mechanism of CGA on mitochondrial biogenesis was correlated with up-regulation AMPK and PGC-1α.

Chlorogenic acid ameliorates endotoxin-induced liver injury by promoting mitochondrial oxidative phosphorylation

International Nuclear Information System (INIS)

Zhou, Yan; Ruan, Zheng; Zhou, Lili; Shu, Xugang; Sun, Xiaohong; Mi, Shumei; Yang, Yuhui; Yin, Yulong

2016-01-01

Acute or chronic hepatic injury is a common pathology worldwide. Mitochondrial dysfunction and the depletion of adenosine triphosphate (ATP) play important roles in liver injury. Chlorogenic acids (CGA) are some of the most abundant phenolic acids in human diet. This study was designed to test the hypothesis that CGA may protect against chronic lipopolysaccharide (LPS)-induced liver injury by modulating mitochondrial energy generation. CGA decreased the activities of serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase. The contents of ATP and adenosine monophosphate (AMP), as well as the ratio of AMP/ATP, were increased after CGA supplementation. The activities of enzymes that are involved in glycolysis were reduced, while those of enzymes involved in oxidative phosphorylation were increased. Moreover, phosphorylated AMP-activated protein kinase (AMPK), and mRNA levels of AMPK-α, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial DNA transcription factor A were increased after CGA supplementation. Collectively, these findings suggest that the hepatoprotective effect of CGA might be associated with enhanced ATP production, the stimulation of mitochondrial oxidative phosphorylation and the inhibition of glycolysis. - Highlights: • Dietary supplementation with chlorogenic acid (CGA) improved endotoxin-induced liver injury. • Chlorogenic acid enhances ATP increase and shifts energy metabolism, which is correlated with up-regulation AMPK and PGC-1α. • The possible mechanism of CGA on mitochondrial biogenesis was correlated with up-regulation AMPK and PGC-1α.

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches.

Science.gov (United States)

Wakai, Satoshi; Masanari, Misa; Ikeda, Takumi; Yamaguchi, Naho; Ueshima, Saori; Watanabe, Kaori; Nishihara, Hirofumi; Sambongi, Yoshihiro

2013-04-01

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

Science.gov (United States)

Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

2015-11-01

Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

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Masahiro Myojo

Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1 preserves oxidative phosphorylation in a yeast model of Barth syndrome

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Maxence de Taffin de Tilques

2017-04-01

Full Text Available Cardiolipin (CL is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS. To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS, presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1Δ yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS.

The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

International Nuclear Information System (INIS)

Ribeiro, Mariana P.C.; Silva, Filomena S.G.; Santos, Armanda E.; Santos, Maria S.; Custódio, José B.A.

2013-01-01

Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30 nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50 nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50 nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10 nmol/mg protein prevented and reversed the Ca 2+ -induced depolarization of ΔΨ and the release of mitochondrial Ca 2+ , similarly to cyclosporine A, indicating that EDX did not affect Ca 2+ uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma. - Highlights: ► Mitochondria are important targets of Endoxifen. ► Endoxifen prevents mitochondrial permeability transition. ► Endoxifen prevents oxidative stress in

The antiestrogen endoxifen protects rat liver mitochondria from permeability transition pore opening and oxidative stress at concentrations that do not affect the phosphorylation efficiency

Energy Technology Data Exchange (ETDEWEB)

Ribeiro, Mariana P.C.; Silva, Filomena S.G.; Santos, Armanda E. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal); Santos, Maria S. [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra (Portugal); Custódio, José B.A., E-mail: custodio@ci.uc.pt [Center for Neuroscience and Cell Biology, University of Coimbra, 3000-354 Coimbra (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra (Portugal)

2013-02-15

Endoxifen (EDX) is a key active metabolite of tamoxifen (TAM) with higher affinity and specificity to estrogen receptors that also inhibits aromatase activity. It is safe and well tolerated by healthy humans, but its use requires toxicological characterization. In this study, the effects of EDX on mitochondria, the primary targets for xenobiotic-induced toxicity, were monitored to clarify its potential side effects. EDX up to 30 nmol/mg protein did not affect the mitochondrial oxidative phosphorylation. At 50 nmol EDX/mg protein, EDX decreased the ADP phosphorylation rate and a partial collapse of mitochondrial membrane potential (Δψ), that parallels a state 4 stimulation, was observed. As the stimulation of state 4 was not inhibited by oligomycin and 50 nmol EDX/mg protein caused a slight decrease in the light scattering of mitochondria, these data suggest that EDX promotes membrane permeabilization to protons, whereas TAM at the same concentration induced mitochondrial membrane disruption. Moreover, EDX at 10 nmol/mg protein prevented and reversed the Ca{sup 2+}-induced depolarization of ΔΨ and the release of mitochondrial Ca{sup 2+}, similarly to cyclosporine A, indicating that EDX did not affect Ca{sup 2+} uptake, but directly interfered with the proteins of the mitochondrial permeability transition (MPT) megacomplex, inhibiting MPT induction. At this concentration, EDX exhibited antioxidant activity that may account for the protective effect against MPT pore opening. In conclusion, EDX within the range of concentrations reached in tissues did not significantly damage the bioenergetic functions of mitochondria, contrarily to the prodrug TAM, and prevented the MPT pore opening and the oxidative stress in mitochondria, supporting that EDX may be a less toxic drug for women with breast carcinoma. - Highlights: ► Mitochondria are important targets of Endoxifen. ► Endoxifen prevents mitochondrial permeability transition. ► Endoxifen prevents oxidative

Phosphorylation prevents C/EBPβ from the calpain-dependent degradation

International Nuclear Information System (INIS)

Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi

2012-01-01

Highlights: ► Phosphorylation protected C/EBPβ from μ-calpain-mediated proteolysis in vitro. ► Phosphorylation mimic C/EBPβ was insensitive to calpain accelerator and inhibitor. ► Phosphorylation on Thr 188 contributed more to the stabilization of C/EBPβ. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) β plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBPβ is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr 188 and subsequently by GSK3β on Ser 184 or Thr 179 . Dual phosphorylation is critical for the gain of DNA binding activity of C/EBPβ. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBPβ. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3β protected C/EBPβ from μ-calpain-mediated proteolysis, while phosphorylation on Thr 188 by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBPβ, Further studies indicated that phosphorylation mimic C/EBPβ was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBPβ.

Parkinson's disease associated with impaired oxidative phosphorylation

International Nuclear Information System (INIS)

Finsterer, J.; Jarius, C.; Baumgartner, M.

2001-01-01

Parkinson's disease may be due to primary or secondary oxidative phosphorylation (OXPHOS) defects. In a 76-year-old man with Parkinson's disease since 1992, slightly but recurrently elevated creatine phosphokinase, recurrently elevated blood glucose, thickening of the left ventricular myocardium, bifascicular block and hypacusis were found. Cerebral MRI showed atrophy, periventricular demyelination, multiple, disseminated, supra- and infratentorial lacunas, and haemosiderin deposits in both posterior horns. Muscle biopsy showed typical features of an OXPHOS defect. Whether the association of Parkinson's disease and impaired OXPHOS was causative or coincidental remains unknown. Possibly, the mitochondrial defect acted as an additional risk factor for Parkinson's disease or the OXPHOS defect worsened the preexisting neurological impairments by a cumulative or synergistic mechanism. In conclusion, this case shows that Parkinson's disease may be associated with a mitochondrially or nuclearly encoded OXPHOS defect, manifesting as hypacusis, myopathy, axonal polyneuropathy, cardiomyopathy and recurrent subclinical ischaemic strokes and haemorrhages. (orig.)

Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato

NARCIS (Netherlands)

Bultema, Jelle B.; Braun, Hans-Peter; Boekema, Egbert J.; Kouřil, Roman

The individual protein complexes of the oxidative phosphorylation system (OXPHOS complexes 1 to V) specifically interact and form defined supramolecular structures, the so-called "respiratory supercomplexes". Some supercomplexes appear to associate into larger structures, or megacomplexes, such as a

The HK2 Dependent "Warburg Effect" and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate.

Science.gov (United States)

Lis, Paweł; Dyląg, Mariusz; Niedźwiecka, Katarzyna; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2016-12-15

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the "Warburg" and "Crabtree" effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the "Warburg effect" and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the "Warburg effect", and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the "Warburg effect", i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as "multiple myeloma". Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

Expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and mitochondrial oxidative phosphorylation in septic patients.

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Laura A Nucci

Full Text Available Sepsis is a complex disease that is characterized by activation and inhibition of different cell signaling pathways according to the disease stage. Here, we evaluated genes involved in the TLR signaling pathway, oxidative phosphorylation and oxidative metabolism, aiming to assess their interactions and resulting cell functions and pathways that are disturbed in septic patients.Blood samples were obtained from 16 patients with sepsis secondary to community acquired pneumonia at admission (D0, and after 7 days (D7, N = 10 of therapy. Samples were also collected from 8 healthy volunteers who were matched according to age and gender. Gene expression of 84 genes was performed by real-time polymerase chain reactions. Their expression was considered up- or down-regulated when the fold change was greater than 1.5 compared to the healthy volunteers. A p-value of ≤ 0.05 was considered significant.Twenty-two genes were differently expressed in D0 samples; most of them were down-regulated. When gene expression was analyzed according to the outcomes, higher number of altered genes and a higher intensity in the disturbance was observed in non-survivor than in survivor patients. The canonical pathways altered in D0 samples included interferon and iNOS signaling; the role of JAK1, JAK2 and TYK2 in interferon signaling; mitochondrial dysfunction; and superoxide radical degradation pathways. When analyzed according to outcomes, different pathways were disturbed in surviving and non-surviving patients. Mitochondrial dysfunction, oxidative phosphorylation and superoxide radical degradation pathway were among the most altered in non-surviving patients.Our data show changes in the expression of genes belonging to the interacting TLR cascades, NADPH-oxidase and oxidative phosphorylation. Importantly, distinct patterns are clearly observed in surviving and non-surviving patients. Interferon signaling, marked by changes in JAK-STAT modulation, had prominent changes in

IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION

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IN VITRO CARDIOTOXICITY OF AIR POLLUTION PARTICLES: ROLE OF BIOAVAILABLE CONSTITUENTS, OXIDATIVE STRESS AND TYROSINE PHOSPHORYLATION.T. L. Knuckles1 R. Jaskot2, J. Richards2, and K.Dreher2.1Department of Molecular and Biomedical Sciences, College of Veterinary Medicin...

The HK2 Dependent “Warburg Effect” and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate

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Paweł Lis

2016-12-01

Full Text Available This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the “Warburg” and “Crabtree” effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2, a major player in both the “Warburg effect” and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the “Warburg effect”, and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP. This small alkylating compound targets both the “Warburg effect”, i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as “multiple myeloma”. Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development.

Phosphorylation prevents C/EBP{beta} from the calpain-dependent degradation

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Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi, E-mail: lixi@shmu.edu.cn

2012-03-16

Highlights: Black-Right-Pointing-Pointer Phosphorylation protected C/EBP{beta} from {mu}-calpain-mediated proteolysis in vitro. Black-Right-Pointing-Pointer Phosphorylation mimic C/EBP{beta} was insensitive to calpain accelerator and inhibitor. Black-Right-Pointing-Pointer Phosphorylation on Thr{sub 188} contributed more to the stabilization of C/EBP{beta}. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) {beta} plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBP{beta} is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr{sub 188} and subsequently by GSK3{beta} on Ser{sub 184} or Thr{sub 179}. Dual phosphorylation is critical for the gain of DNA binding activity of C/EBP{beta}. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBP{beta}. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3{beta} protected C/EBP{beta} from {mu}-calpain-mediated proteolysis, while phosphorylation on Thr{sub 188} by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBP{beta}, Further studies indicated that phosphorylation mimic C/EBP{beta} was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBP{beta}.

Ferroxitosis: A cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma

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Lakhter, Alexander J.; Hamilton, James; Dagher, Pierre C.; Mukkamala, Suresh; Hato, Takashi; Dong, X. Charlie; Mayo, Lindsey D.; Harris, Robert A.; Shekhar, Anantha; Ivan, Mircea; Brustovetsky, Nickolay; Naidu, Samisubbu R.

2014-01-01

Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma. PMID:25587028

Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress

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Claudia Rodrigues

2016-12-01

Full Text Available Aquaporin-5 (AQP5 is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5’s ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics.

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

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Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-01-01

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

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Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

2016-05-06

The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Acyclic retinoid in chemoprevention of hepatocellular carcinoma: Targeting phosphorylated retinoid X receptor-α for prevention of liver carcinogenesis

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Masahito Shimizu

2012-01-01

Full Text Available One of the key features of hepatocellular carcinoma (HCC is the high rate of intrahepatic recurrence that correlates with poor prognosis. Therefore, in order to improve the clinical outcome for patients with HCC, development of a chemopreventive agent that can decrease or delay the incidence of recurrence is a critical issue for urgent investigation. Acyclic retinoid (ACR, a synthetic retinoid, successfully improves HCC patient survival by preventing recurrence and the formation of secondary tumors. A malfunction of the retinoid X receptor-α (RXRα due to phosphorylation by the Ras-MAPK signaling pathway plays a critical role in liver carcinogenesis, and ACR exerts chemopreventive effects on HCC development by inhibiting RXRα phosphorylation. Here, we review the relationship between retinoid signaling abnormalities and liver disease, the mechanisms of how RXRα phosphorylation contributes to liver carcinogenesis, and the detailed effects of ACR on preventing HCC development, especially based on the results of our basic and clinical research. We also outline the concept of "clonal deletion and inhibition" therapy, which is defined as the removal and inhibition of latent malignant clones from the liver before they expand into clinically detectable HCC, because ACR prevents the development of HCC by implementing this concept. Looking toward the future, we discuss "combination chemoprevention" using ACR as a key drug since it can generate a synergistic effect, and may thus be an effective new strategy for the prevention of HCC.

Reliability of maximal mitochondrial oxidative phosphorylation in permeabilized fibers from the vastus lateralis employing high-resolution respirometry

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Cardinale, Daniele A; Gejl, Kasper D; Ørtenblad, Niels

2018-01-01

The purpose was to assess the impact of various factors on methodological errors associated with measurement of maximal oxidative phosphorylation (OXPHOS) in human skeletal muscle determined by high-resolution respirometry in saponin-permeabilized fibers. Biopsies were collected from 25 men...

Hydrocortisone and insulin effect on oxidative phosphorylation in mitochondria of liver and spleen th rats exposed to fast neutrons

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Sutkovoj, D.A.; Alferov, A.N.; Letov, V.N.

1979-01-01

Total-body exposure of rats to fast neutrons (100 rad) elicits an increase in the content of 11-oxycorticosteroids in blood plasma and an appreciable decrease in the respiratory and phosphorylation activity of liver and spleen mitochondria. Administration of hydrocortisone (50 mg/kg) and particularly insulin (2 units/kg) intensifies energy generation in the liver. In the spleen, a beneficial effect was only produced by insulin. Glucocorticoid considerably aggravates the postradiation impairment of oxidative phosphorylation

Mitochondrial oxidative phosphorylation efficiency is upregulated during fasting in two major oxidative tissues of ducklings.

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Monternier, Pierre-Axel; Teulier, Loïc; Drai, Jocelyne; Bourguignon, Aurore; Collin-Chavagnac, Delphine; Hervant, Frédéric; Rouanet, Jean-Louis; Roussel, Damien

2017-10-01

Fasted endothermic vertebrates must develop physiological responses to maximize energy conservation and survival. The aim of this study was to determine the effect of 1-wk. fasting in 5-wk. old ducklings (Cairina moschata) from whole-body resting metabolic rate and body temperature to metabolic phenotype of tissues and mitochondrial coupling efficiency. At the level of whole organism, the mass-specific metabolic rate of ducklings was decreased by 40% after 1-wk. of fasting, which was associated with nocturnal Tb declines and shallow diurnal hypothermia during fasting. At the cellular level, fasting induced a large reduction in liver, gastrocnemius (oxidative) and pectoralis (glycolytic) muscle masses together with a fuel selection towards lipid oxidation and ketone body production in liver and a lower glycolytic phenotype in skeletal muscles. At the level of mitochondria, fasting induced a reduction of oxidative phosphorylation activities and an up-regulation of coupling efficiency (+30% on average) in liver and skeletal muscles. The present integrative study shows that energy conservation in fasted ducklings is mainly achieved by an overall reduction in mitochondrial activity and an increase in mitochondrial coupling efficiency, which would, in association with shallow hypothermia, increase the conservation of endogenous fuel stores during fasting. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes.

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Kellogg, Dean L; McCammon, Karen M; Hinchee-Rodriguez, Kathryn S; Adamo, Martin L; Roman, Linda J

2017-09-01

Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H 2 O 2 ) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H 2 O 2 -induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H 2 O 2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H 2 O 2 -stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H 2 O 2 -activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance. Copyright © 2017. Published by Elsevier Inc.

Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

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Guntur, Anyonya R; Gerencser, Akos A; Le, Phuong T; DeMambro, Victoria E; Bornstein, Sheila A; Mookerjee, Shona A; Maridas, David E; Clemmons, David E; Brand, Martin D; Rosen, Clifford J

2018-06-01

Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

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Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Hepatic mitochondrial oxidative phosphorylation is normal in obese patients with and without type 2 diabetes

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Lund, Michael Taulo; Kristensen, Marianne Dalsgaard; Hansen, Merethe

2016-01-01

INTRODUCTION: Obese patients with (T2DM) and without (OB) type 2 diabetes are characterized by high hepatic lipid content and hepatic insulin resistance. This may be linked to impaired hepatic mitochondrial oxidative phosphorylation (OXPHOS) capacity. The aim of the present study was to investiga...... role in the development of obesity-induced type 2 diabetes. This article is protected by copyright. All rights reserved....

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

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Javier Modrego

Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

Expression of oxidative phosphorylation components in mitochondria of long-living Ames dwarf mice.

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Brown-Borg, Holly M; Johnson, W Thomas; Rakoczy, Sharlene G

2012-02-01

Reduced signaling of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) pathway is associated with extended life span in several species. Ames dwarf mice are GH-deficient and live >50% longer than wild-type littermates. Previously, we have shown that tissues from Ames mice exhibit elevated levels of antioxidative enzymes, less H(2)O(2) production, and lower oxidative damage suggesting that mitochondrial function may differ between genotypes. To explore the relationship between hormone deficiency and mitochondria in mice with extended longevity, we evaluated activity, protein, and gene expression of oxidative phosphorylation components in dwarf and wild-type mice at varying ages. Liver complex I + III activity was higher in dwarf mice compared to wild-type mice. The activity of I + III decreased between 3 and 20 months of age in both genotypes with greater declines in wild-type mice in liver and skeletal muscle. Complex IV activities in the kidney were elevated in 3- and 20-month-old dwarf mice relative to wild-type mice. In Ames mice, protein levels of the 39 kDa complex I subunit were elevated at 20 months of age when compared to wild-type mouse mitochondria for every tissue examined. Kidney and liver mitochondria from 20-month-old dwarf mice had elevated levels of both mitochondrially-encoded and nuclear-encoded complex IV proteins compared to wild-type mice (p dwarf mice. Overall, we found that several components of the oxidative phosphorylation (OXPHOS) system were elevated in Ames mice. Mitochondrial to nuclear DNA ratios were not different between genotypes despite the marked increase in PGC-1α levels in dwarf mice. The increased OXPHOS activities, along with lower ROS production in dwarf mice, predict enhanced mitochondrial function and efficiency, two factors likely contributing to long-life in Ames mice.

Andrographolide Attenuates LPS-Induced Cardiac Malfunctions Through Inhibition of IκB Phosphorylation and Apoptosis in Mice

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Jinlong Zhang

2015-11-01

Full Text Available Background/Aims: Cardiac malfunction is a common complication in sepsis and significantly increases the mortality of patients in septic shock. However, no studies have examined whether andrographolide (And reduces LPS-induced myocardial malfunction. Methods: Left ventricular systolic and diastolic functions were examined using echocardiography. TNF-a and IL-1ß protein levels were detected by an enzyme-linked immunosorbent assay (ELISA. NO oxidation products were determined using Griess reagent. Protein expression levels of inhibitors of NF-κBa (IκB and phospho-IκB were determined via Western blot. Oxidative injury was determined by measuring myocardial lipid peroxidation and superoxide dismutase activity. Cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling (TUNEL and cardiac caspase 3/7 activity. Results: And blunted LPS-induced myocardial malfunctions in mice. LPS induced TNF-a, IL-1ß, and NO production as well as I-κB phosphorylation. Cardiac apoptosis was attenuated via incubation with And, but the extent of oxidative injury remained unaffected. Conclusion: And prevents LPS-induced cardiac malfunctions in mice by inhibiting TNF-a, IL-1ß, and NO production, IκB phosphorylation, and cardiac apoptosis, indicating that And may be a potential agent for preventing myocardial malfunction during sepsis.

Study of oxidative and phosphorylative activity in mitochondria from cereal seedlings

Energy Technology Data Exchange (ETDEWEB)

Plhak, F

1973-01-01

In the present paper the oxidative and phosphorylative activity of mitochondria isolated from rye, wheat, barley and corn seedlings are compared. Mitochondria from the shoots as well as from the roots of rye, wheat and corn oxidized succinate and in the presence of ATP or ADP exhibited the respiratory control which reached the values mostly of about 2. In the presence of ATP or ADP the decrease of inorganic phosphorus was contemporarily remarkable. The P/O ratio reached the values mostly of about 0.8 up to 1.0. The presence of ATP effected in some cases more favorable the respiratory control as well as the P/O ratio in comparison with ADP. With regard to the fact that a trapping hexokinase system was not added, the presence of endogenous hexokinase in mitochondria of experimental plants is presumed. The barley mitochondria exhibited the respiratory control as well, but instead of the decrease in inorganic phosphorus content in reaction mixture, an increase took place. It was caused by the presence of active ATP-ase which was not effectively inhibited by present NaF.

SIRT3 mediates decrease of oxidative damage and prevention of ageing in porcine fetal fibroblasts.

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Xie, Xiaoxian; Wang, Liangliang; Zhao, Binggong; Chen, Yangyang; Li, Jiaqi

2017-05-15

Sirtuin 3 (SIRT3) is a mitochondria-specific protein required for the deacetylation of metabolic enzymes and the action of oxidative phosphorylation by acting as a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase. SIRT3 increases oxidative stress resistance and prevents mitochondrial decay associated with ageing in response to caloric restriction. However, the effects of SIRT3 on oxidative damage and ageing are not well understood. We investigated the physiological functions of porcine SIRT3 on the damage and ageing in porcine fetal fibroblasts (PFFs). Overexpression and knockdown of SIRT3 were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. All cells were treated with three different stress reagents 12-o-tetradecanoylphorbol-13-acetate (TPA), methanesulfonic acid methylester (MMS), and tert-butylhydroperoxide (t-BHP), respectively, and then examined by flow cytometry following JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazol-carbocyanine iodide) staining. SIRT3 overexpression enhanced the ability of superoxide dismutase 2 (SOD2) to reduce cellular reactive oxygen species (ROS), which further decreased the damage to the membranes and the organelles of the cells, especially to mitochondria. It inhibited the initial decrease of mitochondrial membrane potential, and prevented the decrease of adenosine triphosphate (ATP) production and activity of Nampt. In contrast, SIRT3 knockdown reduced the ability of SOD2 to increase cellular ROS which was directly correlated with stress-induced oxidative damage and ageing in PFFs. Our findings identify one function of SIRT3 in PFFs was to dampen cytotoxicity, and, therefore, to decrease oxidative damage and attenuate ageing possibly by enhancing the activity of SOD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial oxidative stress causes hyperphosphorylation of tau.

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Simon Melov

2007-06-01

Full Text Available Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD: tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2 die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576 with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.

Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

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Carmen Clapp

2011-07-01

Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis.

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Feichtinger, René G; Neureiter, Daniel; Skaria, Tom; Wessler, Silja; Cover, Timothy L; Mayr, Johannes A; Zimmermann, Franz A; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas ("intestinal" and "diffuse"), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.

A novel mechanism involved in the coupling of mitochondrial biogenesis to oxidative phosphorylation

Directory of Open Access Journals (Sweden)

Jelena Ostojić

2014-01-01

Full Text Available Mitochondria are essential organelles that are central to a multitude of cellular processes, including oxidative phosphorylation (OXPHOS, which produces most of the ATP in animal cells. Thus it is important to understand not only the mechanisms and biogenesis of this energy production machinery but also how it is regulated in both physiological and pathological contexts. A recent study by Ostojić et al. [Cell Metabolism (2013 18, 567-577] has uncovered a regulatory loop by which the biogenesis of a major enzyme of the OXPHOS pathway, the respiratory complex III, is coupled to the energy producing activity of the mitochondria.

Mitochondrial NAD(PH in vivo: identifying natural indicators of oxidative phosphorylation in the 31P magnetic resonance spectrum.

Directory of Open Access Journals (Sweden)

Kevin eConley

2016-03-01

Full Text Available Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P+ and NAD(PH, which are compartmentalized between cell and mitochondria. Here we provide evidence for detection of NAD(P+ and NAD(PH in separate mitochondrial and cell pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy (31P MRS. These NAD(P pools are identified by chemical standards (NAD+, NADP+ and NADH and by physiological tests. A unique resonance reflecting mitochondrial NAD(PH is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(PH with oxidation is matched by a stoichiometric rise in the NAD(P+ peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(PH peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus non-invasive detection of NAD(P+ and NAD(PH in cell vs. mitochondria yield natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

Calcium dobesilate prevents the oxidative stress and inflammation induced by diabetes in the retina of db/db mice.

Science.gov (United States)

Bogdanov, Patricia; Solà-Adell, Cristina; Hernández, Cristina; García-Ramírez, Marta; Sampedro, Joel; Simó-Servat, Olga; Valeri, Marta; Pasquali, Christian; Simó, Rafael

2017-10-01

Calcium dobesilate (CaD) is beneficial in early stages of diabetic retinopathy (DR), but its mechanisms of action remains to be elucidated. The aim was to investigate the effect of CaD on proinflammatory cytokines and oxidative stress. db/db mice were randomly assigned to daily oral treatment with CaD (200mg/kg/day) or vehicle for 15days. Biomarkers of oxidative stress (dihydroethidium, malondialdehyde), NF-κB, and proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, MCP-1) were examined in the retina by immunohistochemical analysis. Cultures of human retinal endothelial cells (HRECs) were used for complementary experiments. CaD significantly reduced the biomarkers of oxidative stress in the retina of db/db mice. In addition, CaD prevented the increase of NF-κB, IL-6, IL-8, TNF-α and MCP-1 induced by diabetes. CaD inhibited the activation of NF-kβ induced by IL-1β by preventing IKKB-α phosphorylation in HRECs and reduced the upregulation of IL-6 and IL-18 induced by TNF-α in a dose-dependent manner. Our results suggest that antioxidant and antiinflammatory effects are crucial in accounting for the effectiveness of CaD for treating DR. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of testosterone on markers of mitochondrial oxidative phosphorylation and lipid metabolism in muscle of aging men with subnormal bioavailable testosterone

DEFF Research Database (Denmark)

Petersson, Stine J; Christensen, Louise L; Kristensen, Jonas M

2014-01-01

therapy on regulators of mitochondrial biogenesis and markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels. METHODS: Skeletal muscle biopsies were obtained before and after treatment with either testosterone gel (n=12) or placebo (n=13......) for 6 months. Insulin sensitivity and substrate oxidation were assessed by euglycemic-hyperinsulinemic clamp and indirect calorimetry. Muscle mRNA levels and protein abundance and phosphorylation of enzymes involved in mitochondrial biogenesis, OxPhos, and lipid metabolism were examined by quantitative......: The beneficial effect of testosterone treatment on lipid oxidation is not explained by increased abundance or phosphorylation-dependent activity of enzymes known to regulate mitochondrial biogenesis or markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable...

Arsenate uncoupling of oxidative phosphorylation in isolated plant mitochondria

Energy Technology Data Exchange (ETDEWEB)

Wickes, W A; Wiskich, J T

1976-01-01

The uncoupling by arsenate of beetroot and cauliflower bud mitochondria showed the following characteristics: arsenate stimulation of respiration above the rate found with phosphate; inhibition of arsenate-stimulated respiration by phosphate; enhancement of arsenate-stimulated respiration by ADP; only partial prevention of this ADP-enhanced respiration by atractyloside; inhibition by oligomycin of the arsenate-stimulated respiration back to the phosphate rate; and the absence of any stimulatory effect of ADP in the presence of oligomycin. These results are qualitatively analogous to those reported for arsenate uncoupling in rat liver mitochondria. Arsenate stimulated malate oxidation, presumably by stimulating malate entry, in both beetroot and cauliflower bud mitochondria; however, high rates of oxidation, and presumably entry, were only sustained with arsenate in beetroot mitochondria. NADH was oxidized rapidly in cauliflower bud mitochondria in the presence of arsenate, showing that arsenate did not inhibit electron transfer processes.

L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446 in a Nitric Oxide-Dependent Manner in C2C12 Cells

Directory of Open Access Journals (Sweden)

Ruxia Wang

2018-01-01

Full Text Available Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS, the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446 and p70S6K (Thr 389. L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside, an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446/p70S6K signaling pathway in an NO-dependent manner.

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

OpenAIRE

Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

2004-01-01

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

Low intensity exercise prevents disturbances in rat cardiac insulin signaling and endothelial nitric oxide synthase induced by high fructose diet.

Science.gov (United States)

Stanišić, Jelena; Korićanac, Goran; Ćulafić, Tijana; Romić, Snježana; Stojiljković, Mojca; Kostić, Milan; Pantelić, Marija; Tepavčević, Snežana

2016-01-15

Increase in fructose consumption together with decrease in physical activity contributes to the development of metabolic syndrome and consequently cardiovascular diseases. The current study examined the preventive role of exercise on defects in cardiac insulin signaling and function of endothelial nitric oxide synthase (eNOS) in fructose fed rats. Male Wistar rats were divided into control, sedentary fructose (received 10% fructose for 9 weeks) and exercise fructose (additionally exposed to low intensity exercise) groups. Concentration of triglycerides, glucose, insulin and visceral adipose tissue weight were determined to estimate metabolic syndrome development. Expression and/or phosphorylation of cardiac insulin receptor (IR), insulin receptor substrate 1 (IRS1), tyrosine-specific protein phosphatase 1B (PTP1B), Akt, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and eNOS were evaluated. Fructose overload increased visceral adipose tissue, insulin concentration and homeostasis model assessment index. Exercise managed to decrease visceral adiposity and insulin level and to increase insulin sensitivity. Fructose diet increased level of cardiac PTP1B and pIRS1 (Ser307), while levels of IR and ERK1/2, as well as pIRS1 (Tyr 632), pAkt (Ser473, Thr308) and pERK1/2 were decreased. These disturbances were accompanied by reduced phosphorylation of eNOS at Ser1177. Exercise managed to prevent most of the disturbances in insulin signaling caused by fructose diet (except phosphorylation of IRS1 at Tyr 632 and phosphorylation and protein expression of ERK1/2) and consequently restored function of eNOS. Low intensity exercise could be considered as efficient treatment of cardiac insulin resistance induced by fructose diet. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A novel redox-based switch: LMW-PTP oxidation enhances Grb2 binding and leads to ERK activation

International Nuclear Information System (INIS)

Giannoni, Elisa; Raugei, Giovanni; Chiarugi, Paola; Ramponi, Giampietro

2006-01-01

Low molecular weight-PTP has been reported as a redox-sensitive protein during both platelet-derived growth factor and integrin signalling. In response to oxidation the phosphatase undergoes a reversible inactivation, which in turn leads to the increase in tyrosine phosphorylation of its substrates and the properly executed anchorage-dependent proliferation program. Here, we report that an exogenous oxidative stress enhances LMW-PTP tyrosine phosphorylation, through oxidation/inactivation of the enzyme, thus preventing its auto-dephosphorylation activity. In particular, we observed a selective hyper-phosphorylation of Tyr132, that acts as a docking site for the adaptor protein Grb2. The redox-dependent enhancement of Grb2 recruitment to LMW-PTP ultimately leads to an improvement of ERK activation, likely triggering a prosurvival signal against the oxidant environment

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis

Science.gov (United States)

Skaria, Tom; Wessler, Silja; Cover, Timothy L.; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection. PMID:28744336

Rosamines targeting the cancer oxidative phosphorylation pathway.

Directory of Open Access Journals (Sweden)

Siang Hui Lim

Full Text Available Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM, inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6 exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

Time Window Is Important for Adenosine Preventing Cold-induced Injury to the Endothelium.

Science.gov (United States)

Li, Yan; Hu, Xiao-Xia; Fu, Li; Chen, Jing; Lu, Li-He; Liu, Xiang; Xu, Zhe; Zhou, Li; Wang, Zhi-Ping; Zhang, Xi; Ou, Zhi-Jun; Ou, Jing-Song

2017-06-01

Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.

Neuroprotective effects of Arctium lappa L. roots against glutamate-induced oxidative stress by inhibiting phosphorylation of p38, JNK and ERK 1/2 MAPKs in PC12 cells.

Science.gov (United States)

Tian, Xing; Sui, Shuang; Huang, Jin; Bai, Jun-Peng; Ren, Tian-Shu; Zhao, Qing-Chun

2014-07-01

Many studies have shown that glutamate-induced oxidative stress can lead to neuronal cell death involved in the development of neurodegenerative diseases. In this work, protective effects of ethyl acetate extract (EAE) of Arctium lappa L. roots against glutamate-induced oxidative stress in PC12 cells were evaluated. Also, the effects of EAE on antioxidant system, mitochondrial pathway, and signal transduction pathway were explored. Pretreatment with EAE significantly increased cell viability, activities of GSH-Px and SOD, mitochondrial membrane potential and reduced LDH leakage, ROS formation, and nuclear condensation in a dose-dependent manner. Furthermore, western blot results revealed that EAE increased the Bcl-2/Bax ratio, and inhibited the up-regulation of caspase-3, release of cytochrome c, phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). Therefore, our results indicate that EAE may be a promising neuroprotective agent for the prevention and treatment of neurodegenerative diseases implicated with oxidative stress. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

Science.gov (United States)

Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

Science.gov (United States)

Louis, Xavier Lieben; Thandapilly, Sijo Joseph; Kalt, Wilhelmina; Vinqvist-Tymchuk, Melinda; Aloud, Basma Milad; Raj, Pema; Yu, Liping; Le, Hoa; Netticadan, Thomas

2014-08-01

The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies.

Science.gov (United States)

Korzeniewski, Bernard; Zoladz, Jerzy A

2002-07-01

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

Erection capability is potentiated by long-term sildenafil treatment: role of blood flow-induced endothelial nitric-oxide synthase phosphorylation.

Science.gov (United States)

Musicki, Biljana; Champion, Hunter C; Becker, Robyn E; Liu, Tongyun; Kramer, Melissa F; Burnett, Arthur L

2005-07-01

Despite demonstrated clinical efficacy of sildenafil for the temporary treatment of erectile dysfunction, the possibility that sildenafil used long-term durably augments erectile ability remains unclear. We investigated whether continuous long-term administration of sildenafil at clinically relevant levels to aged rats "primes" the penis for improved erectile ability and involves nitric oxide (NO) or RhoA/Rho-kinase signaling pathways. In aged, but not young rats, sildenafil prolonged erection and increased the protein expressions of phosphorylated endothelial NO synthase (eNOS) at serine-1177 and phosphorylated Akt at serine-473 in penes. Only in the young rat penis, protein expressions of phosphodiesterase-5 and phosphomyosin phosphatase target subunit 1, a marker of Rho-kinase activity, were increased by sildenafil. Sildenafil inhibited phosphodiesterase-5 activity in penes of young and aged rats coincident with assayed free plasma levels of the drug equivalent to clinically therapeutic measurements. We conclude that erectile ability can be enhanced under preconditions of erectile impairment by long-term inhibition of phosphodiesterase-5 and that the effect is mediated by Akt-dependent eNOS phosphorylation. The lack of erectile ability enhancement in young rats by long-term phosphodiesterase-5 inhibition may relate to restrained NO signaling by phosphodiesterase-5 up-regulation, lack of incremental Akt and eNOS phosphorylation, and heightened Rho-kinase signaling in the penis.

Analysis of effects of 2,2',5,5'-tetracholorobiphenyl on the flux control in oxidative phosphorylation system in rat liver mitochondria.

NARCIS (Netherlands)

Mildaziene, V.; Nauciene, Z.; Baniene, R.; Demin, O.V.; Krab, K.

2002-01-01

Modular kinetic analysis reveals that the environmental pollutant 2,2′,5,5′tetrachlorobiphenyl (2,2′,5,5′-TCB) affects a large number of steps in oxidative phosphorylation in rat liver mitochondria. 2,2′,5,5′-TCB increases membrane permeability to ions, and inhibits NADH dehydrogenase, cytochrome

Heritable oxidative phosphorylation differences in a pollutant resistant Fundulus heteroclitus population

International Nuclear Information System (INIS)

Du, Xiao; Crawford, Douglas L.; Nacci, Diane E.; Oleksiak, Marjorie F.

2016-01-01

Highlights: • Laboratory reared fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Laboratory reared F3 fish from polluted population displayed higher routine metabolism and complex II activity but lower complex I enzyme activity. • Enhanced OxPhos metabolism and toxicity resistance were retained in laboratory reared F3 fish from the polluted population. - Abstract: Populations can adapt to stress including recent anthropogenic pollution. Our published data suggests heritable differences in hepatocyte oxidative phosphorylation (OxPhos) metabolism in field-caught killifish (Fundulus heteroclitus) from the highly polluted Elizabeth River, VA, USA, relative to fish from a nearby, relatively unpolluted reference site in King’s Creek VA. Consistent with other studies showing that Elizabeth River killifish are resistant to some of the toxic effects of certain contaminants, OxPhos measurements in hepatocytes from field-caught King’s Creek but not field-caught Elizabeth River killifish were altered by acute benzo [a] pyrene exposures. To more definitively test whether the enhanced OxPhos metabolism and toxicity resistance are heritable, we measured OxPhos metabolism in a laboratory-reared F3 generation from the Elizabeth River population versus a laboratory-reared F1 generation from the King’s Creek population and compared these results to previous data from the field-caught fish. The F3 Elizabeth River fish compared to F1 King’s Creek fish had significantly higher State 3 respiration (routine metabolism) and complex II activity, and significantly lower complex I activity. The consistently higher routine metabolism in the F3 and field-caught Elizabeth River fish versus F1 and field-caught King’s Creek fish implies a heritable change in OxPhos function. The observation that LEAK, E-State, Complex I and Complex II were different in laboratory bred

Heritable oxidative phosphorylation differences in a pollutant resistant Fundulus heteroclitus population

Energy Technology Data Exchange (ETDEWEB)

Du, Xiao, E-mail: xdu@rsmas.miami.edu [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States); Crawford, Douglas L. [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States); Nacci, Diane E. [Population Ecology Branch, Atlantic Ecology Division, Office of Research and Development, U.S. Environmental Protection Agency, 27 Tarzwell Dr., Narragansett, RI 02882 (United States); Oleksiak, Marjorie F., E-mail: moleksiak@rsmas.miami.edu [Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149 (United States)

2016-08-15

Highlights: • Laboratory reared fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Laboratory reared F3 fish from polluted population displayed higher routine metabolism and complex II activity but lower complex I enzyme activity. • Enhanced OxPhos metabolism and toxicity resistance were retained in laboratory reared F3 fish from the polluted population. - Abstract: Populations can adapt to stress including recent anthropogenic pollution. Our published data suggests heritable differences in hepatocyte oxidative phosphorylation (OxPhos) metabolism in field-caught killifish (Fundulus heteroclitus) from the highly polluted Elizabeth River, VA, USA, relative to fish from a nearby, relatively unpolluted reference site in King’s Creek VA. Consistent with other studies showing that Elizabeth River killifish are resistant to some of the toxic effects of certain contaminants, OxPhos measurements in hepatocytes from field-caught King’s Creek but not field-caught Elizabeth River killifish were altered by acute benzo [a] pyrene exposures. To more definitively test whether the enhanced OxPhos metabolism and toxicity resistance are heritable, we measured OxPhos metabolism in a laboratory-reared F3 generation from the Elizabeth River population versus a laboratory-reared F1 generation from the King’s Creek population and compared these results to previous data from the field-caught fish. The F3 Elizabeth River fish compared to F1 King’s Creek fish had significantly higher State 3 respiration (routine metabolism) and complex II activity, and significantly lower complex I activity. The consistently higher routine metabolism in the F3 and field-caught Elizabeth River fish versus F1 and field-caught King’s Creek fish implies a heritable change in OxPhos function. The observation that LEAK, E-State, Complex I and Complex II were different in laboratory bred

Vegetation successfully prevents oxidization of sulfide minerals in mine tailings.

Science.gov (United States)

Li, Yang; Sun, Qingye; Zhan, Jing; Yang, Yang; Wang, Dan

2016-07-15

The oxidization of metal sulfide in tailings causes acid mine drainage. However, it remains unclear whether vegetation prevents the oxidization of metal sulfides. The oxidization characteristics and microbial indices of the tailings in the presence of various plant species were investigated to explore the effects of vegetation on the oxidization of sulfide minerals in tailings. The pH, reducing sulfur, free iron oxides (Fed), chemical oxygen consumption (COC) and biological oxygen consumption (BOC) were measured. Key iron- and sulfur-oxidizing bacteria (Acidithiobacillus spp., Leptospirillum spp. and Thiobacillus spp.) were quantified using real-time PCR. The results indicate that vegetation growing on tailings can effectively prevent the oxidization of sulfide minerals in tailings. A higher pH and reducing-sulfur content and lower Fed were observed in the 0-30 cm depth interval in the presence of vegetation compared to bare tailings (BT). The COC gradually decreased with depth in all of the soil profiles; specifically, the COC rapidly decreased in the 10-20 cm interval in the presence of vegetation but gradually decreased in the BT profiles. Imperata cylindrica (IC) and Chrysopogon zizanoides (CZ) profiles contained the highest BOC in the 10-20 cm interval. The abundance of key iron- and sulfur-oxidizing bacteria in the vegetated tailings were significantly lower than in the BT; in particular, IC was associated with the lowest iron- and sulfur-oxidizing bacterial abundance. In conclusion, vegetation successfully prevented the oxidization of sulfide minerals in the tailings, and Imperata cylindrica is the most effective in reducing the number of iron- and sulfur-oxidizing bacteria and helped to prevent the oxidization of sulfide minerals in the long term. Copyright © 2016 Elsevier Ltd. All rights reserved.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Science.gov (United States)

Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, S P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2011-09-01

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

International Nuclear Information System (INIS)

Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

1986-01-01

The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with γ- 32 P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the V600EBRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to V600EBRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably......, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to V...

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

International Nuclear Information System (INIS)

Ghosh, Jyotirmoy; Das, Joydeep; Manna, Prasenjit; Sil, Parames C.

2009-01-01

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As

Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

DEFF Research Database (Denmark)

Guterbaum, Thomas Jeremy; Braunstein, Thomas Hartig; Fossum, A

2013-01-01

Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein end...

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

Science.gov (United States)

Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

2016-06-10

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei

Czech Academy of Sciences Publication Activity Database

Verner, Zdeněk; Čermáková, P.; Škodová, Ingrid; Kováčová, B.; Lukeš, Julius; Horváth, A.

2014-01-01

Roč. 193, č. 1 (2014), s. 55-65 ISSN 0166-6851 R&D Projects: GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032; GA MŠk LH12104 Institutional support: RVO:60077344 Keywords : mitochondrion * oxidative phosphorylation * Trypanosoma * Leishmania * Phytomonas * Crithidia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

DEFF Research Database (Denmark)

Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

2016-01-01

ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... checkpoints, initiating DNA repair and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Energy Technology Data Exchange (ETDEWEB)

Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

2012-11-01

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation. �

Importance of glycolysis and oxidative phosphorylation in advanced melanoma

Directory of Open Access Journals (Sweden)

Ho Jonhan

2012-10-01

Full Text Available Abstract Serum lactate dehydrogenase (LDH is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS, we subsequently determined using tissue microarray (TMA analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

Science.gov (United States)

Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

2016-05-16

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of K_Ca3.1 channel as a novel regulator of Oxidative phosphorylation in a subset of pancreatic carcinoma cell lines

DEFF Research Database (Denmark)

Kovalenko, Ilya; Glasauer, Andrea; Schöckel, Laura

2016-01-01

, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well...

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the (V600E)BRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS......). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes...

Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

Science.gov (United States)

Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

2016-01-01

Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

[Role of green tea in oxidative stress prevention].

Science.gov (United States)

Metro, D; Muraca, U; Manasseri, L

2006-01-01

Oxidative stress is a condition caused by an increase of Reactive Oxygen Species (ROS) or by a shortage of the mechanisms of cellular protection and antioxidant defence. ROS have a potential oxidative effect towards various cellular macromolecules: proteins, nucleic acids, proteoglycans, lipids, with consequent damages in several cellular districts and promotion of the ageing process of the organism. However, some substances are able to prevent and/or reduce the damages caused by ROS; therefore, they are defined antioxidant. The present research studied, in a group of subjects, the antioxidant effects of the green tea, that was administered with fruit and vegetables in a strictly controlled diet. 50 subjects were selected and requested to daily consume 2-3 fruit portions (especially pineapple), 3-5 portions of vegetables (especially tomato) and 2-3 glasses of green tea for about 2 months to integrate the controlled basic diet. Some indicators of the oxidative stress were measured in the plasma before and after the integration period. The integration of a basic diet with supplements of fruit, vegetables and green tea turned out to be able in increasing both plasmatic total antioxidant capacity and endogenous antioxidant levels and to reduce the lipid peroxidation of the membranes, suggesting a reduction of the oxidative stress. These data suggest that an adequate supplement of antioxidants can prevent oxidative stress and correlated pathologies.

SH3 domain tyrosine phosphorylation--sites, role and evolution.

Directory of Open Access Journals (Sweden)

Zuzana Tatárová

Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

Science.gov (United States)

Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

2016-09-01

In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

Science.gov (United States)

Nath, Sunil

2016-12-01

As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

Axin-mediated CKI phosphorylation of beta-catenin at Ser 45

DEFF Research Database (Denmark)

Amit, Sharon; Hatzubai, Ada; Birman, Yaara

2002-01-01

The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin...... and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta......-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta...

Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK-3β

Energy Technology Data Exchange (ETDEWEB)

Song, Jae-Sook; Kim, Eun-Kyung; Choi, Yong-Won [Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588 (Korea, Republic of); Oh, Won Keun [College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University (Korea, Republic of); Kim, Young-Mi, E-mail: ymikim12@hanyang.ac.kr [Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 15588 (Korea, Republic of)

2016-09-15

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level. Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3β through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3β inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3β signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

DEFF Research Database (Denmark)

Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

2016-01-01

of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

Directory of Open Access Journals (Sweden)

Michael Van Meter

2016-09-01

Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

Characterisation and properties of homo- and heterogenously phosphorylated nanocellulose.

Science.gov (United States)

Kokol, Vanja; Božič, Mojca; Vogrinčič, Robert; Mathew, Aji P

2015-07-10

Nano-sized cellulose ester derivatives having phosphoryl side groups were synthesised by phosphorylation of nanofibrilated cellulose (NFC) and nanocrystaline cellulose (NCC), using different heterogeneous (in water) and homogeneous (in molten urea) processes with phosphoric acid as phosphoryl donor. The phosphorylation mechanism, efficacy, stability, as well as its influence on the NC crystallinity and thermal properties, were evaluated using ATR-FTIR and (13)C NMR spectroscopies, potentiometric titration, capillary electrophoresis, X-ray diffraction, colorimetry, thermogravimmetry and SEM. Phosphorylation under both processes created dibasic phosphate and monobasic tautomeric phosphite groups at C6 and C3 positioned hydroxyls of cellulose, yielded 60-fold (∼1,173 mmol/kg) and 2-fold (∼1.038 mmol/kg) higher surface charge density for p-NFC and p-NCC, respectively, under homogenous conditions. None of the phosphorylations affected neither the NC crystallinity degree nor the structure, and noticeably preventing the derivatives from weight loss during the pyrolysis process. The p-NC showed high hydrolytic stability to water at all pH mediums. Reusing of the treatment bath was examined after the heterogeneous process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

DEFF Research Database (Denmark)

Rosenbaek, L L; Assentoft, M; Pedersen, N B

2012-01-01

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antib......The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho......-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and m...

Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

International Nuclear Information System (INIS)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-01-01

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO

Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

Science.gov (United States)

Batchuluun, Battsetseg; Inoguchi, Toyoshi; Sonoda, Noriyuki; Sasaki, Shuji; Inoue, Tomoaki; Fujimura, Yoshinori; Miura, Daisuke; Takayanagi, Ryoichi

2014-01-01

Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Potential of the Antibody Against cis-Phosphorylated Tau in the Early Diagnosis, Treatment, and Prevention of Alzheimer Disease and Brain Injury.

Science.gov (United States)

Lu, Kun Ping; Kondo, Asami; Albayram, Onder; Herbert, Megan K; Liu, Hekun; Zhou, Xiao Zhen

2016-11-01

Alzheimer disease (AD) and chronic traumatic encephalopathy (CTE) share a common neuropathologic signature-neurofibrillary tangles made of phosphorylated tau-but do not have the same pathogenesis or symptoms. Although whether traumatic brain injury (TBI) could cause AD has not been established, CTE is shown to be associated with TBI. Until recently, whether and how TBI leads to tau-mediated neurodegeneration was unknown. The unique prolyl isomerase Pin1 protects against the development of tau-mediated neurodegeneration in AD by converting the phosphorylated Thr231-Pro motif in tau (ptau) from the pathogenic cis conformation to the physiologic trans conformation, thereby restoring ptau function. The recent development of antibodies able to distinguish and eliminate both conformations specifically has led to the discovery of cis-ptau as a precursor of tau-induced pathologic change and an early driver of neurodegeneration that directly links TBI to CTE and possibly to AD. Within hours of TBI in mice or neuronal stress in vitro, neurons prominently produce cis-ptau, which causes and spreads cis-ptau pathologic changes, termed cistauosis. Cistauosis eventually leads to widespread tau-mediated neurodegeneration and brain atrophy. Cistauosis is effectively blocked by the cis-ptau antibody, which targets intracellular cis-ptau for proteasome-mediated degradation and prevents extracellular cis-ptau from spreading to other neurons. Treating TBI mice with cis-ptau antibody not only blocks early cistauosis but also prevents development and spreading of tau-mediated neurodegeneration and brain atrophy and restores brain histopathologic features and functional outcomes. Thus, cistauosis is a common early disease mechanism for AD, TBI, and CTE, and cis-ptau and its antibody may be useful for early diagnosis, treatment, and prevention of these devastating diseases.

Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria.

Science.gov (United States)

Filipović, Dragana; Costina, Victor; Perić, Ivana; Stanisavljević, Andrijana; Findeisen, Peter

2017-03-15

Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics. Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

International Nuclear Information System (INIS)

Gunter, Thomas E.; Gerstner, Brent; Lester, Tobias; Wojtovich, Andrew P.; Malecki, Jon; Swarts, Steven G.; Brookes, Paul S.; Gavin, Claire E.; Gunter, Karlene K.

2010-01-01

Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn 2+ inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn 2+ inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn 2+ inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F 1 F 0 ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn 2+ inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and Caucasians in the arctic winter.

Science.gov (United States)

Gnaiger, E; Boushel, R; Søndergaard, H; Munch-Andersen, T; Damsgaard, R; Hagen, C; Díez-Sánchez, C; Ara, I; Wright-Paradis, C; Schrauwen, P; Hesselink, M; Calbet, J A L; Christiansen, M; Helge, J W; Saltin, B

2015-12-01

During evolution, mitochondrial DNA haplogroups of arctic populations may have been selected for lower coupling of mitochondrial respiration to ATP production in favor of higher heat production. We show that mitochondrial coupling in skeletal muscle of traditional and westernized Inuit habituating northern Greenland is identical to Danes of western Europe haplogroups. Biochemical coupling efficiency was preserved across variations in diet, muscle fiber type, and uncoupling protein-3 content. Mitochondrial phenotype displayed plasticity in relation to lifestyle and environment. Untrained Inuit and Danes had identical capacities to oxidize fat substrate in arm muscle, which increased in Danes during the 42 days of acclimation to exercise, approaching the higher level of the Inuit hunters. A common pattern emerges of mitochondrial acclimatization and evolutionary adaptation in humans at high latitude and high altitude where economy of locomotion may be optimized by preservation of biochemical coupling efficiency at modest mitochondrial density, when submaximum performance is uncoupled from VO2max and maximum capacities of oxidative phosphorylation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

Science.gov (United States)

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of Anthropogenic Pollution on the Oxidative Phosphorylation Pathway of Hepatocytes from Natural Populations of Fundulus heteroclitus

Energy Technology Data Exchange (ETDEWEB)

Du, Xiao; Crawford, Douglas L.; Oleksiak, Marjorie F., E-mail: moleksiak@rsmas.miami.edu

2015-08-15

Highlights: • Fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Polluted fish had lower LEAK, enzyme III and enzyme IV but higher enzyme I. • Exposures to PAH and PCB only affected individuals from the reference population. - Abstract: Persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), potentially target mitochondria and cause toxicity. We compared the effects of POPs on mitochondrial respiration by measuring oxidative phosphorylation (OxPhos) metabolism in hepatocytes isolated from lab-depurated Fundulus heteroclitus from a Superfund site contaminated with PAHs (Elizabeth River VA, USA) relative to OxPhos metabolism in individuals from a relatively clean, reference population (King’s Creek VA, USA). In individuals from the polluted Elizabeth River population, OxPhos metabolism displayed lower LEAK and lower activities in complex III, complex IV, and E State, but higher activity in complex I compared to individuals from the reference King’s Creek population. To test the supposition that these differences were due to or related to the chronic PAH contamination history of the Elizabeth River population, we compared the OxPhos functions of undosed individuals from the polluted and reference populations to individuals from these populations dosed with a PAH {benzo [α] pyrene (BaP)} or a PCB {PCB126 (3,3′,4,4′,5-pentachlorobiphenyl)}, respectively. Exposure to PAH or PCB affected OxPhos in the reference King’s Creek population but had no detectable effects on the polluted Elizabeth River population. Thus, PAH exposure significantly increased LEAK, and exposure to PCB126 significantly decreased State 3, E state and complex I activity in the reference King’s Creek population. These data strongly implicate an evolved tolerance in the Elizabeth River fish where dosed

Effects of Anthropogenic Pollution on the Oxidative Phosphorylation Pathway of Hepatocytes from Natural Populations of Fundulus heteroclitus

International Nuclear Information System (INIS)

Du, Xiao; Crawford, Douglas L.; Oleksiak, Marjorie F.

2015-01-01

Highlights: • Fish from a highly polluted and clean reference population were compared. • Oxidative phosphorylation (e.g., State 3, enzymes, and proton LEAK) was quantified. • Polluted fish had lower LEAK, enzyme III and enzyme IV but higher enzyme I. • Exposures to PAH and PCB only affected individuals from the reference population. - Abstract: Persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), potentially target mitochondria and cause toxicity. We compared the effects of POPs on mitochondrial respiration by measuring oxidative phosphorylation (OxPhos) metabolism in hepatocytes isolated from lab-depurated Fundulus heteroclitus from a Superfund site contaminated with PAHs (Elizabeth River VA, USA) relative to OxPhos metabolism in individuals from a relatively clean, reference population (King’s Creek VA, USA). In individuals from the polluted Elizabeth River population, OxPhos metabolism displayed lower LEAK and lower activities in complex III, complex IV, and E State, but higher activity in complex I compared to individuals from the reference King’s Creek population. To test the supposition that these differences were due to or related to the chronic PAH contamination history of the Elizabeth River population, we compared the OxPhos functions of undosed individuals from the polluted and reference populations to individuals from these populations dosed with a PAH {benzo [α] pyrene (BaP)} or a PCB {PCB126 (3,3′,4,4′,5-pentachlorobiphenyl)}, respectively. Exposure to PAH or PCB affected OxPhos in the reference King’s Creek population but had no detectable effects on the polluted Elizabeth River population. Thus, PAH exposure significantly increased LEAK, and exposure to PCB126 significantly decreased State 3, E state and complex I activity in the reference King’s Creek population. These data strongly implicate an evolved tolerance in the Elizabeth River fish where dosed

Zinc in the prevention of Fe2initiated lipid and protein oxidation

Directory of Open Access Journals (Sweden)

M. PAOLA ZAGO

2000-01-01

Full Text Available In the present study we characterized the capacity of zinc to protect lipids and proteins from Fe2+-initiated oxidative damage. The effects of zinc on lipid oxidation were investigated in liposomes composed of brain phosphatidylcholine (PC and phosphatidylserine (PS at a molar relationship of 60:40 (PC:PS, 60:40. Lipid oxidation was evaluated as the oxidation of cis-parinaric acid or as the formation of 2-thiobarbituric acid-reactive substances (TBARS. Zinc protected liposomes from Fe2+ (2.5-50 muM-supported lipid oxidation. However, zinc (50 muM did not prevent the oxidative inactivation of glutamine synthelase and glucose 6-phosphate dehydrogenase when rat brain superntants were oxidized in the presence of 5 muM Fe2+ and 0.5 mM H2O2 .We also studied the interactions of zinc with epicatechin in the prevention of liid oxidation in liposomes. The simulaneous addition of 0.5 muM epicatechin (EC and 50 muM zinc or EC separately. Zinc (50 muM also protecte liposomes from the stimulatory effect of aluminum on Fe2+-initiated lipid oxidation. Zinc could play an important role as an antioxidant in biological systems, replacing iron and other metals with pro-oxidant activity from binding sites and interacting with other components of the oxidant defense system.

Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

Energy Technology Data Exchange (ETDEWEB)

Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

2013-07-12

Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation

DEFF Research Database (Denmark)

Dzamko, Nicolas; Schertzer, Jonathan D.; Ryall, James G.

2008-01-01

The activation of AMP-activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl-CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser-221 phosphorylation does not always correlate...... with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside (AICAR) and contraction-stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle-specific kinase...... dead (KD) AMPK alpha2. In wild-type (WT) mice, AICAR and contraction increased AMPK alpha2 and alpha1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl-CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained...

Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis

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Michael I. Carr

2016-09-01

Full Text Available ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2S394A knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2S394A mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2S394A mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies.

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

Directory of Open Access Journals (Sweden)

Thomas H Sanderson

Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Heme oxygenase-1 prevents cardiac dysfunction in streptozotocin-diabetic mice by reducing inflammation, oxidative stress, apoptosis and enhancing autophagy.

Directory of Open Access Journals (Sweden)

Yanli Zhao

Full Text Available Heme oxygenase-1 (HO-1 has been implicated in cardiac dysfunction, oxidative stress, inflammation, apoptosis and autophagy associated with heart failure, and atherosclerosis, in addition to its recognized role in metabolic syndrome and diabetes. Numerous studies have presented contradictory findings about the role of HO-1 in diabetic cardiomyopathy (DCM. In this study, we explored the role of HO-1 in myocardial dysfunction, myofibril structure, oxidative stress, inflammation, apoptosis and autophagy using a streptozotocin (STZ-induced diabetes model in mice systemically overexpressing HO-1 (Tg-HO-1 or mutant HO-1 (Tg-mutHO-1. The diabetic mouse model was induced by multiple peritoneal injections of STZ. Two months after injection, left ventricular (LV function was measured by echocardiography. In addition, molecular biomarkers related to oxidative stress, inflammation, apoptosis and autophagy were evaluated using classical molecular biological/biochemical techniques. Mice with DCM exhibited severe LV dysfunction, myofibril structure disarray, aberrant cardiac oxidative stress, inflammation, apoptosis, autophagy and increased levels of HO-1. In addition, we determined that systemic overexpression of HO-1 ameliorated left ventricular dysfunction, myofibril structure disarray, oxidative stress, inflammation, apoptosis and autophagy in DCM mice. Furthermore, serine/threonine-specific protein kinase (Akt and AMP-activated protein kinase (AMPK phosphorylation is normally inhibited in DCM, but overexpression of the HO-1 gene restored the phosphorylation of these kinases to normal levels. In contrast, the functions of HO-1 in DCM were significantly reversed by overexpression of mutant HO-1. This study underlines the unique roles of HO-1, including the inhibition of oxidative stress, inflammation and apoptosis and the enhancement of autophagy, in the pathogenesis of DCM.

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53Ser-15 phosphorylation

International Nuclear Information System (INIS)

Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader; Shahin, Allen; Patel, Mahendra; Galadari, Sehamuddin

2009-01-01

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser 15 . Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53 Ser-15 phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.

Oxidative stress in Alzheimer disease: a possibility for prevention.

Science.gov (United States)

Bonda, David J; Wang, Xinglong; Perry, George; Nunomura, Akihiko; Tabaton, Massimo; Zhu, Xiongwei; Smith, Mark A

2010-01-01

Oxidative stress is at the forefront of Alzheimer disease (AD) research. While its implications in the characteristic neurodegeneration of AD are vast, the most important aspect is that it seems increasingly apparent that oxidative stress is in fact a primary progenitor of the disease, and not merely an epiphenomenon. Moreover, evidence indicates that a long "dormant period" of gradual oxidative damage accumulation precedes and actually leads to the seemingly sudden appearance of clinical and pathological AD symptoms, including amyloid-beta deposition, neurofibrillary tangle formation, metabolic dysfunction, and cognitive decline. These findings provide important insights into the development of potential treatment regimens and even allude to the possibility of a preventative cure. In this review, we elaborate on the dynamic role of oxidative stress in AD and present corresponding treatment strategies that are currently under investigation. Copyright 2010 Elsevier Ltd. All rights reserved.

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Science.gov (United States)

Gan, Fang; Hu, Zhihua; Huang, Yu; Xue, Hongxia; Huang, Da; Qian, Gang; Hu, Junfa; Chen, Xingxiang; Wang, Tian; Huang, Kehe

2016-01-01

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells. PMID:26943035

Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating

DEFF Research Database (Denmark)

Moeller, Hanne B; Macaulay, Nanna; Knepper, Mark A

2009-01-01

demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating......Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites...... in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S...

Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

Science.gov (United States)

Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

2013-07-12

Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.

High-intensity interval training prevents oxidant-mediated diaphragm muscle weakness in hypertensive mice.

Science.gov (United States)

Bowen, T Scott; Eisenkolb, Sophia; Drobner, Juliane; Fischer, Tina; Werner, Sarah; Linke, Axel; Mangner, Norman; Schuler, Gerhard; Adams, Volker

2017-01-01

Hypertension is a key risk factor for heart failure, with the latter characterized by diaphragm muscle weakness that is mediated in part by increased oxidative stress. In the present study, we used a deoxycorticosterone acetate (DOCA)-salt mouse model to determine whether hypertension could independently induce diaphragm dysfunction and further investigated the effects of high-intensity interval training (HIIT). Sham-treated (n = 11), DOCA-salt-treated (n = 11), and DOCA-salt+HIIT-treated (n = 15) mice were studied over 4 wk. Diaphragm contractile function, protein expression, enzyme activity, and fiber cross-sectional area and type were subsequently determined. Elevated blood pressure confirmed hypertension in DOCA-salt mice independent of HIIT (P HIIT. Myosin heavy chain (MyHC) protein expression tended to decrease (∼30%; P = 0.06) in DOCA-salt vs. sham- and DOCA-salt+HIIT mice, whereas oxidative stress increased (P HIIT further prevented direct oxidant-mediated diaphragm contractile dysfunction (P hypertension induces diaphragm contractile dysfunction via an oxidant-mediated mechanism that is prevented by HIIT.-Bowen, T. S., Eisenkolb, S., Drobner, J., Fischer, T., Werner, S., Linke, A., Mangner, N., Schuler, G., Adams, V. High-intensity interval training prevents oxidant-mediated diaphragm muscle weakness in hypertensive mice. © FASEB.

The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

Science.gov (United States)

McCully, Kilmer S

2015-01-01

The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. © 2015 by the Association of Clinical Scientists, Inc.

OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

Science.gov (United States)

Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

2016-02-01

Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. © 2016 American Society of Plant Biologists. All Rights Reserved.

PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

Energy Technology Data Exchange (ETDEWEB)

Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

2014-03-10

Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

Supplementation with Vitis vinifera L. skin extract improves insulin resistance and prevents hepatic lipid accumulation and steatosis in high-fat diet-fed mice.

Science.gov (United States)

Santos, Izabelle Barcellos; de Bem, Graziele Freitas; Cordeiro, Viviane Silva Cristino; da Costa, Cristiane Aguiar; de Carvalho, Lenize Costa Reis Marins; da Rocha, Ana Paula Machado; da Costa, Gisele França; Ognibene, Dayane Teixeira; de Moura, Roberto Soares; Resende, Angela Castro

2017-07-01

Nonalcoholic fatty liver disease is one of the most common complications of obesity. The Vitis vinifera L. grape skin extract (ACH09) is an important source of polyphenols, which are related to its antioxidant and antihyperglycemic activities. We hypothesized that ACH09 could also exert beneficial effects on metabolic disorders associated with obesity and evaluated ACH09's influence on high-fat (HF) diet-induced hepatic steatosis and insulin resistance in C57BL/6 mice. The animals were fed a standard diet (10% fat, control) or an HF diet (60% fat, HF) with or without ACH09 (200mg/[kg d]) for 12weeks. Our results showed that ACH09 reduced HF diet-induced body weight gain, prevented hepatic lipid accumulation and steatosis, and improved hyperglycemia and insulin resistance. The underlying mechanisms of these beneficial effects of ACH09 may involve the activation of hepatic insulin-signaling pathway because the expression of phosphorylated insulin receptor substrate-1, phosphatidylinositol 3-kinase, phosphorylated Akt serine/threonine kinase 1, and glucose transporter 2 was increased by ACH09 and correlated with improvement of hyperglycemia, hyperinsulinemia, and insulin resistance. ACH09 reduced the expression of the lipogenic factor sterol regulatory-element binding protein-1c in the liver and upregulated the lipolytic pathway (phosphorylated liver kinase B1/phosphorylated adenosine-monophosphate-activated protein kinase), which was associated with normal hepatic levels of triglyceride and cholesterol and prevention of steatosis. ACH09 prevented the hepatic oxidative damage in HF diet-fed mice probably by restoration of antioxidant activity. In conclusion, ACH09 protected mice from HF diet-induced obesity, insulin resistance, and hepatic steatosis. The regulation of hepatic insulin signaling pathway, lipogenesis, and oxidative stress may contribute to ACH09's protective effect. Copyright © 2017 Elsevier Inc. All rights reserved.

Preventing Bacterial Infections using Metal Oxides Nanocoatings on Bone Implant

Science.gov (United States)

Duceac, L. D.; Straticiuc, S.; Hanganu, E.; Stafie, L.; Calin, G.; Gavrilescu, S. L.

2017-06-01

Nowadays bone implant removal is caused by infection that occurs around it possibly acquired after surgery or during hospitalization. The purpose of this study was to reveal some metal oxides applied as coatings on bone implant thus limiting the usual antibiotics-resistant bacteria colonization. Therefore ZnO, TiO2 and CuO were synthesized and structurally and morphologically analized in order to use them as an alternative antimicrobial agents deposited on bone implant. XRD, SEM, and FTIR characterization techniques were used to identify structure and texture of these nanoscaled metal oxides. These metal oxides nanocoatings on implant surface play a big role in preventing bacterial infection and reducing surgical complications.

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei, E-mail: fchen@wayne.edu

2014-05-01

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: a role for nitric oxide and hydrogen sulfide

Science.gov (United States)

2012-01-01

Background In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic value. The current study explores the potential of skin and flesh extracts from a hard-necked Rocambole variety of purple garlic in preventing cardiomyocyte hypertrophy and cell death. Methods Norepinephrine (NE) was used to induce hypertrophy in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Cell death was measured as ratio of rod to round shaped cardiomyocytes. Fluorescent probes were used to measure apoptosis and oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO) and hydrogen sulfide (H2S) were used to elucidate the mechanism of action of garlic extracts. Garlic extract samples were also tested for alliin and allicin concentrations. Results Exposure of cardiomyocytes to NE induced an increase in cell size and cell death; this increase was significantly prevented upon treatment with garlic skin and flesh extracts. Norepinephrine increased apoptosis and oxidative stress in cardiomyocytes which was prevented upon pretreatment with skin and flesh extracts; NO, and H2S blockers significantly inhibited this beneficial effect. Allicin and alliin concentration were significantly higher in garlic flesh extract when compared to the skin extract. Conclusion These results suggest that both skin and flesh garlic extracts are effective in preventing NE induced cardiomyocyte hypertrophy and cell death. Reduction in oxidative stress may also play an important role in the anti-hypertrophic and anti-apoptotic properties of garlic extracts. These beneficial effects may in part be mediated by NO and H2S. PMID:22931510

Increased oxidative stress and anaerobic energy release, but blunted Thr172-AMPKα phosphorylation, in response to sprint exercise in severe acute hypoxia in humans.

Science.gov (United States)

Morales-Alamo, David; Ponce-González, Jesús Gustavo; Guadalupe-Grau, Amelia; Rodríguez-García, Lorena; Santana, Alfredo; Cusso, Maria Roser; Guerrero, Mario; Guerra, Borja; Dorado, Cecilia; Calbet, José A L

2012-09-01

AMP-activated protein kinase (AMPK) is a major mediator of the exercise response and a molecular target to improve insulin sensitivity. To determine if the anaerobic component of the exercise response, which is exaggerated when sprint is performed in severe acute hypoxia, influences sprint exercise-elicited Thr(172)-AMPKα phosphorylation, 10 volunteers performed a single 30-s sprint (Wingate test) in normoxia and in severe acute hypoxia (inspired Po(2): 75 mmHg). Vastus lateralis muscle biopsies were obtained before and immediately after 30 and 120 min postsprint. Mean power output and O(2) consumption were 6% and 37%, respectively, lower in hypoxia than in normoxia. O(2) deficit and muscle lactate accumulation were greater in hypoxia than in normoxia. Carbonylated skeletal muscle and plasma proteins were increased after the sprint in hypoxia. Thr(172)-AMPKα phosphorylation was increased by 3.1-fold 30 min after the sprint in normoxia. This effect was prevented by hypoxia. The NAD(+)-to-NADH.H(+) ratio was reduced (by 24-fold) after the sprints, with a greater reduction in hypoxia than in normoxia (P exercise in human skeletal muscle is altered in severe acute hypoxia, which abrogated Thr(172)-AMPKα phosphorylation, likely due to lower LKB1 activation by SIRT1.

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Science.gov (United States)

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

Directory of Open Access Journals (Sweden)

Weckwerth Wolfram

2005-11-01

Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase-2

Science.gov (United States)

Andersen, Joshua L; Johnson, Carrie E; Freel, Christopher D; Parrish, Amanda B; Day, Jennifer L; Buchakjian, Marisa R; Nutt, Leta K; Thompson, J Will; Moseley, M Arthur; Kornbluth, Sally

2009-01-01

The apoptotic initiator caspase-2 has been implicated in oocyte death, in DNA damage- and heat shock-induced death, and in mitotic catastrophe. We show here that the mitosis-promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase-2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase-2 interdomain, prevents caspase-2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase-2 detected during interphase was lost in mitosis. Expression of S340A non-phosphorylatable caspase-2 abrogated mitotic suppression of caspase-2 and apoptosis in various settings, including oocytes induced to undergo cdk1-dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase-2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase-2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase-2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur. PMID:19730412

Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

DEFF Research Database (Denmark)

Powell, K A; Valova, V A; Malladi, C S

2000-01-01

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding ...... assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine...... the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus...

Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

DEFF Research Database (Denmark)

Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra

2015-01-01

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes......) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation...... of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association...

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL

Directory of Open Access Journals (Sweden)

Hastie C James

2006-01-01

Full Text Available Abstract Background Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. Results Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-XL. Conclusion All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

International Nuclear Information System (INIS)

Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

2011-01-01

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N G -nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ► Puerarin induced the phosphorylation of eNOS and the production of NO. ► Puerarin activated eNOS through ER-dependent PI3-kinase and Ca 2+ -dependent AMPK. ► Puerarin-induced NO was involved in the inhibition of NF-kB activation. ► Puerarin may help for prevention of vascular dysfunction and diabetes.

Genistein suppresses adhesion-induced protein tyrosine phosphorylation and invasion of B16-BL6 melanoma cells.

Science.gov (United States)

Yan, C; Han, R

1998-07-03

Protein tyrosine phosphorylation occurs as one of the earlier events in cancer cell-extracellular matrix (ECM) interaction. With immunoblot analysis and immunofluorescence microscopy, genistein was found to suppress the tyrosine phosphorylation of proteins located at the cell periphery, including a 125 kDa protein, when B16-BL6 melanoma cells attached to and interacted with ECM. When accompanied by the suppression of adhesion-induced protein tyrosine phosphorylation, the invasive potential of B16-BL6 cells through reconstituted basement membrane was decreased significantly. However, neither adhesive capability nor cell growth was significantly affected by genistein. Therefore, the interruption of cancer cell-ECM interaction by suppression of protein tyrosine phosphorylation may contribute to invasion prevention of genistein.

The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

Science.gov (United States)

Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

2004-05-04

A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

Science.gov (United States)

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

2016-05-10

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

Science.gov (United States)

Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

2016-11-01

Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

Prevention by lactic acid bacteria of the oxidation of human LDL.

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Terahara, M; Kurama, S; Takemoto, N

2001-08-01

Ether extracts of lactic acid bacteria were analyzed for prevention of the oxidation of erythrocyte membrane and human low-density lipoprotein in vivo. Streptococcus thermophilus 1131 and Lactobacillus delbrueckii subsp. bulgaricus 2038, yogurt starters, were chosen as test-strains, and ether extracts of these cultures were used as samples. Both strain 1131 and strain 2038 produced radical scavengers and inhibited oxidation of erythrocyte membranes and low-density lipoproteins. The antioxidative activity of strain 2038 was higher than that of strain 1131.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

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Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

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Kanae Ando

2016-03-01

Full Text Available Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD. β-amyloid (Aβ lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

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Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

2017-01-01

ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

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Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

2015-11-18

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

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Mohan Babu

Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

Mitochondrial toxicity of selective COX-2 inhibitors via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria

DEFF Research Database (Denmark)

Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

2016-01-01

Cyclooxygenase-2 (COX-2) inhibitors (coxibs) are non-steroidal anti-inflammatory drugs (NSAIDs) designed to selectively inhibit COX-2. However, drugs of this therapeutic class are associated with drug induced liver injury (DILI) and mitochondrial injury is likely to play a role. The effects...... of selective COX-2 inhibitors on inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria were investigated. The order of potency of inhibition of ATP synthesis was: lumiracoxib (IC50: 6.48 ± 2.74 μM)>celecoxib (IC50: 14.92 ± 6.40 μM)>valdecoxib (IC50: 161.4 ± 28.6 μM)>rofecoxib (IC50...... correlation (with r(2)=0.921) was observed between the potency of inhibition of ATP synthesis and the log P values. The in vitro metabolism of coxibs in rat liver mitochondria yielded for each drug substance a major single metabolite and identified a hydroxy metabolite with each of the coxibs...

The Stromal Microenvironment Modulates Mitochondrial Oxidative Phosphorylation in Chronic Lymphocytic Leukemia Cells

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Hima V. Vangapandu

2017-10-01

Full Text Available Peripheral blood chronic lymphocytic leukemia (CLL cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow–derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively. Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.

Electron transport phosphorylation in rumen butyrivibrios: unprecedented ATP yield for glucose fermentation to butyrate

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Timothy eHackmann

2015-06-01

Full Text Available From a genomic analysis of rumen butyrivibrios (Butyrivibrio and Pseudobutyrivibrio spp., we have re-evaluated the contribution of electron transport phosphorylation to ATP formation in this group. This group is unique in that most (76% genomes were predicted to possess genes for both Ech and Rnf transmembrane ion pumps. These pumps act in concert with the NifJ and Bcd-Etf to form a electrochemical potential (ΔμH+ and ΔμNa+, which drives ATP synthesis by electron transport phosphorylation. Of the 62 total butyrivibrio genomes currently available from the Hungate 1000 project, all 62 were predicted to possess NifJ, which reduces oxidized ferredoxin (Fdox during pyruvate conversion to acetyl-CoA. All 62 possessed all subunits of Bcd-Etf, which reduces Fdox and oxidizes reduced NAD (NADred during crotonyl-CoA reduction. Additionally, 61 genomes possessed all subunits of the Rnf, which generates ΔμH+ or ΔμNa+ from oxidation of reduced Fd and reduction of oxidized NAD (NADox. Further, 47 genomes possessed all 6 subunits of the Ech, which generates ΔμH+ from oxidation of reduced Fd (Fdred. For glucose fermentation to butyrate and H2, the electrochemical potential established should drive synthesis of ~1.5 ATP by the F0F1-ATP synthase (possessed by all 62 genomes. The total yield is ~4.5 ATP/glucose after accounting for 3 ATP formed by classic substrate-level phosphorylation, and it is one the highest yields for any glucose fermentation. The yield was the same when unsaturated fatty acid bonds, not H+, served as the electron acceptor (as during biohydrogenation. Possession of both Ech and Rnf had been previously documented in only a few sulfate-reducers, was rare in other rumen prokaryotic genomes in our analysis, and may confer an energetic advantage to rumen butyrivibrios. This unique energy conservation system might enhance the butyrivibrios’ ability to overcome growth inhibition by unsaturated fatty acids, as postulated herein.

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

Energy Technology Data Exchange (ETDEWEB)

Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

ERK phosphorylation regulates sleep and plasticity in Drosophila.

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William M Vanderheyden

Full Text Available Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK(SEM pan-neuronally in the adult fly using GeneSwitch (Gsw Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

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Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

2011-11-01

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

Energy Technology Data Exchange (ETDEWEB)

Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2011-11-15

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

Thymosin-β4 (Tβ4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells

Science.gov (United States)

Reyes-Gordillo, Karina; Shah, Ruchi; Popratiloff, Anastas; Fu, Sidney; Hindle, Anna; Brody, Frederick; Rojkind, Marcos

2011-01-01

Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)-β receptor and the proliferative response to platelet-derived growth factor-ββ (PDGF-ββ) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-β4 (Tβ4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that Tβ4 inhibited cell proliferation and migration and prevented the expression of PDGF-β receptor (PDGF-βr), α-smooth muscle actin and α1(I) collagen mRNAs. Tβ4 also inhibited the reappearance of PDGF-βr after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-βr phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that Tβ4 prevented this effect. Tβ4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco's modified Eagle's medium or Dulbecco's minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that Tβ4 by sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, Tβ4 has the potential to be an antifibrogenic agent. PMID:21514425

Haptoglobin is required to prevent oxidative stress and muscle atrophy.

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Enrico Bertaggia

Full Text Available BACKGROUND: Oxidative stress (OS plays a major role on tissue function. Several catabolic or stress conditions exacerbate OS, inducing organ deterioration. Haptoglobin (Hp is a circulating acute phase protein, produced by liver and adipose tissue, and has an important anti-oxidant function. Hp is induced in pro-oxidative conditions such as systemic inflammation or obesity. The role of systemic factors that modulate oxidative stress inside muscle cells is still poorly investigated. RESULTS: We used Hp knockout mice (Hp-/- to determine the role of this protein and therefore, of systemic OS in maintenance of muscle mass and function. Absence of Hp caused muscle atrophy and weakness due to activation of an atrophy program. When animals were stressed by acute exercise or by high fat diet (HFD, OS, muscle atrophy and force drop were exacerbated in Hp-/-. Depending from the stress condition, autophagy-lysosome and ubiquitin-proteasome systems were differently induced. CONCLUSIONS: Hp is required to prevent OS and the activation of pathways leading to muscle atrophy and weakness in normal condition and upon metabolic challenges.

Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans

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Veronica Codoni

2016-10-01

Full Text Available Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules. Six modules were found preserved (P < 10−4 in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR = 8.9 × 10−11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS pathway. This pathway was also found significantly (FDR < 10−4 enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10−8 the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans.

Respiration and phosphorylation in liver and kidney mitochondria of rats exposed to high-energy gamma and beta radiation

Energy Technology Data Exchange (ETDEWEB)

Mokhoreva, S I; Vetlugina, N S

1973-01-01

The effect of whole-body irradiation with ..gamma.. rays (radiation source /sup 60/Co) at 40 rad and ..beta.. rays (source, a linear accelerator, electron energy 25 MeV) at 43 rad on oxidative phosphorylation in liver and kidney mitochondria was studied in rats. Gamma radiation gradually slowed the esterification of phosphate and respiratory rate during the oxidation of succinate in the liver and kidney mitochondria. The decrease was largest on day 15 after irradiation. However, the P/O ratio did not decrease by more than 10 to 12 percent. Despite the oxidation of glutamate in the mitochondria, respiration, phosphate consumption, and P/O ratio scarcely changed. Irradiation with electrons slowed the rate of oxidation of succinate and glutamate in liver mitochondria within 3 to 7 days. Phosphate consumption decreased at the same time so that the P/O ratio remained unchanged. Beta irradiation had virtually no effect on liver mitochondria. There is a discussion of the mechanism of action of high-energy radiation on the phosphorylation system of the mitochondria.

SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

DEFF Research Database (Denmark)

Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

2008-01-01

spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

Science.gov (United States)

Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

2010-08-01

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

USING POLYMERIC HYDROGEN GETTERS TO PREVENT COMBUSTIBLE ATMOSPHERES DURING INTERIM SAFE STORAGE OF PLUTONIUM OXIDE

International Nuclear Information System (INIS)

Woodsmall, T

2007-01-01

Nuclear Materials Management (NMM) of WSRC has recently installed the capability to perform both non-destructive and destructive examination of 3013 containers of Pu oxide in accordance with DOE-STD-3013. The containers will be opened and the oxide will be sampled for analysis. The remaining bulk oxide must then be safely stored in a non-3013-compliant configuration. Available processing equipment and controls cannot prevent the oxide from adsorbing moisture during this process. Subsequent radiolysis of moisture during storage may generate combustible quantities of gases while waiting final processing, and satisfying DOE Interim Safe Storage Criteria (ISSC) would require that storage containers be vented at impractical frequencies. With support from an independent National Laboratory, WSRC/NMM has demonstrated that a commercial hydrogen getter material will effectively prevent the accumulation of combustible gas concentrations. A project overview, including storage requirements and strategies, as well as getter technology, current test results, and anticipated future developments will be addressed

Photoassist-phosphorylated TiO2 as a catalyst for direct formation of 5-(hydroxymethyl)furfural from glucose.

Science.gov (United States)

Hattori, Masashi; Kamata, Keigo; Hara, Michikazu

2017-02-01

Photo-assisted phosphorylation of an anatase TiO 2 catalyst was examined to improve its catalytic performance for the direct production of 5-(hydroxymethyl)furfural (HMF), a versatile chemical platform, from glucose. In phosphorylation based on simple esterification between phosphoric acid and surface OH groups on anatase TiO 2 with water-tolerant Lewis acid sites, the density of phosphates immobilized on TiO 2 is limited to 2 phosphates nm -2 , which limits selective HMF production. Phosphorylation of the TiO 2 surface under fluorescent light irradiation increases the surface phosphate density to 50%, which is higher than the conventional limit, thus preventing the adsorption of hydrophilic glucose molecules on TiO 2 and resulting in a more selective HMF production over photoassist-phosphorylated TiO 2 .

Gallic acid prevents nonsteroidal anti-inflammatory drug-induced gastropathy in rat by blocking oxidative stress and apoptosis.

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Pal, Chinmay; Bindu, Samik; Dey, Sumanta; Alam, Athar; Goyal, Manish; Iqbal, Mohd Shameel; Maity, Pallab; Adhikari, Susanta S; Bandyopadhyay, Uday

2010-07-15

Nonsteroidal anti-inflammatory drug (NSAID)-induced oxidative stress plays a critical role in gastric mucosal cell apoptosis and gastropathy. NSAIDs induce the generation of hydroxyl radical ((*)OH) through the release of free iron, which plays an important role in developing gastropathy. Thus, molecules having both iron-chelating and antiapoptotic properties will be beneficial in preventing NSAID-induced gastropathy. Gallic acid (GA), a polyphenolic natural product, has the capacity to chelate free iron. Here, we report that GA significantly prevents, as well as heals, NSAID-induced gastropathy. In vivo, GA blocks NSAID-mediated mitochondrial oxidative stress by preventing mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. In vitro, GA scavenges free radicals and blocks (*)OH-mediated oxidative damage. GA also attenuates gastric mucosal cell apoptosis in vivo as well as in vitro in cultured gastric mucosal cells as evident from the TUNEL assay. GA prevents NSAID-induced activation of caspase-9, a marker for the mitochondrial pathway of apoptosis, and restores NSAID-mediated collapse of the mitochondrial transmembrane potential and dehydrogenase activity. Thus, the inhibition of mitochondrial oxidative stress by GA is associated with the inhibition of NSAID-induced mitochondrial dysfunction and activation of apoptosis in gastric mucosal cells, which are responsible for gastric injury or gastropathy. Copyright 2010 Elsevier Inc. All rights reserved.

Biochemistry of storage lesions of red cell and platelet concentrates: A continuous fight implying oxidative/nitrosative/phosphorylative stress and signaling.

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Rinalducci, Sara; Zolla, Lello

2015-06-01

The mechanisms responsible for the reduced lifespan of transfused red blood cells (RBCs) and platelets (PLTs) are still under investigation, however one explanation refers to the detrimental biochemical changes occurring during ex vivo storage of these blood products. A myriad of historical and more recent studies has contributed to advance our understanding of storage lesion. Without any doubts, proteomics had great impact on transfusion medicine by profiling the storage-dependent changes in the total detectable protein pool of both RBCs and PLTs. This review article focuses on the role of oxidative/nitrosative stress in developing RBC and PLT storage lesions, with a special glance at its biochemistry and cross-talk with phosphorylative signal transduction. In this sense, we enlighten the potential contribution of new branches of proteomics in identifying novel points of intervention for the improvement of blood product quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bee products prevent agrichemical-induced oxidative damage in fish.

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Daiane Ferreira

Full Text Available In southern South America and other parts of the world, aquaculture is an activity that complements agriculture. Small amounts of agrichemicals can reach aquaculture ponds, which results in numerous problems caused by oxidative stress in non-target organisms. Substances that can prevent or reverse agrichemical-induced oxidative damage may be used to combat these effects. This study includes four experiments. In each experiment, 96 mixed-sex, 6-month-old Rhamdia quelen (118±15 g were distributed into eight experimental groups: a control group that was not exposed to contaminated water, three groups that were exposed to various concentrations of bee products, three groups that were exposed to various concentrations of bee products plus tebuconazole (TEB; Folicur 200 CE™ and a group that was exposed to 0.88 mg L(-1 of TEB alone (corresponding to 16.6% of the 96-h LC50. We show that waterborne bee products, including royal jelly (RJ, honey (H, bee pollen (BP and propolis (P, reversed the oxidative damage caused by exposure to TEB. These effects were likely caused by the high polyphenol contents of these bee-derived compounds. The most likely mechanism of action for the protective effects of bee products against tissue oxidation and the resultant damage is that the enzymatic activities of superoxide dismutase (SOD, catalase (CAT and glutathione-S-transferase (GST are increased.

Bee products prevent agrichemical-induced oxidative damage in fish.

Science.gov (United States)

Ferreira, Daiane; Rocha, Helio Carlos; Kreutz, Luiz Carlos; Loro, Vania Lucia; Marqueze, Alessandra; Koakoski, Gessi; da Rosa, João Gabriel Santos; Gusso, Darlan; Oliveira, Thiago Acosta; de Abreu, Murilo Sander; Barcellos, Leonardo José Gil

2013-01-01

In southern South America and other parts of the world, aquaculture is an activity that complements agriculture. Small amounts of agrichemicals can reach aquaculture ponds, which results in numerous problems caused by oxidative stress in non-target organisms. Substances that can prevent or reverse agrichemical-induced oxidative damage may be used to combat these effects. This study includes four experiments. In each experiment, 96 mixed-sex, 6-month-old Rhamdia quelen (118±15 g) were distributed into eight experimental groups: a control group that was not exposed to contaminated water, three groups that were exposed to various concentrations of bee products, three groups that were exposed to various concentrations of bee products plus tebuconazole (TEB; Folicur 200 CE™) and a group that was exposed to 0.88 mg L(-1) of TEB alone (corresponding to 16.6% of the 96-h LC50). We show that waterborne bee products, including royal jelly (RJ), honey (H), bee pollen (BP) and propolis (P), reversed the oxidative damage caused by exposure to TEB. These effects were likely caused by the high polyphenol contents of these bee-derived compounds. The most likely mechanism of action for the protective effects of bee products against tissue oxidation and the resultant damage is that the enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) are increased.

Exercise increases TBC1D1 phosphorylation in human skeletal muscle

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Jessen, Niels; An, Ding; Lihn, Aina S.; Nygren, Jonas; Hirshman, Michael F.; Thorell, Anders

2011-01-01

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS

Xanthine Oxidase Inhibitor, Allopurinol, Prevented Oxidative Stress, Fibrosis, and Myocardial Damage in Isoproterenol Induced Aged Rats.

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Sagor, Md Abu Taher; Tabassum, Nabila; Potol, Md Abdullah; Alam, Md Ashraful

2015-01-01

We evaluated the preventive effect of allopurinol on isoproterenol (ISO) induced myocardial infarction in aged rats. Twelve- to fourteen-month-old male Long Evans rats were divided into three groups: control, ISO, and ISO + allopurinol. At the end of the study, all rats were sacrificed for blood and organ sample collection to evaluate biochemical parameters and oxidative stress markers analyses. Histopathological examinations were also conducted to assess inflammatory cell infiltration and fibrosis in heart and kidneys. Our investigation revealed that the levels of oxidative stress markers were significantly increased while the level of cellular antioxidants, catalase activity, and glutathione concentration in ISO induced rats decreased. Treatment with allopurinol to ISO induced rats prevented the elevated activities of AST, ALT, and ALP enzymes, and the levels of lipid peroxidation products and increased reduced glutathione concentration. ISO induced rats also showed massive inflammatory cells infiltration and fibrosis in heart and kidneys. Furthermore, allopurinol treatment prevented the inflammatory cells infiltration and fibrosis in ISO induced rats. In conclusion, the results of our study suggest that allopurinol treatment is capable of protecting heart of ISO induced myocardial infarction in rats probably by preventing oxidative stress, inflammation, and fibrosis.

Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS Production

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Abrar Ul Haq Khan

2016-01-01

Full Text Available Tumor cell metabolism is altered during leukemogenesis. Cells performing oxidative phosphorylation (OXPHOS generate reactive oxygen species (ROS through mitochondrial activity. To limit the deleterious effects of excess ROS, certain gene promoters contain antioxidant response elements (ARE, e.g. the genes NQO-1 and HO-1. ROS induces conformational changes in KEAP1 and releases NRF2, which activates AREs. We show in vitro and in vivo that OXPHOS induces, both in primary leukemic cells and cell lines, de novo expression of NQO-1 and HO-1 and also the MAPK ERK5 and decreases KEAP1 mRNA. ERK5 activates the transcription factor MEF2, which binds to the promoter of the miR-23a–27a–24-2 cluster. Newly generated miR-23a destabilizes KEAP1 mRNA by binding to its 3′UTR. Lower KEAP1 levels increase the basal expression of the NRF2-dependent genes NQO-1 and HO-1. Hence, leukemic cells performing OXPHOS, independently of de novo ROS production, generate an antioxidant response to protect themselves from ROS.

Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

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Nisr, Raid B; Affourtit, Charles

2014-02-01

Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

International Nuclear Information System (INIS)

Smiley, R.M.; Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J.

1990-01-01

The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32 PO 4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M r 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

Phosphorylation coexists with O-GlcNAcylation in a plant virus protein and influences viral infection.

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Martínez-Turiño, Sandra; Pérez, José De Jesús; Hervás, Marta; Navajas, Rosana; Ciordia, Sergio; Udeshi, Namrata D; Shabanowitz, Jeffrey; Hunt, Donald F; García, Juan Antonio

2018-06-01

Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O-GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N-terminus and the beginning of the core region. In contrast with the 'yin-yang' mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O-GlcNAcylated (serines Ser-25, Ser-81, Ser-101 and Ser-118). Our findings show that PPV CP can be concurrently phosphorylated and O-GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser-25 in virions recovered from O-GlcNAcylation-deficient plants, suggesting that crosstalk between O-GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O-GlcNAcylation in the N-proximal segment of CP allows a fine-tuning of protein stability, providing the amount of CP required in each step of viral infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Glucose oxidation is critical for CD4+ T cell activation in a mouse model of systemic lupus erythematosus1

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Yin, Yiming; Choi, Seung-Chul; Xu, Zhiwei; Zeumer, Leilani; Kanda, Nathalie; Croker, Byron P.; Morel, Laurence

2015-01-01

We have previously shown that CD4+ T cells from B6.Sle1.Sle2.Sle3 (TC) lupus mice and patients present a high cellular metabolism, and a treatment combining 2-deoxyglucose (2DG), which inhibits glucose metabolism, and metformin, which inhibits oxygen consumption, normalized lupus T cell functions in vitro and reverted disease in mice. We obtained similar results with B6.lpr mice, another model of lupus, and showed that a continuous treatment is required to maintain the beneficial effect of metabolic inhibitors. Further, we investigated the relative roles of glucose oxidation and pyruvate reduction into lactate in this process.. Treatments of TC mice with either 2DG or metformin were sufficient to prevent autoimmune activation, while their combination was necessary to reverse the process. Treatment of TC mice with dichloroacetate (DCA), an inhibitor of lactate production, failed to effectively prevent or reverse autoimmune pathology. In vitro, CD4+ T cell activation upregulated the expression of genes that favor oxidative phosphorylation. Blocking glucose oxidation inhibited both IFNγ and IL-17 production, which could not be achieved by blocking pyruvate reduction. Overall, our data shows that targeting glucose oxidation is required to prevent or reverse lupus development in mice, which cannot be achieved by simply targeting the pyruvate-lactate conversion. PMID:26608911

Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

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Anat Elmann

2016-03-01

Full Text Available Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures was determined by lactate dehydrogenase (LDH activity, intracellular ROS levels were detected using 2′,7′-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS. These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1 and p44/42 mitogen-activated protein kinases (MAPK, and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

Phosphorylation of chicken growth hormone

International Nuclear Information System (INIS)

Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

1990-01-01

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

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Hossain, Ekhtear [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Karnan, Sivasundaram; Damdindorj, Lkhagvasuren [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Takahashi, Miyuki [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan)

2013-12-15

Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

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Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

2014-01-01

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

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Zhang, Jun-Xia; Qu, Xin-Liang; Chu, Peng; Xie, Du-Jiang; Zhu, Lin-Lin; Chao, Yue-Lin; Li, Li; Zhang, Jun-Jie; Chen, Shao-Liang

2018-05-01

Uncoupled endothelial nitric oxide synthase (eNOS) produces O 2 - instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O 2 - production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O 2 - burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O 2 - releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

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Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

2013-03-01

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis.

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Pino, María Teresa Luján; Marotte, Clarisa; Verstraeten, Sandra Viviana

2017-03-01

We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF - cells) or the presence (EGF + cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 μM) decreased cell viability in EGF - but not in EGF + cells. In EGF - cells, Tl(I) decreased mitochondrial potential, enhanced H 2 O 2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF - cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF - and EGF + cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF - cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF - and EGF + cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

Molecular mechanism of APC/C activation by mitotic phosphorylation.

Science.gov (United States)

Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-05-12

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.

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Harper, M E; Brand, M D

1994-08-01

Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for -2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (delta psi m) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

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Chang, Chun-Yu [Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Chao-Yu [School of Medical Imaging and Radiological Sciences, Chung Shan Medical University, Taichung, Taiwan (China); Department of Medical Imaging, Chung Shan Medical University Hospital, Taichung, Taiwan (China); School of Medicine, Chung Shan Medical University, Taichung, Taiwan (China); Kang, Chao-Kai [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, (China); Sher, Yuh-Pyng [Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan (China); Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan (China); Sheu, Wayne H.-H. [Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Division of Endocrinology and Metabolism, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan (China); School of Medicine, National Yang Ming University, Taipei, Taiwan (China); School of Medicine, National Defense Medical Center, Taipei, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Lee, Tsung-Han, E-mail: thlee@email.nchu.edu.tw [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, (China); Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Department of Biological Science and Technology, China Medical University, Taichung, Taiwan (China)

2014-09-15

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation.

Taurine protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways

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Chang, Chun-Yu; Shen, Chao-Yu; Kang, Chao-Kai; Sher, Yuh-Pyng; Sheu, Wayne H.-H.; Chang, Chia-Che; Lee, Tsung-Han

2014-01-01

Oxidized LDL (oxLDL) induces a pro-oxidative environment and promotes apoptosis, causing the progression of renal diseases in humans. Taurine is a semi-essential amino acid in mammals and has been shown to be a potent endogenous antioxidant. The kidney plays a pivotal role in maintaining the balance of taurine. However, the mechanisms underlying the protective effects of taurine against oxLDL-induced injury in renal epithelial cells have not been clarified. In the present study, we investigated the anti-apoptotic effects of taurine on human proximal tubular epithelial (HK-2) cells exposed to oxLDL and explored the related mechanisms. We observed that oxLDL increased the contents of ROS and of malondialdehyde (MDA), which is a lipid peroxidation by-product that acts as an indicator of the cellular oxidation status. In addition, oxLDL induced cell death and apoptosis in HK-2 cells. Pretreatment with taurine at 100 μM significantly attenuated the oxLDL-induced cytotoxicity. We determined that oxLDL triggered the phosphorylation of ERK and, in turn, the activation of p53 and other apoptosis-related events, including calcium accumulation, destabilization of the mitochondrial permeability and disruption of the balance between pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins. The malfunctions induced by oxLDL were effectively blocked by taurine. Thus, our results suggested that taurine exhibits potential therapeutic activity by preventing oxLDL-induced nephrotoxicity. The inhibition of oxLDL-induced epithelial apoptosis by taurine was at least partially due to its anti-oxidant activity and its ability to modulate the ERK and p53 apoptotic pathways. - Highlights: • Oxidized LDL induced cytotoxicity and apoptosis in HK-2 cells. • Pretreatment with taurine attenuated oxLDL-induced nephrotoxicity. • Taurine protected against renal damages through inhibition of ROS generation. • Taurine prevented apoptosis through modulation of the p53 phosphorylation

Radiation oxidative stress in cancer induction and prevention

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Meher, Prabodha Kumar; Mishra, Kaushala Prasad

2017-01-01

Exposure of cells to ionizing radiation causes generation of intracellular reactive oxygen species (ROS) which are implicated in the mechanism of carcinogenesis. Molecular steps involved in the transformation of normal cells to cancer cells have been enigmatic but generally believed to arise from aberration in cellular redox homeostasis. In normal cell function, a delicate balance is maintained between ROS generated in the metabolic process and level of endogenous antioxidant defense. ROS are known to regulate various cellular functions, such as cell division, signal transduction, and apoptosis. Cells experience oxidative stress when excess production of ROS occurs inside a cell upon exposure to external stress or agents. This redox imbalance affects the cellular functions due to DNA strand breaks, chromosomal aberrations, gene mutations, alteration in signal transduction, and inhibition of apoptosis leading to induction of cancer and other diseases. Radiation-induced ROS are involved in initiation and promotion of carcinogenesis. Therefore, detoxification of ROS by exogenous antioxidants including dietary polyphenols offers an important strategy for cancer prevention. Recent research results have shown that resistance of cancer stem cells to therapies is linked to low level of ROS. Interestingly, in vitro and in vivo experiments have reported that radiotherapy- and chemotherapy-induced ROS in cytosol sensitize the tumor cells to death, resulting in tumor growth retardation. This review is an attempt to delineate mechanisms of ROS in carcinogenesis and prevention by dietary compounds. Natural polyphenols and dietary antioxidants hold potential to prevent cancer. Interventions in ROS-mediated signal alteration, apoptosis activation, and modulation of epigenetic processes may offer effective cancer prevention strategy. (author)

Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

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So Ra Yoon

2017-01-01

Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

Properties of phosphorylated thymidylate synthase

DEFF Research Database (Denmark)

Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

2015-01-01

by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

Medroxyprogesterone acetate attenuates estrogen-induced nitric oxide production in human umbilical vein endothelial cells

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Oishi, Akira; Ohmichi, Masahide; Takahashi, Kazuhiro; Takahashi, Toshifumi; Mori-Abe, Akiko; Kawagoe, Jun; Otsu, Reiko; Mochizuki, Yoshiko; Inaba, Noriyuki; Kurachi, Hirohisa

2004-01-01

We report the novel observation that medroxyprogesterone acetate (MPA) attenuates the induction by 17β estradiol (E2) of both nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells. Although MPA had no effect on basal NO production or basal eNOS phosphorylation or activity, it attenuated the E2-induced NO production and eNOS phosphorylation and activity. Moreover, we examined the mechanism by which MPA attenuated the E2-induced NO production and eNOS phosphorylation. MPA attenuated the E2-induced phosphorylation of Akt, a kinase that phosphorylates eNOS. Treatment with pure progesterone receptor (PR) antagonist RU486 completely abolished the inhibitory effect of MPA on E2-induced Akt phosphorylation and eNOS phosphorylation. In addition, the effects of actinomycin D were tested to rule out the influence of genomic events mediated by nuclear PRs. Actinomycin D did not affect the inhibitory effect of MPA on E2-induced Akt phosphorylation. Furthermore, the potential roles of PRA and PRB were evaluated. In COS cells transfected with either PRA or PRB, MPA attenuated E2-induced Akt phosphorylation. These results indicate that MPA attenuated E2-induced NO production via an Akt cascade through PRA or PRB in a non-genomic manner

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

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Yadav, N; Kumar, S; Marlowe, T; Chaudhary, A K; Kumar, R; Wang, J; O'Malley, J; Boland, P M; Jayanthi, S; Kumar, T K S; Yadava, N; Chandra, D

2015-11-05

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency

Gene expression patterns of oxidative phosphorylation complex I subunits are organized in clusters.

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Yael Garbian

Full Text Available After the radiation of eukaryotes, the NUO operon, controlling the transcription of the NADH dehydrogenase complex of the oxidative phosphorylation system (OXPHOS complex I, was broken down and genes encoding this protein complex were dispersed across the nuclear genome. Seven genes, however, were retained in the genome of the mitochondrion, the ancient symbiote of eukaryotes. This division, in combination with the three-fold increase in subunit number from bacteria (N = approximately 14 to man (N = 45, renders the transcription regulation of OXPHOS complex I a challenge. Recently bioinformatics analysis of the promoter regions of all OXPHOS genes in mammals supported patterns of co-regulation, suggesting that natural selection favored a mechanism facilitating the transcriptional regulatory control of genes encoding subunits of these large protein complexes. Here, using real time PCR of mitochondrial (mtDNA- and nuclear DNA (nDNA-encoded transcripts in a panel of 13 different human tissues, we show that the expression pattern of OXPHOS complex I genes is regulated in several clusters. Firstly, all mtDNA-encoded complex I subunits (N = 7 share a similar expression pattern, distinct from all tested nDNA-encoded subunits (N = 10. Secondly, two sub-clusters of nDNA-encoded transcripts with significantly different expression patterns were observed. Thirdly, the expression patterns of two nDNA-encoded genes, NDUFA4 and NDUFA5, notably diverged from the rest of the nDNA-encoded subunits, suggesting a certain degree of tissue specificity. Finally, the expression pattern of the mtDNA-encoded ND4L gene diverged from the rest of the tested mtDNA-encoded transcripts that are regulated by the same promoter, consistent with post-transcriptional regulation. These findings suggest, for the first time, that the regulation of complex I subunits expression in humans is complex rather than reflecting global co-regulation.

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

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Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

2011-01-01

Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

International Nuclear Information System (INIS)

Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

2013-01-01

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Novel oxindole derivatives prevent oxidative stress-induced cell death in mouse hippocampal HT22 cells.

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Hirata, Yoko; Yamada, Chika; Ito, Yuki; Yamamoto, Shotaro; Nagase, Haruna; Oh-Hashi, Kentaro; Kiuchi, Kazutoshi; Suzuki, Hiromi; Sawada, Makoto; Furuta, Kyoji

2018-03-15

The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22 cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca 2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22 cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Alpha-1 Antitrypsin Prevents the Development of Preeclampsia Through Suppression of Oxidative Stress.

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Feng, Yaling; Xu, Jianjuan; Zhou, Qin; Wang, Rong; Liu, Nin; Wu, Yanqun; Yuan, Hua; Che, Haisha

2016-01-01

Preeclampsia (PE) and its complications have become the leading cause of maternal and fetal morbidity and mortality in the world. And the development of PE is still barely predictable and thus challenging to prevent and manage clinically. Oxidative stress contributes to the development of the disease. Our previous study demonstrated that exogenous Alpha-1 antitrypsin (AAT) played a cytoprotective role in vascular endothelial cell by suppressing oxidative stress. In this study, we aim to investigate whether AAT contributes to the development of PE, and to identify the mechanism behind these effects. We found that AAT levels were significantly decreased in placenta tissues from women with PE compared that of healthy women. Notably, we demonstrate that AAT injection is able to relieve the high blood pressure and reduce urine protein levels in a dose-dependent manner in PE mice. In addition, our results showed that AAT injection exhibited an anti-oxidative stress role by significantly reducing PE mediated-upregulation of ROS, MMP9 and MDA, and increasing the levels of SOD, eNOS, and GPx with increased dosage of AAT. Furthermore, we found that AAT injection inactivated PE mediated activation of PAK/STAT1/p38 signaling. These findings were confirmed in human samples. In conclusion, our study suggests that exogenous AAT injection increases the antioxidants and suppresses oxidative stress, and subsequent prevention of PE development through inactivation of STAT1/p38 signaling. Thus, AAT would become a potential strategy for PE therapy.

Alpha-1 antitrypsin prevents the development of preeclampsia through suppression of oxidative stress

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Yaling eFeng

2016-05-01

Full Text Available Preeclampsia (PE and its complications have become the leading cause of maternal and fetal morbidity and mortality in the world. And the development of PE is still barely predictable and thus challenging to prevent and manage clinically. Oxidative stress contributes to the development of the disease. Our previous study demonstrated that exogenous Alpha-1 antitrypsin (AAT played a cytoprotective role in vascular endothelial cell by suppressing oxidative stress. In this study, we aim to investigate whether AAT contributes to the development of PE, and to identify the mechanism behind these effects. We found that AAT levels were significantly decreased in placenta tissues from women with PE compared that of healthy women. Notably, we demonstrate that AAT injection is able to relieve the high blood pressure and reduce urine protein levels in a dose-dependent manner in PE mice. In addition, our results showed that AAT injection exhibited an anti-oxidative stress role by significantly reducing PE mediated-upregulation of ROS, MMP9 and MDA, and increasing the levels of SOD, eNOS and GPx with increased dosage of AAT. Furthermore, we found that AAT injection inactivated PE mediated activation of PAK/STAT1/p38 signaling. These findings were confirmed in human samples. In conclusion, our study suggests that exogenous AAT injection increases the antioxidants and suppresses oxidative stress, and subsequent prevention of PE development through inactivation of STAT1/p38 signaling. Thus, AAT would become a potential strategy for PE therapy.

Mcm2 phosphorylation and the response to replicative stress

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Stead Brent E

2012-05-01

Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

Energy Technology Data Exchange (ETDEWEB)

Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

2013-04-15

The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

Science.gov (United States)

Patel, Anant B; Lai, James C K; Chowdhury, Golam M I; Hyder, Fahmeed; Rothman, Douglas L; Shulman, Robert G; Behar, Kevin L

2014-04-08

Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

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Philippe Régnier

2015-05-01

Full Text Available Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI+/ion trap-MS characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM. No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

International Nuclear Information System (INIS)

Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

2008-01-01

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

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Nicola V. Huck

2017-12-01

Full Text Available Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

International Nuclear Information System (INIS)

Wu Defeng; Cederbaum, Arthur

2006-01-01

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N G -Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 ± 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 ± 5%, while, SNAP or DETA-NONO increased viability to 66 ± 8 or 71 ± 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and

Phosphorylation of human INO80 is involved in DNA damage tolerance

International Nuclear Information System (INIS)

Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

2012-01-01

Highlights: ► Depletion of hINO80 significantly reduced PCNA ubiquitination. ► Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. ► Western blot analyses showed phosphorylated hINO80 C-terminus. ► Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

Astragaloside IV attenuates experimental autoimmune encephalomyelitis of mice by counteracting oxidative stress at multiple levels.

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Yixin He

Full Text Available Multiple sclerosis (MS is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI, a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.

Astragaloside IV attenuates experimental autoimmune encephalomyelitis of mice by counteracting oxidative stress at multiple levels.

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He, Yixin; Du, Min; Gao, Yan; Liu, Hongshuai; Wang, Hongwei; Wu, Xiaojun; Wang, Zhengtao

2013-01-01

Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease found mostly in young adults in the western world. Oxidative stress induced neuronal apoptosis plays an important role in the pathogenesis of MS. In current study, astragaloside IV (ASI), a natural saponin molecule isolated from Astragalus membranceus, given at 20 mg/kg daily attenuated the severity of experimental autoimmune encephalomyelitis (EAE) in mice significantly. Further studies disclosed that ASI treatment inhibited the increase of ROS and pro-inflammatory cytokine levels, down-regulation of SOD and GSH-Px activities, and elevation of iNOS, p53 and phosphorylated tau in central nervous system (CNS) as well as the leakage of BBB of EAE mice. Meanwhile, the decreased ratio of Bcl-2/Bax was reversed by ASI. Moreover, ASI regulated T-cell differentiation and infiltration into CNS. In neuroblast SH-SY5Y cells, ASI dose-dependently reduced cellular ROS level and phosphorylation of tau in response to hydrogen peroxide challenge by modulation of Bcl-2/Bax ratio. ASI also inhibited activation of microglia both in vivo and in vitro. iNOS up-regulation induced by IFNγ stimulation was abolished by ASI dose-dependently in BV-2 cells. In summary, ASI prevented the severity of EAE progression possibly by counterbalancing oxidative stress and its effects via reduction of cellular ROS level, enhancement of antioxidant defense system, increase of anti-apoptotic and anti-inflammatory pathways, as well as modulation of T-cell differentiation and infiltration into CNS. The study suggested ASI may be effective for clinical therapy/prevention of MS.

Quercetin prevents chronic unpredictable stress induced behavioral dysfunction in mice by alleviating hippocampal oxidative and inflammatory stress.

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Mehta, Vineet; Parashar, Arun; Udayabanu, Malairaman

2017-03-15

It is now evident that chronic stress is associated with anxiety, depression and cognitive dysfunction and very few studies have focused on identifying possible methods to prevent these stress-induced disorders. Previously, we identified abundance of quercetin in Urtica dioica extract, which efficiently attenuated stress related complications. Therefore, current study was designed to investigate the effect of quercetin on chronic unpredicted stress (CUS) induced behavioral dysfunction, oxidative stress and neuroinflammation in the mouse hippocampus. Animals were subjected to unpredicted stress for 21days, during which 30mg/kg quercetin was orally administered to them. Effect of CUS and quercetin treatment on animal behavior was assessed between day 22-26. Afterward, the hippocampus was processed to evaluate neuronal damage, oxidative and inflammatory stress. Results revealed that stressed animals were highly anxious (Elevated Plus Maze and Open Field), showed depressive-like behavior (sucrose preference task), performed poorly in short-term and long-term associative memory task (passive avoidance step-through task) and displayed reduced locomotion (open field). Quercetin alleviated behavioral dysfunction in chronically stressed animals. Compared to CUS, quercetin treatment significantly reduced anxiety, attenuated depression, improved cognitive dysfunction and normalized locomotor activity. Further, CUS elevated the levels of oxidative stress markers (TBARS, nitric oxide), lowered antioxidants (total thiol, catalase), enhanced expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β and COX-2) in the hippocampus and damaged hippocampal neurons. Quercetin treatment significantly lowered oxidative and inflammatory stress and prevented neural damage. In conclusion, quercetin can efficiently prevent stress induced neurological complications by rescuing brain from oxidative and inflammatory stress. Copyright © 2017 Elsevier Inc. All rights reserved.

De Novo Synthesis of Phosphorylated Triblock Copolymers with Pathogen Virulence-Suppressing Properties That Prevent Infection-Related Mortality

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Mao, Jun; Zaborin, Alexander; Poroyko, Valeriy; Goldfeld, David; Lynd, Nathaniel A. [McKetta; Chen, Wei [Institute; Tirrell, Matthew V. [Institute; Zaborina, Olga; Alverdy, John C.

2017-07-31

Phosphate is a key and universal "cue" in response to which bacteria either enhance their virulence when local phosphate is scarce or downregulate it when phosphate is adundant. Phosphate becomes depleted in the mammalian gut following physiologic stress and serves as a major trigger for colonizing bacteria to express virulence. This process cannot be reversed with oral supplementation of inorganic phosphate because it is nearly completely absorbed in the proximal small intestine. In the present study, we describe the de novo synthesis of phosphorylated polyethylene glycol compounds with three defined ABA (hydrophilic/-phobic/-philic) structures, ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20, and linear polymer PEG20k-Pi20 absent of the hydrophobic block. The 10k, 16k, and 20k demonstrate the molecular weights of the poly(ethylene glycol) block, and Pi10, Pi14, and Pi20 represent the repeating units of phosphate. Polymers were tested for their efficacy against Pseudomonas aeruginosa virulence in vitro and in vivo by assessing the expression of the phosphate sensing protein PstS, the production of key virulence factor pyocyanin, and Caenorhabditis elegans killing assays. Results indicate that all phosphorylated polymers suppressed phosphate sensing, virulence expression, and lethality in P. aeruginosa. Among all of the phosphorylated polymers, ABA-PEG20kPi20 displayed the greatest degree of protection against P. aeruginosa. To define the role of the hydrophobic core in ABA-PEG20k-Pi20 in the above response, we synthesized PEG20k-Pi20 in which the hydrophobic core is absent. Results indicate that the hypdrophobic core of ABA-PEG20k-Pi20 is a key structure in its protective effect against P. aeruginosa, in part due to its ability to coat the surface of bacteria. Taken together, the synthesis of novel polymers with defined structures and levels of phosphorylation may elucidate their antivirulence action against clinically important and lethal pathogens such as

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

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Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

2014-09-15

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM. © 2014. Published by The Company of Biologists Ltd.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

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Yan, C; Han, R

1997-01-01

Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds.

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Stier, Antoine; Massemin, Sylvie; Criscuolo, François

2014-12-01

Endotherms have evolved two major types of thermogenesis that allow them to actively produce heat in response to cold exposure, either through muscular activity (i.e. shivering thermogenesis) or through futile electro-chemical cycles (i.e. non-shivering thermogenesis). Amongst the latter, mitochondrial uncoupling is of key importance because it is suggested to drive heat production at a low cost in terms of oxidative stress. While this has been experimentally shown in mammals, the oxidative stress consequences of cold exposure and mitochondrial uncoupling are clearly less understood in the other class of endotherms, the birds. We compared metabolic and oxidative stress responses of zebra finches chronically treated with or without a chemical mitochondrial uncoupler (2,4-dinitrophenol: DNP), undergoing an acute (24 h) and a chronic (4 weeks) cold exposure (12 °C). We predicted that control birds should present at least a transient elevation of oxidative stress levels in response to cold exposure. This oxidative stress cost should be more pronounced in control birds than in DNP-treated birds, due to their lower basal uncoupling state. Despite similar increase in metabolism, control birds presented elevated levels of DNA oxidative damage in response to acute (but not chronic) cold exposure, while DNP-treated birds did not. Plasma antioxidant capacity decreased overall in response to chronic cold exposure. These results show that acute cold exposure increases oxidative stress in birds. However, uncoupling mitochondrial functioning appears as a putative compensatory mechanism preventing cold-induced oxidative stress. This result confirms previous observations in mice and underlines non-shivering thermogenesis as a putative key mechanism for endotherms in mounting a response to cold at a low oxidative cost.

Blockade of Drp1 rescues oxidative stress-induced osteoblast dysfunction

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Gan, Xueqi; Huang, Shengbin; Yu, Qing [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States); State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yu, Haiyang [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yan, Shirley ShiDu, E-mail: shidu@ku.edu [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States)

2015-12-25

Osteoblast dysfunction, induced by oxidative stress, plays a critical role in the pathophysiology of osteoporosis. However, the underlying mechanisms remain unclarified. Imbalance of mitochondrial dynamics has been closely linked to oxidative stress. Here, we reveal an unexplored role of dynamic related protein 1(Drp1), the major regulator in mitochondrial fission, in the oxidative stress-induced osteoblast injury model. We demonstrate that levels of phosphorylation and expression of Drp1 significantly increased under oxidative stress. Blockade of Drp1, through pharmaceutical inhibitor or gene knockdown, significantly protected against H{sub 2}O{sub 2}-induced osteoblast dysfunction, as shown by increased cell viability, improved cellular alkaline phosphatase (ALP) activity and mineralization and restored mitochondrial function. The protective effects of blocking Drp1 in H{sub 2}O{sub 2}-induced osteoblast dysfunction were evidenced by increased mitochondrial function and suppressed production of reactive oxygen species (ROS). These findings provide new insights into the role of the Drp1-dependent mitochondrial pathway in the pathology of osteoporosis, indicating that the Drp1 pathway may be targetable for the development of new therapeutic approaches in the prevention and the treatment of osteoporosis. - Highlights: • Oxidative stress is an early pathological event in osteoporosis. • Imbalance of mitochondrial dynamics are linked to oxidative stress in osteoporosis. • The role of the Drp1-dependent mitochondrial pathway in osteoporosis.

Glycogen phosphorylation and Lafora disease.

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Roach, Peter J

2015-12-01

Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes.

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Chen, Mingcang; Gu, Honggang; Ye, Yiyi; Lin, Bing; Sun, Lijuan; Deng, Weiping; Zhang, Jingzhe; Liu, Jianwen

2010-10-01

Increasing evidence regarding free radical generating agents and the inflammatory process suggest that accumulation of reactive oxygen species (ROS) could involve hepatotoxicity. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, antihypercholesterolemic, and anticarcinogenic actions. However, the cytoprotection and mechanism of hesperidin to neutralize oxidative stress in human hepatic L02 cells remain unclear. In this work, we assessed the capability of hesperidin to prevent tert-butyl hydroperoxide (t-BuOOH)-induced cell damage by augmenting cellular antioxidant defense. Hesperidin significantly protected hepatocytes against t-BuOOH-induced cell cytotoxicity, such as mitochondrial membrane potential (MMP) deplete and lactate dehydrogenase (LDH) release. Hesperidin also remarkably prevented indicators of oxidative stress, such as the ROS and lipid peroxidation level in a dose-dependent manner. Western blot showed that hesperidin facilitated ERK/MAPK phosphorylation which appeared to be responsible for nuclear translocation of Nrf2, thereby inducing cytoprotective heme oxygenase-1 (HO-1) expression. Based on the results described above, it suggested that hesperidin has potential as a therapeutic agent in the treatment of oxidative stress-related hepatocytes injury and liver dysfunctions. Copyright © 2010 Elsevier Ltd. All rights reserved.

A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

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Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

2016-01-01

ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

Fibronectin phosphorylation by ecto-protein kinase

International Nuclear Information System (INIS)

Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

1988-01-01

The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

Threonine phosphorylation of rat liver glycogen synthase

International Nuclear Information System (INIS)

Arino, J.; Arro, M.; Guinovart, J.J.

1985-01-01

32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation.

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Pallavi Sontakke

Full Text Available The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

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Baronian, Grégory; Ginda, Katarzyna; Berry, Laurence; Cohen-Gonsaud, Martin; Zakrzewska-Czerwińska, Jolanta; Jakimowicz, Dagmara; Molle, Virginie

2015-01-01

Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

Directory of Open Access Journals (Sweden)

Grégory Baronian

Full Text Available Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

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Heo, Jeongyeon; Kim, Jae Geun; Kim, Sunghwan; Kang, Hara

2017-12-23

Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy. © 2017 Wiley Periodicals, Inc.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

Science.gov (United States)

Jang, Hoon; Na, Younghwa; Hong, Kwonho; Lee, Sangho; Moon, Sohyeon; Cho, Minha; Park, Miseon; Lee, Ok-Hee; Chang, Eun Mi; Lee, Dong Ryul; Ko, Jung Jae; Lee, Woo Sik; Choi, Youngsok

2017-10-01

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27 Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27 Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y.

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Vidau, Cyril; González-Polo, Rosa A; Niso-Santano, Mireia; Gómez-Sánchez, Rubén; Bravo-San Pedro, José M; Pizarro-Estrella, Elisa; Blasco, Rafael; Brunet, Jean-Luc; Belzunces, Luc P; Fuentes, José M

2011-12-01

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

Science.gov (United States)

Pang, Yefei; Dong, Jing; Thomas, Peter

2015-05-15

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways. Copyright © 2015 the American Physiological Society.

Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

International Nuclear Information System (INIS)

Pendergast, A.M.

1986-01-01

The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

Effect of Curcumin on Phosphorylation of AMPK and ACC in C2C12 Skeletal Muscle Cells

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F Ghanbarzadeh

2013-10-01

Full Text Available Introduction: AMP activated protein kinase (AMPK as key regulators of cell metabolism, plays a major role in the activation of catabolic pathways, such as glucose transport and fatty acid oxidation. Thus, activation of this pathway can be used in the treatment of diabetes and metabolic syndrome. Many studied proposed the effectiveness of the polyphenols present in rhizomes of turmeric (curcumin on diabetes and its related complications. Therefore, this study investigated the effects of curcumin as an activator of AMPK pathway in C2C12 muscle cells. Methods: This study was done on C2C12 skeletal muscle cell line. The cells were classified into two distinct groups: first group was treated with 40µM curcumin and the second one with 0.1% DMSO as a negative control. The phosphorylated (AMPK and phosphorylated acetyl COA carboxylase (ACC were evaluated and compared by Western blotting technique. Results: intracellular phosphorylated AMPK protein content in Curcumin-treated group was 132.6% and ACC protein phosphorylated was 366.47%. Conclusion: This study showed that the levels of phosphorylated AMPK and ACC protein in cells treated with curcumin are higher than the negative control. Thus curcumin can be regarded as an activator of AMPK activity in these cells and can assist as a potential target for making anti diabetic medecine that has a synergistic activity with insulin.

Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger.

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Wang, Lu; Zhang, Jianhua; Cao, Zhanglei; Wang, Yajun; Gao, Qiang; Zhang, Jian; Wang, Depei

2015-01-16

The spore germination rate and growth characteristics were compared between the citric acid high-yield strain Aspergillus niger CGMCC 5751 and A. niger ATCC 1015 in media containing antimycin A or DNP. We inferred that differences in citric acid yield might be due to differences in energy metabolism between these strains. To explore the impact of energy metabolism on citric acid production, the changes in intracellular ATP, NADH and NADH/NAD+ were measured at various fermentation stages. In addition, the effects of antimycin A or DNP on energy metabolism and citric acid production was investigated by CGMCC 5751. By comparing the spore germination rate and the extent of growth on PDA plates containing antimycin A or DNP, CGMCC 5751 was shown to be more sensitive to antimycin A than ATCC 1015. The substrate-level phosphorylation of CGMCC 5751 was greater than that of ATCC 1015 on PDA plates with DNP. DNP at tested concentrations had no apparent effect on the growth of CGMCC 5751. There were no apparent effects on the mycelial morphology, the growth of mycelial pellets or the dry cell mass when 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP was added to medium at the 24-h time point. The concentrations of intracellular ATP, NADH and NADH/NAD+ of CGMCC 5751 were notably lower than those of ATCC 1015 at several fermentation stages. Moreover, at 96 h of fermentation, the citric acid production of CGMCC 5751 reached up to 151.67 g L(-1) and 135.78 g L(-1) by adding 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP, respectively, at the 24-h time point of fermentation. Thus, the citric acid production of CGMCC 5751 was increased by 19.89% and 7.32%, respectively. The concentrations of intracellular ATP, NADH and NADH/NAD+ of the citric acid high-yield strain CGMCC 5751 were notably lower than those of ATCC 1015. The excessive ATP has a strong inhibitory effect on citric acid accumulation by A. niger. Increasing NADH oxidation and appropriately reducing the concentration of

Eicosapentaenoic acid abolishes inhibition of insulin-induced mTOR phosphorylation by LPS via PTP1B downregulation in skeletal muscle.

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Wei, Hong-Kui; Deng, Zhao; Jiang, Shu-Zhong; Song, Tong-Xing; Zhou, Yuan-Fei; Peng, Jian; Tao, Ya-Xiong

2017-01-05

Dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) increase insulin signaling in skeletal muscle. In the current study, we investigated the effect of eicosapentaenoic acid (EPA) on insulin-induced mammalian target of rapamycin (mTOR) phosphorylation in myotubes. We showed that EPA did not affect basal and insulin-induced mTOR phosphorylation in myotubes. However, EPA abolished lipopolysaccharide (LPS) -induced deficiency in insulin signaling (P  0.05). In myotubes, LPS stimulated PTP1B expression via NF-κB and activation protein-1 (AP1). Pre-incubation of 50 μM EPA prevented the LPS-induced activation of AP1 and NF-κΒ as well as PTP1B expression (P < 0.05). Interestingly, incubation of peroxisome proliferator-activated receptor γ (PPARγ) antagonist (GW9662) prior to EPA treatment, the effect of EPA on insulin-induced mTOR phosphorylation was blocked. Accordingly, EPA did not inhibit the LPS-induced activation of AP1 or NF-κΒ as well as PTP1B expression when incubation of GW9662 prior to EPA treatment. The in vivo study showed that EPA prevented LPS-induced PTPT1B expression and a decrease in insulin-induced mTOR phosphorylation in muscle of mice. In summary, EPA abolished LPS inhibition of insulin-induced mTOR phosphorylation in myotubes, and one of the key mechanisms was to inhibit AP1 and NF-κB activation and PTP1B transcription. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

International Nuclear Information System (INIS)

Callahan, K.S.; Harlan, J.M.

1986-01-01

Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H 2 O 2 or glucose/glucose oxidase (GO)-generated H 2 O 2 . EC injury was assessed by 51 Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60 0 C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H 2 O 2 as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo

Phosphorylation of human skeletal muscle myosin

International Nuclear Information System (INIS)

Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

1986-01-01

Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

Science.gov (United States)

Choi, Sung E; Kwon, Sanghoon; Seok, Sunmi; Xiao, Zhen; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Shinoda, Kosaku; Kajimura, Shingo; Kemper, Byron; Kemper, Jongsook Kim

2017-08-01

Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases. Copyright © 2017 American Society for Microbiology.

Prevention of dopaminergic neurotoxicity by targeting nitric oxide and peroxynitrite: implications for the prevention of methamphetamine-induced neurotoxic damage.

Science.gov (United States)

Imam, S Z; Islam, F; Itzhak, Y; Slikker, W; Ali, S F

2000-09-01

Methamphetamine (METH) is a neurotoxic psychostimulant that produces catecholaminergic brain damage by producing oxidative stress and free radical generation. The role of oxygen and nitrogen radicals is well documented as a cause of METH-induced neurotoxic damage. In this study, we have obtained evidence that METH-induced neurotoxicity is the resultant of interaction between oxygen and nitrogen radicals, and it is mediated by the production of peroxynitrite. We have also assessed the effects of inhibitors of neuronal nitric oxide synthase (nNOS) as well as scavenger of nitric oxide and a peroxynitrite decomposition catalyst. Significant protective effects were observed with the inhibitor of nNOS, 7-nitroindazole (7-NI), as well as by the selective peroxynitrite scavenger or decomposition catalyst, 5,10,15,20-tetrakis(2,4,6-trimethyl-3,5-sulfonatophenyl)porphyrinato iron III (FeTPPS). However, the use of a nitric oxide scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), did not provide any significant protection against METH-induced hyperthermia or peroxynitrite generation and the resulting dopaminergic neurotoxicity. In particular, treatment with FeTPPS completely prevented METH-induced hyperthermia, peroxynitrite production, and METH-induced dopaminergic depletion. Together, these data demonstrate that METH-induced dopaminergic neurotoxicity is mediated by the generation of peroxynitrite, which can be selectively protected by nNOS inhibitors or peroxynitrite scavenger or decomposition catalysts.

SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation of ASK-1 and dissociation of the ASK-1-Trx complex.

Science.gov (United States)

Madan, Esha; Gogna, Rajan; Kuppusamy, Periannan; Bhatt, Madan; Mahdi, Abbas Ali; Pati, Uttam

2013-04-01

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

DEFF Research Database (Denmark)

Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

2000-01-01

Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

DEFF Research Database (Denmark)

Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

2016-01-01

Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN...

SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

Energy Technology Data Exchange (ETDEWEB)

JOHN C WALKER

2011-11-01

Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

Physical exercise is effective in preventing cigarette smoke-induced pulmonary oxidative response in mice

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Nesi RT

2016-03-01

Full Text Available Renata Tiscoski Nesi,1 Priscila Soares de Souza,1 Giulia Pedroso dos Santos,1 Anand Thirupathi,1 Bruno T Menegali,1 Paulo Cesar Lock Silveira,1 Luciano Acordi da Silva,1 Samuel Santos Valença,2 Ricardo Aurino Pinho11Laboratory of Exercise Biochemistry and Physiology, Graduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense, Criciúma, SC, Brazil; 2Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, BrazilAbstract: Reactive oxygen species (ROS are important in the pathogenesis of pulmonary injury induced by cigarette smoke (CS exposure, and physical exercise (Ex is useful in combating impaired oxidative process. We verified the preventive effects of Ex on lung oxidative markers induced by smoking. In this study, 36 mice (C57BL-6, 30–35 g were split into four groups: control, CS, Ex, and CS plus Ex. Ex groups were given prior physical training in water (2×30 min/d, 5 days/wk, 8 weeks. After training, the CS groups were subjected to passive exposure to four cigarettes, 3 × per day, for 60 consecutive days. After 24 hours from the last exposure, CS animals were sacrificed, and lung samples were collected for further analysis. Left lung sample was prepared for histological analysis, and right lung was used for biochemical analysis (superoxide, hydroxyproline, lipid peroxidation [thiobarbituric acid reactive species], protein carbonylation [carbonyl groups formation], superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx] activities. Group comparisons were evaluated by analysis of variance (ANOVA. Results were expressed as mean ± standard deviation, with P<0.05 considered significantly different. Preventive Ex impeded histological changes and increased the enzymatic defense system (SOD and GPx by reducing oxidative damage in lipids and proteins. This preventive effect of prior physical Ex alleviates damage caused by CS exposure.Keywords: exercise

Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells

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Jiamin Li

2018-02-01

Full Text Available Background/Aims: Endothelial-mesenchymal transition (EndMT has been shown to take part in the generation and progression of diverse diseases, involving a series of changes leading to a loss of their endothelial characteristics and an acquirement of properties typical of mesenchymal cells. Low-intensity pulsed ultrasound (LIPUS is a new therapeutic option that has been successfully used in fracture healing. However, whether LIPUS can inhibit oxidative stress-induced endothelial cell damages through inhibiting EndMT remained unknown. This study aimed to investigate the protective effects of LIPUS against oxidative stress-induced endothelial cell damages and the underlying mechanisms. Methods: EndMT was induced by H2O2 (100 µm for seven days. Human aortic endothelial cells (HAECs were exposed to H2O2 with or without LIPUS treatment for seven days. The expression of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA were analyzed. The levels of total and phosphorylated PI3K and AKT proteins were detected by Western Blot analysis. Cell chemotaxis was determined by wound healing and transwell assay. Results: LIPUS relieved EndMT by decreasing ROS accumulation and increasing activation of the PI3K signaling cascade. LIPUS alleviated the migration of EndMT-derived mesenchymal-like cells through reducing extracellular matrix (ECM deposition that is associated with matrix metallopeptidase (MMP proteolytic activity and collagen production. Conclusion: LIPUS produces cytoprotective effects against oxidative injuries to endothelial cells through suppressing the oxidative stress-induced EndMT, activating the PI3K/AKT pathway under oxidative stress, and limiting cell migration and excessive ECM deposition.

Mapping of p140Cap phosphorylation sites

DEFF Research Database (Denmark)

Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

2013-01-01

phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

Combination of PKCε Activation and PTP1B Inhibition Effectively Suppresses Aβ-Induced GSK-3β Activation and Tau Phosphorylation.

Science.gov (United States)

Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

2016-09-01

Glycogen synthase kinase-3β (GSK-3β) is a key element to phosphorylate tau and form neurofibrillary tangles (NFTs) found in tauopathies including Alzheimer's disease (AD). A current topic for AD therapy is focused upon how to prevent tau phosphorylation. In the present study, PKCε activated Akt and inactivated GSK-3β by directly interacting with each protein. Inhibition of protein tyrosine phosphatase 1B (PTP1B), alternatively, caused an enhancement in the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), allowing activation of Akt through a pathway along an IRS-1/phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, to phosphorylate and inactivate GSK-3β. Combination of PKCε activation and PTP1B inhibition more sufficiently activated Akt and inactivated GSK-3β than each independent treatment, to suppress amyloid β (Aβ)-induced tau phosphorylation and ameliorate spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. This may represent an innovative strategy for AD therapy.

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

Science.gov (United States)

Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-03-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

Protein-Tyrosine Phosphorylation in Bacillus subtilis

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

2005-01-01

phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

Tyrosine phosphorylation of Grb14 by Tie2

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Dumont Daniel J

2010-10-01

Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

Inhaled Nitric Oxide for the Prevention of Impaired Arterial Oxygenation during Myocardial Revascularization with Extracorporeal Circulation

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I. A. Kozlov

2011-01-01

Full Text Available Objective: to study the efficacy of inhaled nitric oxide used intraoperatively to prevent lung oxygenating dysfunction in patients with coronary heart disease after myocardial revascularization under extracorporeal circulation (EC. Subjects and methods. Thirty-two patients aged 55.0±2.0 years were examined. The inclusion criteria were the standard course of surgical intervention (the absence of hemorrhage, acute cardiovascular insufficiency, perioperative myocardial infarction, etc., a pulmonary artery wedge pressure of less than 15 – mm Hg throughout the study, and the baseline arterial partial oxygen tension/inspired mixture oxygen fraction (PaO2/FiO2 ratio of at least 350 mm Hg. There was a control group (n=21; Group 1 that used no special measures to prevent and/or to correct lung oxygenating dysfunction and Group 2 (n=11 that received inhaled nitric oxide. Ihe administration of inhaled nitric oxide at a concentration of 10 ppm was initiated after water anesthesia, stopped during EC, and resumed in the postperfusion period. Results. At the end, PaO2/FiO2 and intrapulmonary shunt fraction did not differ between the groups (p>0.05. Before EC, the patients receiving inhaled nitric oxide had a lower intrapulmonary blood shunting (8.9±0.7 and 11.7±1.0%; p<0.05. There were no intergroup differences in the values of PaO2/FiO2 at this stage. In the earliest postperfusion period, PaO2/FiO2 was higher in Group 2 than that in Group 1. At the end of operations, Groups 1 and 2 had a PaO2/FiO2 of 336.0±16.8 and 409.0±24.3 mm Hg, respectively (p<0.05 and an intrapulmonary shunt fraction of 14.5±1.0 and 10.4±1.0% (p<0.05. At the end of surgery, the rate of a reduction in PaO2/FiO2 to the level below 350 mm Hg was 52.4±11.1% in Group 1 and 18.2±11.6% in Group 2 (p<0.05. Six hours after surgery, PaO2/FiO2 values less than 300 mm Hg were diagnosed in 61.9±10.5% of Group 1 patients and in 27.3±13.4% of Group 2 ones (p<0.05. Conclusion. The

Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

Science.gov (United States)

Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

2016-02-15

Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

Propofol directly increases tau phosphorylation.

Directory of Open Access Journals (Sweden)

Robert A Whittington

2011-01-01

Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

PhosphoBase: a database of phosphorylation sites

DEFF Research Database (Denmark)

Blom, Nikolaj; Kreegipuu, Andres; Brunak, Søren

1998-01-01

PhosphoBase is a database of experimentally verified phosphorylation sites. Version 1.0 contains 156 entries and 398 experimentally determined phosphorylation sites. Entries are compiled and revised from the literature and from major protein sequence databases such as SwissProt and PIR. The entries...... provide information about the phosphoprotein and the exact position of its phosphorylation sites. Furthermore, part of the entries contain information about kinetic data obtained from enzyme assays on specific peptides. To illustrate the use of data extracted from PhosphoBase we present a sequence logo...... displaying the overall conservation of positions around serines phosphorylated by protein kinase A (PKA). PhosphoBase is available on the WWW at http://www.cbs.dtu.dk/databases/PhosphoBase/....

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

Science.gov (United States)

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

Science.gov (United States)

Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-01-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

Vitamin E and Lycopene Reduce Coal Burning Fluorosis-induced Spermatogenic Cell Apoptosis via Oxidative Stress-mediated JNK and ERK Signaling Pathways.

Science.gov (United States)

Tian, Yuan; Xiao, Yuehai; Wang, Bolin; Sun, Chao; Tang, Kaifa; Sun, Fa

2017-12-22

Although fluoride has been widely used in toothpaste, mouthwash, and drinking water to prevent dental caries, the excessive intake of fluoride can cause fluorosis which is associated with dental, skeletal, and soft tissue fluorosis. Recent evidences have drawn the attention to its adverse effects on male reproductive system that include spermatogenesis defect, sperm count loss, and sperm maturation impairment. Fluoride induces oxidative stress through the activation of mitogen activated protein kinase (MAPK) cascade which can lead to cell apoptosis. Vitamin E (VE) and lycopene are two common anti-oxidants, being protective to reactive oxygen species (ROS)-induced toxic effects. However, whether and how these two anti-oxidants prevent fluoride-induced spermatogenic cell apoptosis are largely unknown. In the present study, a male rat model for coal burning fluorosis was established and the histological lesions and spermatogenic cell apoptosis in rat testes were observed. The decreased expression of clusterin, a heterodimeric glycoprotein reported to regulate spermatogenic cell apoptosis, is detected in fluoride-treated rat testes. Interestingly, the co-administration with VE or lycopene reduced fluorosis-mediated testicular toxicity and rescued clusterin expression. Further, fluoride caused the enhanced Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) phosphorylation, which was reduced by VE or lycopene. Thus, VE and lycopene prevent coal burning fluorosis-induced spermatogenic cell apoptosis through the suppression of oxidative stress-mediated JNK and ERK signaling pathway, which could be an alternative therapeutic strategy for the treatment of fluorosis. ©2017 The Author(s).

Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts

Directory of Open Access Journals (Sweden)

Jong Yuh Cherng

2012-08-01

Full Text Available UVC irradiation induces oxidative stress and leads to cell death through an apoptotic pathway. This apoptosis is caused by activation of caspase-3 and formation of poly(ADP-ribose polymerase-1 (PARP-1. In this study, the underlying mechanisms of Chlorella derived peptide (CDP activity against UVC-induced cytotoxicity were investigated. Human skin fibroblasts were treated with CDP, vitamin C, or vitamin E after UVC irradiation for a total energy of 15 J/cm². After the UVC exposure, cell proliferation and caspase-3 activity were measured at 12, 24, 48, and 72 h later. Expression of phosphorylated FADD and cleaved PARP-1 were measured 16 h later. DNA damage (expressed as pyrimidine (6-4 pyrimidone photoproducts DNA concentration and fragmentation assay were performed 24 h after the UVC exposure. Results showed that UVC irradiation induced cytotoxicity in all groups except those treated with CDP. The caspase-3 activity in CDP-treated cells was inhibited from 12 h onward. Expression of phosphorylated FADD and cleaved PARP-1 were also reduced in CDP-treated cells. Moreover, UVC-induced DNA damage and fragmentation were also prevented by the CDP treatment. This study shows that treatment of CDP provides protective effects against UVC-induced cytotoxicity through the inhibition of caspase-3 activity and the reduction of phosphorylated FADD and cleaved PARP-1 expression.

Ir catalysts: Preventing CH3COOH formation in ethanol oxidation

Science.gov (United States)

Miao, Bei; Wu, Zhipeng; Xu, Han; Zhang, Minhua; Chen, Yifei; Wang, Lichang

2017-11-01

Current catalysts used for ethanol oxidation reaction (EOR) cannot effectively prevent CH3COOH formation, and thus become a major hindrance for direct ethanol fuel cell applications. We report an Ir catalyst that shows great promise for a complete EOR based on density functional theory calculations using PBE functional. The reaction barrier on Ir(1 0 0) was found to be 2.10 eV for CH3COOH formation, which is much higher than currently used Pd and Pt, and 0.57 eV for Csbnd C bond cleavage in CHCO species, which are comparable to Pd and Pt. The result suggests future directions for studying optimal complete EOR catalysts.

Olfactory Dysfunctions and Decreased Nitric Oxide Production in the Brain of Human P301L Tau Transgenic Mice.

Science.gov (United States)

Hu, Yang; Ding, Wenting; Zhu, Xiaonan; Chen, Ruzhu; Wang, Xuelan

2016-04-01

Different patterns of olfactory dysfunction have been found in both patients and mouse models of Alzheimer's Disease. However, the underlying mechanism of the dysfunction remained unknown. Deficits of nitric oxide production in brain can cause olfactory dysfunction by preventing the formation of olfactory memory. The aim of this study was to investigate the behavioral changes in olfaction and alterations in metabolites of nitric oxide, nitrate/nitrite concentration, in the brain of human P301L tau transgenic mice. The tau mice showed impairments in olfaction and increased abnormal phosphorylation of Tau protein at AT8 in different brain areas, especially in olfactory bulb. We now report that these olfactory deficits and Tau pathological changes were accompanied by decreased nitrate/nitrite concentration in the brain, especially in the olfactory bulb, and reduced expression of nNOS in the brain of tau mice. These findings provided evidence of olfactory dysfunctions correlated with decreased nitric oxide production in the brain of tau mice.

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2012-01-01

in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

Science.gov (United States)

Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

2016-09-02

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Mahalingan, Krishna K.; Baskaran†, Sulochanadevi; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med)

2017-01-10

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

Effects of Ca2+ on oxidative phosphorylation in mitochondria from the thermogenic organ of marlin.

Science.gov (United States)

O'Brien, J; Block, B A

1996-12-01

Mitochondria from the muscle-derived thermogenic (heater) organ and oxidative red muscle of the blue marlin (Makaira nigricans) were studied in order to evaluate aspects of the mechanism of thermogenesis in heater tissue. We investigated whether short-term Ca(2+)-induced uncoupling of mitochondria contributes to the thermogenic cycle of the heater organ by enhancing the respiration rate. Specific electrodes were used to obtain simultaneous measurements of oxygen consumption and Ca2+ fluxes on isolated mitochondria, and the effects of various concentrations of Ca2+ on respiration rates and the ADP phosphorylated/atomic oxygen consumed (P/O) ratio were examined. Addition of Ca2+ in excess of 10 mumol l-1 to respiring heater organ or red muscle mitochondria partially inhibited state 3 respiration and reduced the P/O ratio, indicating that the mitochondria were partially uncoupled. These effects were blocked by 2 mumol l-1 Ruthenium Red. In heater organ mitochondria, state 3 respiration rate and the P/O ratio were not significantly reduced by 1 mumol l-1 free Ca2+, a concentration likely to be near the maximum achieved in a stimulated cell. This indicates that transient increases in cytosolic Ca2+ concentration may not significantly reduce the P/O ratio of heater organ mitochondria. The activity of 2-oxoglutarate dehydrogenase in heater organ mitochondria was stimulated by approximately 15% by Ca2+ concentrations between 0.2 and 1 mumol l-1. These results suggest that heater organ mitochondria are able to maintain a normal P/O ratio and should maintain ATP output during transient increases in Ca2+ concentration, supporting a model in which an ATP-consuming process drives thermogenesis. Activation of mitochondrial dehydrogenases by low levels of Ca2+ may also enhance respiration and contribute to thermogenesis.

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation.

Science.gov (United States)

Kang, Kyoung Ah; Lee, Kyoung Hwa; Chae, Sungwook; Zhang, Rui; Jung, Myung Sun; Ham, Young Min; Baik, Jong Seok; Lee, Nam Ho; Hyun, Jin Won

2006-02-15

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway. (c) 2005 Wiley-Liss, Inc.

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

Science.gov (United States)

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Stathmin mediates hepatocyte resistance to death from oxidative stress by down regulating JNK.

Directory of Open Access Journals (Sweden)

Enpeng Zhao

Full Text Available Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK. The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth.

Membrane phosphorylation and nerve cell function

International Nuclear Information System (INIS)

Baer, P.R.

1982-01-01

This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Urra, Félix A., E-mail: felix.urra@qf.uchile.cl [Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 7, Santiago (Chile); Córdova-Delgado, Miguel [Departamento de Química Orgánica y Físico-Química, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1 (Chile); Lapier, Michel; Orellana-Manzano, Andrea [Programa de Farmacología Molecular y Clínica, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 7, Santiago (Chile); Acevedo-Arévalo, Luis; Pessoa-Mahana, Hernán; González-Vivanco, Jaime M. [Departamento de Química Orgánica y Físico-Química, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1 (Chile); Martínez-Cifuentes, Maximiliano [Instituto de Química de Recursos Naturales, Universidad de Talca, Casilla 747, Talca (Chile); and others

2016-01-15

Mitochondria participate in several distinctiveness of cancer cell, being a promising target for the design of anti-cancer compounds. Previously, we described that ortho-carbonyl hydroquinone scaffold 14 inhibits the complex I-dependent respiration with selective anti-proliferative effect on mouse mammary adenocarcinoma TA3/Ha cancer cells; however, the structural requirements of this hydroquinone scaffold to affect the oxidative phosphorylation (OXPHOS) of cancer cells have not been studied in detail. Here, we characterize the mitochondrial metabolism of TA3/Ha cancer cells, which exhibit a high oxidative metabolism, and evaluate the effect of small structural changes of the hydroquinone scaffold 14 on the respiration of this cell line. Our results indicate that these structural changes modify the effect on OXPHOS, obtaining compounds with three alternative actions: inhibitors of complex I-dependent respiration, uncoupler of OXPHOS and compounds with both actions. To confirm this, the effect of a bicyclic hydroquinone (9) was evaluated in isolated mitochondria. Hydroquinone 9 increased mitochondrial respiration in state 4o without effects on the ADP-stimulated respiration (state 3{sub ADP}), decreasing the complexes I and II-dependent respiratory control ratio. The effect on mitochondrial respiration was reversed by 6-ketocholestanol addition, indicating that this hydroquinone is a protonophoric uncoupling agent. In intact TA3/Ha cells, hydroquinone 9 caused mitochondrial depolarization, decreasing intracellular ATP and NAD(P)H levels and GSH/GSSG ratio, and slightly increasing the ROS levels. Moreover, it exhibited selective NAD(P)H availability-dependent anti-proliferative effect on cancer cells. Therefore, our results indicate that the ortho-carbonyl hydroquinone scaffold offers the possibility to design compounds with specific actions on OXPHOS of cancer cells. - Highlights: • Small changes on a hydroquinone scaffold modify the action on OXPHOS of cancer

Importance of tyrosine phosphorylation in receptor kinase complexes.

Science.gov (United States)

Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

2015-05-01

Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tyrosine Phosphorylation in Toll-Like Receptor Signaling

Science.gov (United States)

Chattopadhyay, Saurabh; Sen, Ganes C.

2014-01-01

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

Systematic inference of functional phosphorylation events in yeast metabolism.

Science.gov (United States)

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

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Curran, Jerry; Tang, Lifei; Roof, Steve R; Velmurugan, Sathya; Millard, Ashley; Shonts, Stephen; Wang, Honglan; Santiago, Demetrio; Ahmad, Usama; Perryman, Matthew; Bers, Donald M; Mohler, Peter J; Ziolo, Mark T; Shannon, Thomas R

2014-01-01

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is

Protein phosphorylation during coconut zygotic embryo development

International Nuclear Information System (INIS)

Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

Science.gov (United States)

Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

2011-01-01

One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

Science.gov (United States)

Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...

Identification of the protein kinase C phosphorylation site in neuromodulin

International Nuclear Information System (INIS)

Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

1990-01-01

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

Nitric oxide generated by ionizing radiation and EGF is implicated in EGF receptor phosphorylation in A549 lung carcinoma cells

International Nuclear Information System (INIS)

Park, In Chul; Lee, Hyung Chahn; Rhee, Chang Hun; Hong, Seok Il

2004-01-01

Although it has been demonstrated that ionizing radiation (IR) control various cell functions in a different cell types, the mechanisms of its action via NO are not well understood. NO may potentially affect every type of mammalian cells, owing to its ubiquitous production and participate in the control of cell proliferation in a great variety of cell types. The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of Mr 170,000. When EGF binds to its receptor, the receptor is dimerized and autophosphorylated at the carboxyl-terminal tyrosine 992, 1608, 1086, 1148 and 1173. This phosphorylated receptor initiates a series of signal tranduction events through interacting proteins of SH2 family including Shc, Grb2 and Sos, which in turn trigger ativation of MAPK cascades. Although the number of signaling events mediated by IR-induced NO is growing, it is still unclear how NO activate cellular signaling events. Thus, we examined the effect of NO on cellular phosphorylation and found that NO was produced by ionizing radiation in A549 lung adenocarcinoma cells and enhances the unique tyrosine phosphorylation on EGF receptor

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

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Selbig Joachim

2009-04-01

Full Text Available Abstract Background Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites. Results We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D structural information available in the protein data bank (PDB and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information. Conclusion While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as

Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition.

Science.gov (United States)

Chia, Karin K M; Liu, Chia-Chi; Hamilton, Elisha J; Garcia, Alvaro; Fry, Natasha A; Hannam, William; Figtree, Gemma A; Rasmussen, Helge H

2015-08-15

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O2 (·-)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease β1-Na(+)-K(+) pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. Copyright © 2015 the American Physiological Society.

Streptozotocin induced activation of oxidative stress responsive splenic cell signaling pathways: Protective role of arjunolic acid

International Nuclear Information System (INIS)

Manna, Prasenjit; Ghosh, Jyotirmoy; Das, Joydeep; Sil, Parames C.

2010-01-01

Present study investigates the beneficial role of arjunolic acid (AA) against the alteration in the cytokine levels and simultaneous activation of oxidative stress responsive signaling pathways in spleen under hyperglycemic condition. Diabetes was induced by injection of streptozotocin (STZ) (at a dose of 70 mg/kg body weight, injected in the tail vain). STZ administration elevated the levels of IL-2 as well as IFN-γ and attenuated the level of TNF-α in the sera of diabetic animals. In addition, hyperglycemia is also associated with the increased production of intracellular reactive intermediates resulting with the elevation in lipid peroxidation, protein carbonylation and reduction in intracellular antioxidant defense. Investigating the oxidative stress responsive cell signaling pathways, increased expressions (immunoreactive concentrations) of phosphorylated p65 as well as its inhibitor protein phospho IκBα and phosphorylated mitogen activated protein kinases (MAPKs) have been observed in diabetic spleen tissue. Studies on isolated splenocytes revealed that hyperglycemia caused disruption of mitochondrial membrane potential, elevation in the concentration of cytosolic cytochrome c as well as activation of caspase 3 leading to apoptotic cell death. Histological examination revealed that diabetic induction depleted the white pulp scoring which is in agreement with the reduced immunological response. Treatment with AA prevented the hyperglycemia and its associated pathogenesis in spleen tissue. Results suggest that AA might act as an anti-diabetic and immunomodulatory agent against hyperglycemia.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

International Nuclear Information System (INIS)

Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

2012-01-01

Highlights: ► Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. ► Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. ► Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. ► Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of β-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

Energy Technology Data Exchange (ETDEWEB)

Srisuttee, Ratakorn [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Jhun, Byung Hak [Department of Applied Nanoscience, Pusan National University, Busan 609-735 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

2012-12-07

Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats.

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Yafei Feng

Full Text Available BACKGROUND: Clinical evidence indicates that late acute renal failure (ARF predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved. MATERIALS AND METHODS: Rats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area. Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn. RESULTS: Renal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn. CONCLUSIONS: These results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.

Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

LENUS (Irish Health Repository)

Yates, Darran M

2009-04-01

Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls

International Nuclear Information System (INIS)

Majkova, Zuzana; Layne, Joseph; Sunkara, Manjula; Morris, Andrew J.; Toborek, Michal; Hennig, Bernhard

2011-01-01

Coplanar polychlorinated biphenyls (PCBs) may facilitate development of atherosclerosis by stimulating pro-inflammatory pathways in the vascular endothelium. Nutrition, including fish oil-derived long-chain omega-3 fatty acids, such as docosahexaenoic acid (DHA, 22:6ω-3), can reduce inflammation and thus the risk of atherosclerosis. We tested the hypothesis that cyclopentenone metabolites produced by oxidation of DHA can protect against PCB-induced endothelial cell dysfunction. Oxidized DHA (oxDHA) was prepared by incubation of the fatty acid with the free radical generator 2,2-azo-bis(2-amidinopropane) dihydrochloride (AAPH). Cellular pretreatment with oxDHA prevented production of superoxide induced by PCB77, and subsequent activation of nuclear factor-κB (NF-κB). A 4 /J 4 -neuroprostanes (NPs) were identified and quantitated using HPLC ESI tandem mass spectrometry. Levels of these NPs were markedly increased after DHA oxidation with AAPH. The protective actions of oxDHA were reversed by treatment with sodium borohydride (NaBH 4 ), which concurrently abrogated A 4 /J 4 -NP formation. Up-regulation of monocyte chemoattractant protein-1 (MCP-1) by PCB77 was markedly reduced by oxDHA, but not by un-oxidized DHA. These protective effects were proportional to the abundance of A 4 /J 4 NPs in the oxidized DHA sample. Treatment of cells with oxidized eicosapentaenoic acid (EPA, 20:5ω-3) also reduced MCP-1 expression, but less than oxDHA. Treatment with DHA-derived cyclopentenones also increased DNA binding of NF-E2-related factor-2 (Nrf2) and downstream expression of NAD(P)H:quinone oxidoreductase (NQO1), similarly to the Nrf-2 activator sulforaphane. Furthermore, sulforaphane prevented PCB77-induced MCP-1 expression, suggesting that activation of Nrf-2 mediates the observed protection against PCB77 toxicity. Our data implicate A 4 /J 4 -NPs as mediators of omega-3 fatty acid-mediated protection against the endothelial toxicity of coplanar PCBs.

Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

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Danming Tang

2012-09-01

GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

Myosin light chain kinase phosphorylation in tracheal smooth muscle

International Nuclear Information System (INIS)

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

1990-01-01

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

Phosphorylation of the Epstein-Barr virus nuclear antigen 2

DEFF Research Database (Denmark)

Grässer, F A; Göttel, S; Haiss, P

1992-01-01

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

p-Benzoquinone initiates non-invasive urothelial cancer through aberrant tyrosine phosphorylation of EGFR, MAP kinase activation and cell cycle deregulation: Prevention by vitamin C

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Shinjini Ganguly

Full Text Available According to WHO classification system, non-invasive urothelial carcinoma represents urothelial carcinoma in situ (CIS and dysplasia. Dysplastic urothelium often progresses to CIS that further advances to urothelial carcinoma (UC. The strongest risk factor for UC is cigarette smoking. However, the pathogenesis of cigarette smoke (CS-induced UC is poorly understood. Earlier we had shown that p-benzoquinone (p-BQ, a major toxic quinone derived from p-benzosemiquinone of CS in vivo, is a causative factor for various CS-induced diseases. Here, using a guinea pig model we showed that prolonged treatment with p-BQ led to non-invasive UC, specifically carcinoma in situ (CIS of the renal pelvis and dysplasia in the ureter and bladder. The mechanisms of carcinogenesis were p-BQ-induced oxidative damage and apoptosis that were later suppressed and followed by activation of epidermal growth factor receptor, aberrant phosphorylation of intracellular tyrosine residues, activation of MAP kinase pathway and persistent growth signaling. This was accompanied by deregulation of cell cycle as shown by marked decrease in the expression of p21waf1/cip1 and cyclin D1 proteins as well as hyperphosphorylation of pRb. UC has been characterised by histopathology and immunohistochemistry showing aberrant CK20, increased Ki-67, and marked p53 nuclear immunopositivity with uniformly negative labelling of CD44. Oral supplementation of vitamin C (30 mg/kg body weight/day prevented CIS of the renal pelvis and dysplasia in the ureter and bladder. Since majority of non-invasive UC progresses to invasive cancer with increased risk of mortality, our preclinical study might help to devise effective strategies for early intervention of the disease. Abbreviations: Bax, Bcl-2, CS, DNPH, GAPDH, IARC, p-BQ, p-BSQ, PAHs, PBS, ROS, SDS PAGE, TUNEL, WHO, UC, CIS, EGFR, MAPK, Keywords: p-Benzoquinone, Carcinoma in situ, Dysplasia, Aberrant EGFR activation, Cell cycle deregulation

Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells.

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Suda, Jo; Rockey, Don C; Karvar, Serhan

2016-02-01

The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and

MST4 kinase phosphorylates ACAP4 protein to orchestrate apical membrane remodeling during gastric acid secretion.

Science.gov (United States)

Yuan, Xiao; Yao, Phil Y; Jiang, Jiying; Zhang, Yin; Su, Zeqi; Yao, Wendy; Wang, Xueying; Gui, Ping; Mullen, McKay; Henry, Calmour; Ward, Tarsha; Wang, Wenwen; Brako, Larry; Tian, Ruijun; Zhao, Xuannv; Wang, Fengsong; Cao, Xinwang; Wang, Dongmei; Liu, Xing; Ding, Xia; Yao, Xuebiao

2017-09-29

Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,K-ATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr 545 Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts with MST4 and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr 545 by mass spectrometric analyses. Importantly, phosphorylation of Thr 545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylation-deficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr 545 enables ACAP4 to interact with ezrin. Given the location of Thr 545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

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Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

2009-11-30

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

Science.gov (United States)

Yun, Young Sook; Tajima, Miki; Takahashi, Shigeru; Takahashi, Yuji; Umemura, Mariko; Nakano, Haruo; Park, Hyun Sun; Inoue, Hideshi

2016-10-01

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element-binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea M AXIM. var. kiushiana Makino was found to suppress cAMP-enhanced PEPCK-C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin-stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK-C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway

International Nuclear Information System (INIS)

Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-01-01

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis.

Phosphorylation of Akt by SC79 Prevents Iron Accumulation and Ameliorates Early Brain Injury in a Model of Experimental Subarachnoid Hemorrhage

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Shuangying Hao

2016-03-01

Full Text Available Previous studies have demonstrated that activation of Akt may alleviate early brain injury (EBI following subarachnoid hemorrhage (SAH. This study is undertaken to determine whether iron metabolism is involved in the beneficial effect of Akt activation after SAH. Therefore, we used a novel molecule, SC79, to activate Akt in an experimental Sprague–Dawley rat model of SAH. Rats were randomly divided into four groups as follows: sham, SAH, SAH + vehicle, SAH + SC79. The results confirmed that SC79 effectively enhanced the defense against oxidative stress and alleviated EBI in the temporal lobe after SAH. Interestingly, we found that phosphorylation of Akt by SC79 reduced cell surface transferrin receptor-mediated iron uptake and promoted ferroportin-mediated iron transport after SAH. As a result, SC79 administration diminished the iron content in the brain tissue. Moreover, the impaired Fe-S cluster biogenesis was recovered and loss of the activities of the Fe-S cluster-containing enzymes were regained, indicating that injured mitochondrial functions are restored to healthy levels. These findings suggest that disrupted iron homeostasis could contribute to EBI and Akt activation may regulate iron metabolism to relieve iron toxicity, further protecting neurons from EBI after SAH.

Alternative waste residue materials for passive in situ prevention of sulfide-mine tailings oxidation: a field evaluation.

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Nason, Peter; Johnson, Raymond H; Neuschütz, Clara; Alakangas, Lena; Öhlander, Björn

2014-02-28

Novel solutions for sulfide-mine tailings remediation were evaluated in field-scale experiments on a former tailings repository in northern Sweden. Uncovered sulfide-tailings were compared to sewage-sludge biosolid amended tailings over 2 years. An application of a 0.2m single-layer sewage-sludge amendment was unsuccessful at preventing oxygen ingress to underlying tailings. It merely slowed the sulfide-oxidation rate by 20%. In addition, sludge-derived metals (Cu, Ni, Fe, and Zn) migrated and precipitated at the tailings-to-sludge interface. By using an additional 0.6m thick fly-ash sealing layer underlying the sewage sludge layer, a solution to mitigate oxygen transport to the underlying tailings and minimize sulfide-oxidation was found. The fly-ash acted as a hardened physical barrier that prevented oxygen diffusion and provided a trap for sludge-borne metals. Nevertheless, the biosolid application hampered the application, despite the advances in the effectiveness of the fly-ash layer, as sludge-borne nitrate leached through the cover system into the underlying tailings, oxidizing pyrite. This created a 0.3m deep oxidized zone in 6-years. This study highlights that using sewage sludge in unconventional cover systems is not always a practical solution for the remediation of sulfide-bearing mine tailings to mitigate against sulfide weathering and acid rock drainage formation. Copyright © 2014 Elsevier B.V. All rights reserved.

Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

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Zhao, Kong-Nan; Masci, Paul P.; Lavin, Martin F.

2011-01-01

Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex. PMID:22163289

Prevention of lipid oxidation in omega-3 enriched oofds by antioxidants and the use of delivery systems

DEFF Research Database (Denmark)

Jacobsen, Charlotte

Due to the health beneficial effects of marine omega-3 fatty acids there is an increasing interest in developing functional foods containing these healthy fatty acids. However, such foods are very susceptible to lipid oxidation, which will give rise to undesirable off-flavours and unhealthy...... oxidation products. Efficients strategies to prevent lipid oxidation are therefore required. Such strategies include addition of antioxidants or the use of omega-3 delivery emulsions. However, antioxidant efficacy in complex omega-3 enriched foods are influenced by many factors including the lipophilicity...... of the antioxidants. Selection of the optimal antioxidant system is therefore a major challenge. Likewise, a range of factors can influence the ability of omega-3 delivery systems to protect the omega-3 fatty acids against oxidation after addition to food systems. These challenges will be discussed...

Proteasome phosphorylation regulates cocaine-induced sensitization.

Science.gov (United States)

Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

Caffeine prevents d-galactose-induced cognitive deficits, oxidative stress, neuroinflammation and neurodegeneration in the adult rat brain.

Science.gov (United States)

Ullah, Faheem; Ali, Tahir; Ullah, Najeeb; Kim, Myeong Ok

2015-11-01

d-galactose has been considered a senescent model for age-related neurodegenerative disease. It induces oxidative stress which triggers memory impairment, neuroinflammation and neurodegeneration. Caffeine act as anti-oxidant and has been used in various model of neurodegenerative disease. Nevertheless, the effect of caffeine against d-galactose aging murine model of age-related neurodegenerative disease elucidated. Here, we investigated the neuroprotective effect of caffeine against d-galactose. We observed that chronic treatment of caffeine (3 mg/kg/day intraperitoneally (i.p) for 60 days) improved memory impairment and synaptic markers (Synaptophysin and PSD95) in the d-galactose treated rats. Chronic caffeine treatment reduced the oxidative stress via the reduction of 8-oxoguanine through immunofluorescence in the d-galactose-treated rats. Consequently caffeine treatment suppressed stress kinases p-JNK. Additionally, caffeine treatment significantly reduced the d-galactose-induced neuroinflammation through alleviation of COX-2, NOS-2, TNFα and IL-1β. Furthermore we also analyzed that caffeine reduced cytochrome C, Bax/Bcl2 ratio, caspase-9, caspase-3 and PARP-1 level. Moreover by evaluating the immunohistochemical results of Nissl and Fluro-Jade B staining showed that caffeine prevented the neurodegeneration in the d-galactose-treated rats. Our results showed that caffeine prevents the d-galactose-induced oxidative stress and consequently alleviated neuroinflammation and neurodegeneration; and synaptic dysfunction and memory impairment. Therefore, we could suggest that caffeine might be a dietary anti-oxidant agent and a good candidate for the age-related neurodegenerative disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

A grammar inference approach for predicting kinase specific phosphorylation sites.

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

O-GlcNAc modification: why so intimately associated with phosphorylation?

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Ande Sudharsana R

2011-01-01

Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

Immobilization of Deoxyadenosine Kinase from Dictyostelium discoideum (DddAK) and Its Application in the 5’-Phosphorylation of Arabinosyladenine and Arabinosyl-2-fluoroadenine

DEFF Research Database (Denmark)

Serra, Immacolata; Ubiali, Daniela; Piskur, Jure

2017-01-01

Deoxyadenosine kinase from Dictyostelium discoideum (DddAK) phosphorylates its natural substrate (2’‐deoxyadenosine, dAdo) as well as the arabinosyladenine analogues vidarabine (araA) and fludarabine (F‐araA) to their corresponding 5’‐monophosphates. DddAK has been here immobilized by ionic...... interaction on an aminated epoxy‐functionalized support (SepabeadsTM EC‐EP), and cross‐linked with oxidized dextran. The final activity recovery was 33–42 %, depending on the protein loading. Immobilization enhanced the stability of DddAK at pH 10 and, to a lesser extent, at 45 °C. Phosphorylation of d...

Protein phosphorylation systems in postmortem human brain

International Nuclear Information System (INIS)

Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P.

1989-01-01

Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders

Novel Role of Src in Priming Pyk2 Phosphorylation.

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Ming Zhao

Full Text Available Proline-rich tyrosine kinase 2 (Pyk2 is a member of the focal adhesion kinase (FAK family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating

DEFF Research Database (Denmark)

Assentoft, Mette; Kaptan, Shreyas; Fenton, Robert A.

2013-01-01

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is theref......Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4...... is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic...... of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations...

Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells

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Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

2015-01-01

Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress. PMID:26234813

Protein kinase A governs oxidative phosphorylation kinetics and oxidant emitting potential at complex I

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Daniel Stephen Lark

2015-11-01

Full Text Available The mitochondrial electron transport system (ETS is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC/cyclic AMP (cAMP/Protein kinase A (PKA axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduces complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowers both the Km and Vmax for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS.

Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation.

Science.gov (United States)

Ramseyer, Vanesa D; Gonzalez-Vicente, Agustin; Carretero, Oscar A; Garvin, Jeffrey L

2015-01-15

Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% (P thick ascending limbs from ANG II-infused animals [ANG II -0.01 ± 0.06 arbitrary fluorescence units (AFU)/min vs. 0.17 ± 0.02 AFU/min in controls; P thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation. Copyright © 2015 the American Physiological Society.

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation.

Science.gov (United States)

García García, Tránsito; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Correia Santos, Sara; Henry, Céline; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise; Poncet, Sandrine

2018-01-01

Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

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Ayswarya Ravi

2018-02-01

Full Text Available Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN, a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT and Gallium-immobilized metal affinity chromatography (Ga-IMAC were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.

Secoisolariciresinol diglucoside prevents the oxidative stress-induced apoptosis of myocardial cells through activation of the JAK2/STAT3 signaling pathway.

Science.gov (United States)

Huang, Guiqiong; Huang, Xiaofang; Liu, Min; Hua, Yue; Deng, Bo; Jin, Wen; Yan, Wen; Tan, Zhangbin; Wu, Yifen; Liu, Bin; Zhou, Yingchun

2018-06-01

Myocardial cell apoptosis mediated by oxidative stress has previously been identified as a key process in ischemic heart disease. Secoisolariciresinol diglucoside (SDG), a polyphenolic plant lignan primarily found in flaxseed, has been demonstrated to effectively protect myocardial cells from apoptosis. In the present study, the role of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) was investigated in mediating the protective effect of SDG. Findings of the present study revealed that treatment with H2O2 reduced cell viability and induced apoptosis in H9C2 rat cardiomyocytes. However, SDG was able to reduce the effect of H2O2 in a dose‑dependent manner. H2O2 reduced the expression level of phosphorylated STAT3 and inhibited the levels of B‑cell lymphoma‑extra‑large and induced myeloid leukemia cell differentiation protein, which are the STAT3 target genes. Conversely, SDG rescued phosphorylation of STAT3 and increased the levels of STAT3 target genes. Treatment with SDG alone led to a dose‑dependent increased phosphorylation of JAK2 and STAT3, without activating Src. Furthermore, the anti‑apoptotic effects of SDG were partially abolished by a JAK2/STAT3 inhibitor. In addition, molecular docking revealed that SDG may bind to the protein kinase domain of JAK2, at a binding energy of ‑8.258 kcal/mol. Molecular dynamics simulations revealed that JAK2‑SDG binding was stable. In conclusion, activation of the JAK2/STAT3 signaling pathway contributed to the anti‑apoptotic activity of SDG, which may be a potential JAK2 activator.

Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

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Giovanna Damia

2001-01-01

Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

Regulation of protein phosphorylation in oat mitochondria

International Nuclear Information System (INIS)

Pike, C.; Kopeck, K.; Sceppa, E.

1989-01-01

We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1

DEFF Research Database (Denmark)

Kjaerulff, Søren; Andersen, Nicoline Resen; Borup, Mia Trolle

2007-01-01

Eukaryotic cells normally differentiate from G(1); here we investigate the mechanism preventing expression of differentiation-specific genes outside G(1). In fission yeast, induction of the transcription factor Ste11 triggers sexual differentiation. We find that Ste11 is only active in G(1) when...... Cdk activity is low. In the remaining part of the cell cycle, Ste11 becomes Cdk-phosphorylated at Thr 82 (T82), which inhibits its DNA-binding activity. Since the ste11 gene is autoregulated and the Ste11 protein is highly unstable, this Cdk switch rapidly extinguishes Ste11 activity when cells enter...... S phase. When we mutated T82 to aspartic acid, mimicking constant phosphorylation, cells no longer underwent differentiation. Conversely, changing T82 to alanine rendered Ste11-controlled transcription constitutive through the cell cycle, and allowed mating from S phase with increased frequency...

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

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Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Inhibitory effects of myricitrin on oxidative stress-induced endothelial damage and early atherosclerosis in ApoE −/− mice

International Nuclear Information System (INIS)

Sun, Gui-bo; Qin, Meng; Ye, Jing-xue; Pan, Rui-le; Meng, Xiang-bao; Wang, Min; Luo, Yun; Li, Zong-yang; Wang, Hong-wei; Sun, Xiao-bo

2013-01-01

Atherosclerosis (AS) is a state of heightened oxidative stress characterized by lipid and protein oxidation in vascular walls. Oxidative stress-induced vascular endothelial cell (VEC) injury is a major factor in the pathogenesis of AS. Myricitrin, a natural flavonoid isolated from the root bark of Myrica cerifera, was recently found to have a strong antioxidative effect. However, its use for treating cardiovascular diseases, especially AS is still unreported. Consequently, we evaluated the cytoprotective effect of myricitrin on AS by assessing oxidative stress-induced VEC damage. The in vivo study using an ApoE −/− mouse model of AS demonstrated that myricitrin treatment protects against VEC damage and inhibits early AS plaque formation. This effect is associated with the antioxidative effect of myricitrin, as observed in a hydrogen peroxide (H 2 O 2 )-induced rat model of artery endothelial injury and primary cultured human VECs. Myricitrin treatment also prevents and attenuates H 2 O 2 -induced endothelial injury. Further investigation of the cytoprotective effects of myricitrin demonstrated that myricitrin exerts its function by scavenging for reactive oxygen species, as well as reducing lipid peroxidation, blocking NO release, and maintaining mitochondrial transmembrane potential. Myricitrin treatment also significantly decreased H 2 O 2 -induced apoptosis in VECs, which was associated with significant inhibition of p53 gene expression, activation of caspase-3 and the MAPK signaling pathway, and alteration of the patterns of pro-apoptotic and anti-apoptotic gene expression. The resulting significantly increased bcl-2/bax ratio indicates that myricitrin may prevent the apoptosis induced by oxidative stress injury. - Highlights: • Myricitrin prevents early atherosclerosis in ApoE−/− mice. • Myricitrin protects endothelial cell from H 2 O 2 induced injury in rat and HUVECs. • Myricitrin enhanced NO release and up regulates eNOS activity in HUVECs. �

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

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Laura Civiero

2017-12-01

Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

Rosemary as natural antioxidant to prevent oxidation in chicken burgers

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Daiane PEREIRA

Full Text Available Abstract Rosemary (Rosmarinus officinalis is known for their sensory characteristics and antioxidant properties, mainly due to the presence of several phenolic compounds. The aim of this work, was determine the antioxidant activity and apply the Rosemary lyophilized extract (RLE in chicken burger, for assess their ability to reduce the lipid oxidation. Total antioxidant capacity and phenolic compounds profile were analyzed by colorimetric tests and liquid chromatography analysis, respectively. Thiobarbituric acid reactive substances assay was used to evaluate the ability of the RLE to prevent lipid peroxidation in chicken burger stored at 4 °C. Three treatments of chicken burgers were prepared (T1 – control, without addition of synthetic antioxidant BHT: butylated hydroxytoluene or RLE, T2 – with addition of BHT, and T3 – experimental, containing RLE. The high contents of total phenolic compounds (40.91 mg GAE g-1: Gallic Acid Equivalent and total flavonoids (24.26 mg QE g-1: Quercetin Equivalents were found in RLE. Rutin was the major phenolic compound identified in the RLE. The RLE showed strong antioxidant capacity and inhibited 48.29% of lipid oxidation (21 days of storage in comparison to the control (T1, with low production of malonaldehyde, which has potential to be used in chicken burgers.

Src kinase regulation by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Roskoski, Robert

2005-01-01

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

Curcumin prevents the oxidation and lipid modification of LDL and its inhibition of prostacyclin generation by endothelial cells in culture.

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Mahfouz, Mohamedain M; Zhou, Sherry Q; Kummerow, Fred A

2009-11-01

Low-density lipoprotein (LDL) was isolated from human plasma and oxidized by 5microM copper sulfate for 4h at 37 degrees C in the absence and presence of 1, 3, 5, 10, or 20microM of curcumin. LDL oxidized in the absence of curcumin (oxLDL) showed an increased levels of conjugated dienes, lipid peroxides (TBARS) and lysolecithin (lysoPC) and a significant loss of polyunsaturated fatty acids (PUFA). LDL oxidized with 5microM copper sulfate in the presence of curcumin caused a significant decrease of conjugated diene, lipid peroxides, lysoPC and significant increase of PUFA compared to oxLDL. These changes were dose dependent and reached a maximum at 5microM curcumin. Incubation of human endothelial cells (EC) with 200microg protein/ml of oxLDL caused a significant decrease of prostacyclin (PGI(2)) generation. LDL oxidized in presence of 5microM curcumin did not show any inhibition of PGI(2) generation compared to the control cells. These results indicate that curcumin is an effective chain-breaking antioxidant which prevents oxidation and lipid modification of LDL. The inhibition of oxLDL on PGI(2) is considered a contributing factor in the pathogenesis of thrombosis and atherosclerosis. Curcumin supplementation could be an effective strategy in preventing LDL oxidation and its impact on atherosclerosis and lesion formation.

Phototropism and Protein Phosphorylation in Higher Plants: Unilateral Blue Light Irradiation Generates a Directional Gradient of Protein Phosphorylation Across the Oat Coleoptile

International Nuclear Information System (INIS)

Salomon, M.; Zacherl, M.; Rüdiger, W.

1997-01-01

Blue light induces the phosphorylation of a 116 kDa oat protein found in plasma membrane preparations from coleoptile tips. We developed a very sensitive in vitro method that allowed us to determine the tissue distribution of protein phosphorylation after applying unilateral and bilateral blue light pulses in vivo. We found that following unilateral in vivo irradiation the degree in phosphorylation of the 116 kDa protein is significantly higher at the irradiated than at the shaded side of the coleoptile tip. This asymmetry can be considered as previously missing criterion that protein phosphorylation represents an early event within the transduction chain for phototropism. (author)

Amino acid chirality breaking by N-phosphorylation

International Nuclear Information System (INIS)

Zhao Yufen; Yan Qingjin.

1995-01-01

The chirality breaking of amino acid is a focus issue in the origin of life. For chemists, there are some interesting chemical approaches to solve the symmetry breaking problem. Our previous experiments indicated that when amino acids were phosphorylated, there were many bio-mimic reactions happened. In this paper, it was found that there had significant difference between the N-phosphoryl L- and D- amino acids such as serine and threonine. The optical rotation tracing experiments of the racemic N-phosphoamino acids also showed the similar results. The chirality breaking of amino acids by N-phosphorylation was a novel phenomena. (author). 3 refs, 1 fig. Abstract only

Selol, an organic selenium donor, prevents lipopolysaccharide-induced oxidative stress and inflammatory reaction in the rat brain.

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Dominiak, Agnieszka; Wilkaniec, Anna; Jęśko, Henryk; Czapski, Grzegorz A; Lenkiewicz, Anna M; Kurek, Eliza; Wroczyński, Piotr; Adamczyk, Agata

2017-09-01

Neuroinflammation and oxidative stress are key intertwined pathological factors in many neurological, particularly neurodegenerative diseases, such as Alzheimer's and Parkinson's disorders as well as autism. The present study was conducted to evaluate the protective effects of Selol, an organic selenium donor, against lipopolysaccharide (LPS)-mediated inflammation in rat brain. The results demonstrated that the peripheral administration of LPS in a dose of 100 μg/kg b.w. evoked typical pathological reaction known as systemic inflammatory response. Moreover, we observed elevated blood levels of thiobarbituric acid-reactive substances (TBARS), a marker of oxidative stress, as well as increased concentration of tumor necrosis factor-α (TNF-α) in LPS-treated animals. Selol significantly prevented these LPS-evoked changes. Subsequently, Selol protected against LPS-induced up-regulation of proinflammatory cytokines (Tnfa, Ifng, Il6) in rat brain cortex. The molecular mechanisms through which Selol prevented the neuroinflammation were associated with the inhibition of oxidized glutathione (GSSG) accumulation and with an increase of glutathione-associated enzymes: glutathione peroxidase (Se-GPx), glutathione reductase (GR) as well as thioredoxin reductase (TrxR) activity and expression. Finally, we observed that Selol administration effectively protected against LPS-induced changes in the expression of brain-derived neurotrophic factor (Bdnf). In conclusion, our studies indicated that Selol effectively protects against LPS-induced neuroinflammation by inhibiting pro-inflammatory cytokine release, by boosting antioxidant systems, and by augmenting BDNF level. Therefore, Selol could be a multi-potent and effective drug useful in the treatment and prevention of brain disorders associated with neuroinflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

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Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

2009-09-10

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

Aging and oxidative stress reduce the response of human articular chondrocytes to insulin-like growth factor 1 and osteogenic protein 1.

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Loeser, Richard F; Gandhi, Uma; Long, David L; Yin, Weihong; Chubinskaya, Susan

2014-08-01

To determine the effects of aging and oxidative stress on the response of human articular chondrocytes to insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Chondrocytes isolated from normal articular cartilage obtained from tissue donors were cultured in alginate beads or monolayer. Cells were stimulated with 50-100 ng/ml of IGF-1, OP-1, or both. Oxidative stress was induced using tert-butyl hydroperoxide. Sulfate incorporation was used to measure proteoglycan synthesis, and immunoblotting of cell lysates was performed to analyze cell signaling. Confocal microscopy was performed to measure nuclear translocation of Smad4. Chondrocytes isolated from the articular cartilage of tissue donors ranging in age from 24 years to 81 years demonstrated an age-related decline in proteoglycan synthesis stimulated by IGF-1 and IGF-1 plus OP-1. Induction of oxidative stress inhibited both IGF-1- and OP-1-stimulated proteoglycan synthesis. Signaling studies showed that oxidative stress inhibited IGF-1-stimulated Akt phosphorylation while increasing phosphorylation of ERK, and that these effects were greater in cells from older donors. Oxidative stress also increased p38 phosphorylation, which resulted in phosphorylation of Smad1 at the Ser(206) inhibitory site and reduced nuclear accumulation of Smad1. Oxidative stress also modestly reduced OP-1-stimulated nuclear translocation of Smad4. These results demonstrate an age-related reduction in the response of human chondrocytes to IGF-1 and OP-1, which are 2 important anabolic factors in cartilage, and suggest that oxidative stress may be a contributing factor by altering IGF-1 and OP-1 signaling. Copyright © 2014 by the American College of Rheumatology.

Sildenafil Prevents Apoptosis of Human First-Trimester Trophoblast Cells Exposed to Oxidative Stress

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Bolnick, Jay M.; Kilburn, Brian A.; Bolnick, Alan D.; Diamond, Michael P.; Singh, Manvinder; Hertz, Michael; Dai, Jing

2015-01-01

Human first-trimester trophoblast cells proliferate at low O2, but survival is compromised by oxidative stress, leading to uteroplacental insufficiency. The vasoactive drug, sildenafil citrate (Viagra, Sigma, St Louis, Missouri), has proven useful in reducing adverse pregnancy outcomes. An important biological function of this pharmaceutical is its action as an inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type 5 activity, which suggests that it could have beneficial effects on trophoblast survival. To investigate whether sildenafil can prevent trophoblast cell death, human first-trimester villous explants and the HTR-8/SVneo cytotrophoblast cell line were exposed to hypoxia and reoxygenation (H/R) to generate oxidative stress, which induces apoptosis. Apoptosis was optimally inhibited during H/R by 350 ng/mL sildenafil. Sildenafil-mediated survival was reversed by l-NG-nitro-l-arginine methyl ester hydrochloride or cGMP antagonist, indicating a dependence on both nitric oxide (NO) and cGMP. Indeed, either a cGMP agonist or an NO generator was cytoprotective independent of sildenafil. These findings suggest a novel intervention route for patients with recurrent pregnancy loss or obstetrical placental disorders. PMID:25431453

Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation.

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Abbey P Theiss

Full Text Available Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR, but shorter ischemic intervals (less than 17 minutes facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

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Finelli, Mattéa J; Murphy, Kevin J; Chen, Lei; Zou, Hongyan

2013-05-30

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Sevoflurane posttreatment prevents oxidative and inflammatory injury in ventilator-induced lung injury.

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Julie Wagner

Full Text Available Mechanical ventilation is a life-saving clinical treatment but it can induce or aggravate lung injury. New therapeutic strategies, aimed at reducing the negative effects of mechanical ventilation such as excessive production of reactive oxygen species, release of pro-inflammatory cytokines, and transmigration as well as activation of neutrophil cells, are needed to improve the clinical outcome of ventilated patients. Though the inhaled anesthetic sevoflurane is known to exert organ-protective effects, little is known about the potential of sevoflurane therapy in ventilator-induced lung injury. This study focused on the effects of delayed sevoflurane application in mechanically ventilated C57BL/6N mice. Lung function, lung injury, oxidative stress, and inflammatory parameters were analyzed and compared between non-ventilated and ventilated groups with or without sevoflurane anesthesia. Mechanical ventilation led to a substantial induction of lung injury, reactive oxygen species production, pro-inflammatory cytokine release, and neutrophil influx. In contrast, sevoflurane posttreatment time dependently reduced histological signs of lung injury. Most interestingly, increased production of reactive oxygen species was clearly inhibited in all sevoflurane posttreatment groups. Likewise, the release of the pro-inflammatory cytokines interleukin-1β and MIP-1β and neutrophil transmigration were completely prevented by sevoflurane independent of the onset of sevoflurane administration. In conclusion, sevoflurane posttreatment time dependently limits lung injury, and oxidative and pro-inflammatory responses are clearly prevented by sevoflurane irrespective of the onset of posttreatment. These findings underline the therapeutic potential of sevoflurane treatment in ventilator-induced lung injury.

Radical Cation Salt-initiated Aerobic C-H Phosphorylation of N-Benzylanilines: Synthesis of a-Aminophosphonates.

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Jia, Xiao Dong; Liu, Xiaofei; Yuan, Yu; Li, Pengfei; Hou, Wentao; He, Kaixuan

2018-06-03

A radical cation salt-initiated phosphorylation of N-benzylanilines was realized through the aerobic oxidation of sp3 C-H bond, providing a series of α-aminophosphonates in high yields. The investigation of the reaction scope revealed that this mild catalyst system is superior in good functional group tolerance and high reaction efficiency. The mechanistic study implied that the cleavage of the sp3 C-H bond was involved in the rate-determining step. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kefir Peptides Prevent Hyperlipidemia and Obesity in High-Fat-Diet-Induced Obese Rats via Lipid Metabolism Modulation.

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Tung, Yu-Tang; Chen, Hsiao-Ling; Wu, Hsin-Shan; Ho, Mei-Hsuan; Chong, Kowit-Yu; Chen, Chuan-Mu

2018-02-01

Obesity has reached epidemic proportions worldwide. Obesity is a complex metabolic disorder that is linked to numerous serious health complications with high morbidity. The present study evaluated the effects of kefir peptides on high fat diet (HFD)-induced obesity in rats. Kefir peptides markedly improved obesity, including body weight gain, inflammatory reactions and the formation of adipose tissue fat deposits around the epididymis and kidney, and adipocyte size. Treating high fat diet (HFD)-induced obese rats with kefir peptides significantly reduced the fatty acid synthase protein and increased the p-acetyl-CoA carboxylase protein to block lipogenesis in the livers. Kefir peptides also increased fatty acid oxidation by increasing the protein expressions of phosphorylated AMP-activated protein kinase, peroxisome proliferator-activated receptor-α, and hepatic carnitine palmitoyltransferase-1 in the livers. In addition, administration of kefir peptides significantly decreased the inflammatory response (TNF-α, IL-1β, and TGF-β) to modulate oxidative damage. These results demonstrate that kefir peptides treatment improves obesity via inhibition of lipogenesis, modulation of oxidative damage, and stimulation of lipid oxidation. Therefore, kefir peptides may act as an anti-obesity agent to prevent body fat accumulation and obesity-related metabolic diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

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Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

2014-02-15

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

International Nuclear Information System (INIS)

Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

2008-01-01

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

Protein phosphorylation and its role in archaeal signal transduction

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Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

2016-01-01

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

Cannabinoid CB2 receptors are involved in the protection of RAW264.7 macrophages against the oxidative stress: an in vitro study

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Sabrina Giacoppo

2017-01-01

Full Text Available Research in the last decades has widely investigated the anti-oxidant properties of natural products as a therapeutic approach for the prevention and the treatment of oxidative-stress related disorders. In this context, several studies were aimed to evaluate the therapeutic potential of phytocannabinoids, the bioactive compounds of Cannabis sativa. Here, we examined the anti-oxidant ability of Cannabigerol (CBG, a non-psychotropic cannabinoid, still little known, into counteracting the hydrogen peroxide (H2O2-induced oxidative stress in murine RAW264.7 macrophages. In addition, we tested selective receptor antagonists for cannabinoid receptors and specifically CB1R (SR141716A and CB2R (AM630 in order to investigate through which CBG may exert its action. Taken together, our in vitro results showed that CBG is able to counteract oxidative stress by activation of CB2 receptors. CB2 antagonist pre-treatment indeed blocked the protective effects of CBG in H2O2 stimulated macrophages, while CB1R was not involved. Specifically, CBG exhibited a potent action in inhibiting oxidative stress, by down-regulation of the main oxidative markers (iNOS, nitrotyrosine and PARP-1, by preventing IκB-α phosphorylation and translocation of the nuclear factor-κB (NF-κB and also via the modulation of MAP kinases pathway. On the other hand, CBG was found to increase anti-oxidant defense of cells by modulating superoxide dismutase-1 (SOD-1 expression and thus inhibiting cell death (results focused on balance between Bax and Bcl-2. Based on its antioxidant activities, CBG may hold great promise as an anti-oxidant agent and therefore used in clinical practice as a new approach in oxidative-stress related disorders.

Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation.

Science.gov (United States)

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Merlo, Kleison C; Romão, Tatiany P; Rocha, Pollyanna O; Assis, Ludmila A; Nascimento, Larissa M; Xavier, Camila C; Rezende, Antonio M; Reis, Christian R S; Papadopoulou, Barbara

2018-03-23

The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.

Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

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Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

2014-04-25

The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

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Peng, Xiaoli; Gan, Jing; Wang, Qian; Shi, Zhenqiang; Xia, Xiaodong

2016-11-30

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Lipotoxicity induces hepatic protein inclusions through TBK1-mediated p62/SQSTM1 phosphorylation.

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Cho, Chun-Seok; Park, Hwan-Woo; Ho, Allison; Semple, Ian A; Kim, Boyoung; Jang, Insook; Park, Haeli; Reilly, Shannon; Saltiel, Alan R; Lee, Jun Hee

2017-12-18

Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause non-alcoholic steatohepatitis (NASH). NASH in turn is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). The formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here we show that upon saturated fatty acid-induced lipotoxicity, Tank-binding protein kinase 1 (TBK1) is activated and phosphorylates p62. The TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and the formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in the lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of the ubiquitin-p62 aggregates, not only in cultured hepatocytes, but also in mouse models of obesity and NASH. These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases.

Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc-

International Nuclear Information System (INIS)

Gu Li; Hu Xiaoling; Xue Zhanxia; Yang Jun; Wan Lishu; Ren Yan; Hertz, Leif; Peng Liang

2010-01-01

Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK 1/2 ) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by , that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a ∼ 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK 1/2 phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X c - could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK 1/2 phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X c - prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK 1/2 phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X c - like quisqualate (Venkatraman et al. 1994), by 'self-sensitization' with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

DEFF Research Database (Denmark)

Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

2007-01-01

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

3-phosphorylated and -thiophosphorylated 2-thiazolidine- and 2-oxazolidine-thiones

International Nuclear Information System (INIS)

Vorob'eva, N.N.; Razvodovskaya, L.V.; Negrebetskii, V.V.; Grapov, A.F.; Mel'nikov, N.N.

1987-01-01

We investigated the phosphorylation and thiophosphorylation of 2-thiazolidine- and 2-oxazolidine-thiones. The presence in the heterocycle of the ambident triad HN-C=S can also lead to two series of phosphorylation products formed at the nitrogen and at the sulfur atom. It was therefore of interest to determine the dependence of the site of the phosphorylation on the structures of the heterocycle and off the phosphorylating agent. The formation of the N-phosphorylation products is confirmed by the 1 H NMR spectra, in which the signals of protons of the methylene group of the heteroring (C 4 H 2 ) are split on account of interaction with the phosphorus atom ( 3 JPH 0.5-2.3 Hz). We observed analogous values of 3 JPH constants for 2-aminothiazolines phosphorylated on the endocyclic nitrogen atom. In the 13 C NMR spectra of these compounds there are also coupling constants for the interaction of the carbon atoms C 4 and C 5 of the heterocycle with the phosphorus atom. The existence of the compounds as N-phosphorylated heterocycles is evidenced also by the 31 P chemical shifts

Phosphorylation of p37 is important for Golgi disassembly at mitosis

International Nuclear Information System (INIS)

Kaneko, Yayoi; Tamura, Kaori; Totsukawa, Go; Kondo, Hisao

2010-01-01

Research highlights: → p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. → Phosphorylated p37 does not bind to Golgi membranes. → p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

Melatonin Therapy Prevents Programmed Hypertension and Nitric Oxide Deficiency in Offspring Exposed to Maternal Caloric Restriction

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You-Lin Tain

2014-01-01

Full Text Available Nitric oxide (NO deficiency is involved in the development of hypertension, a condition that can originate early in life. We examined whether NO deficiency contributed to programmed hypertension in offspring from mothers with calorie-restricted diets and whether melatonin therapy prevented this process. We examined 3-month-old male rat offspring from four maternal groups: untreated controls, 50% calorie-restricted (CR rats, controls treated with melatonin (0.01% in drinking water, and CR rats treated with melatonin (CR + M. The effect of melatonin on nephrogenesis was analyzed using next-generation sequencing. The CR group developed hypertension associated with elevated plasma asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor, decreased L-arginine, decreased L-arginine-to-ADMA ratio (AAR, and decreased renal NO production. Maternal melatonin treatment prevented these effects. Melatonin prevented CR-induced renin and prorenin receptor expression. Renal angiotensin-converting enzyme 2 protein levels in the M and CR + M groups were also significantly increased by melatonin therapy. Maternal melatonin therapy had long-term epigenetic effects on global gene expression in the kidneys of offspring. Conclusively, we attributed these protective effects of melatonin on CR-induced programmed hypertension to the reduction of plasma ADMA, restoration of plasma AAR, increase of renal NO level, alteration of renin-angiotensin system, and epigenetic changes in numerous genes.

Highly Conductive and Reliable Copper-Filled Isotropically Conductive Adhesives Using Organic Acids for Oxidation Prevention

Science.gov (United States)

Chen, Wenjun; Deng, Dunying; Cheng, Yuanrong; Xiao, Fei

2015-07-01

The easy oxidation of copper is one critical obstacle to high-performance copper-filled isotropically conductive adhesives (ICAs). In this paper, a facile method to prepare highly reliable, highly conductive, and low-cost ICAs is reported. The copper fillers were treated by organic acids for oxidation prevention. Compared with ICA filled with untreated copper flakes, the ICA filled with copper flakes treated by different organic acids exhibited much lower bulk resistivity. The lowest bulk resistivity achieved was 4.5 × 10-5 Ω cm, which is comparable to that of commercially available Ag-filled ICA. After 500 h of 85°C/85% relative humidity (RH) aging, the treated ICAs showed quite stable bulk resistivity and relatively stable contact resistance. Through analyzing the results of x-ray diffraction, x-ray photoelectron spectroscopy, and thermogravimetric analysis, we found that, with the assistance of organic acids, the treated copper flakes exhibited resistance to oxidation, thus guaranteeing good performance.

Phosphorylation of the Bacillus subtilis Replication Controller YabA Plays a Role in Regulation of Sporulation and Biofilm Formation

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Tránsito García García

2018-03-01

Full Text Available Bacillus subtilis cells can adopt different life-styles in response to various environmental cues, including planktonic cells during vegetative growth, sessile cells during biofilm formation and sporulation. While switching life-styles, bacteria must coordinate the progression of their cell cycle with their physiological status. Our current understanding of the regulatory pathways controlling the decision-making processes and triggering developmental switches highlights a key role of protein phosphorylation. The regulatory mechanisms that integrate the bacterial chromosome replication status with sporulation involve checkpoint proteins that target the replication initiator DnaA or the kinase phosphorelay controlling the master regulator Spo0A. B. subtilis YabA is known to interact with DnaA to prevent over-initiation of replication during vegetative growth. Here, we report that YabA is phosphorylated by YabT, a Ser/Thr kinase expressed during sporulation and biofilm formation. The phosphorylation of YabA has no effect on replication initiation control but hyper-phosphorylation of YabA leads to an increase in sporulation efficiency and a strong inhibition of biofilm formation. We also provide evidence that YabA phosphorylation affects the level of Spo0A-P in cells. These results indicate that YabA is a multifunctional protein with a dual role in regulating replication initiation and life-style switching, thereby providing a potential mechanism for cross-talk and coordination of cellular processes during adaptation to environmental change.

Taenia crassiceps: fatty acids oxidation and alternative energy source in in vitro cysticerci exposed to anthelminthic drugs.

Science.gov (United States)

Vinaud, Marina Clare; Ferreira, Cirlane Silva; Lino Junior, Ruy de Souza; Bezerra, José Clecildo Barreto

2009-07-01

Cysticerci metabolic studies demonstrate alternative pathways responsible for its survival, such as energy sources, fatty acids oxidation and excretion of beta-hydroxybutyrate, which indicates the capability of energy production from proteins. The aim of this study was to detect alternative metabolic pathways for energy production and its end products in Taenia crassiceps cysticerci in vitro exposed to praziquantel and albendazole, in sub-lethal doses. Spectrophotometer and chromatographic analysis were performed to detect: propionate, acetate, beta-hydroxybutyrate, total proteins, urea and creatinine, SE by cysticerci in vitro exposed to praziquantel and albendazole. The drugs influenced the metabolism by inducing the creatinine phosphate phosphorylation as an alternative energy source, inhibiting the use of proteins and amino acids in the acid nucleic synthesis; and preventing the budding and replication of the cysticerci. This study also highlights the description of urea excretion, which is an important metabolic pathway to excrete toxic products such as ammonia, and the fatty acid oxidation as an alternative energy source in cysticerci exposed to anthelmintic drugs.

Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

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Ndong Christian

2012-04-01

Full Text Available Abstract Background Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide. Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. Conclusions Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

Effects of protein phosphorylation on color stability of ground meat.

Science.gov (United States)

Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

2017-03-15

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Occurrence and prevention of enhanced oxide deposition in boiler flow control orifices

International Nuclear Information System (INIS)

Woolsey, I.S.; Thomas, D.M.; Garbett, K.; Bignold, G.J.

1989-10-01

Once-through boilers, such as those of the AGRs, incorporate flow control orifices at the boiler inlet to ensure a satisfactory flow distribution and stability in the parallel flow paths of the boiler. Deposition of corrosion products in the flow control orifice leads to changes in the orifice pressure loss characteristics, which could lead to problems of flow maldistribution within the boiler, and any adverse consequences resulting from this, such as tube overheating. To date, AGR boiler inlet orifices have not suffered significant fouling due to corrosion products in the boiler feedwater. However, oxide deposition in orifices has been observed in other plants, and in experimental loops operating under conditions very similar to those at inlet to AGR boilers. The lack of deposition in AGR flow control orifices is therefore somewhat surprising. This Report describes studies carried out to examine the factors controlling oxide deposition in flow control orifices, the intention of the work being to explain why deposition has not occurred in AGR boilers to date, and to provide means of preventing deposition in the future should this prove necessary. (author)

Stevia Prevents Acute and Chronic Liver Injury Induced by Carbon Tetrachloride by Blocking Oxidative Stress through Nrf2 Upregulation

Science.gov (United States)

Ramos-Tovar, Erika; Hernández-Aquino, Erika; Casas-Grajales, Sael; Buendia-Montaño, Laura D.; Tsutsumi, Víctor

2018-01-01

The effect of stevia on liver cirrhosis has not been previously investigated. In the present study, the antioxidant and anti-inflammatory properties of stevia leaves were studied in male Wistar rats with carbon tetrachloride- (CCl4-) induced acute and chronic liver damage. Acute and chronic liver damage induced oxidative stress, necrosis, and cholestasis, which were significantly ameliorated by stevia. Chronic CCl4 treatment resulted in liver cirrhosis, as evidenced by nodules of hepatocytes surrounded by thick bands of collagen and distortion of the hepatic architecture, and stevia significantly prevented these alterations. Subsequently, the underlying mechanism of action of the plant was analyzed. Our study for the first time shows that stevia upregulated Nrf2, thereby counteracting oxidative stress, and prevented necrosis and cholestasis through modulation of the main proinflammatory cytokines via NF-κB inhibition. These multitarget mechanisms led to the prevention of experimental cirrhosis. Given the reasonable safety profile of stevia, our results indicated that it may be useful for the clinical treatment of acute and chronic liver diseases. PMID:29849889

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

International Nuclear Information System (INIS)

Tengan, Celia H.; Kiyomoto, Beatriz H.; Godinho, Rosely O.; Gamba, Juliana; Neves, Afonso C.; Schmidt, Beny; Oliveira, Acary S.B.; Gabbai, Alberto A.

2007-01-01

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle

The in vivo phosphorylation sites of rat brain dynamin I

DEFF Research Database (Denmark)

Graham, Mark E; Anggono, Victor; Bache, Nicolai

2007-01-01

-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation...... of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results...

Phosphorylation of mouse serine racemase regulates D-serine synthesis

DEFF Research Database (Denmark)

Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

2010-01-01

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

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Neil Arvin Bretaña

Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Science.gov (United States)

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

Skeletal Muscle Pyruvate Dehydrogenase Phosphorylation and Lactate Accumulation During Sprint Exercise in Normoxia and Severe Acute Hypoxia: Effects of Antioxidants

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David Morales-Alamo

2018-03-01

Full Text Available Compared to normoxia, during sprint exercise in severe acute hypoxia the glycolytic rate is increased leading to greater lactate accumulation, acidification, and oxidative stress. To determine the role played by pyruvate dehydrogenase (PDH activation and reactive nitrogen and oxygen species (RNOS in muscle lactate accumulation, nine volunteers performed a single 30-s sprint (Wingate test on four occasions: two after the ingestion of placebo and another two following the intake of antioxidants, while breathing either hypoxic gas (PIO2 = 75 mmHg or room air (PIO2 = 143 mmHg. Vastus lateralis muscle biopsies were obtained before, immediately after, 30 and 120 min post-sprint. Antioxidants reduced the glycolytic rate without altering performance or VO2. Immediately after the sprints, Ser293- and Ser300-PDH-E1α phosphorylations were reduced to similar levels in all conditions (~66 and 91%, respectively. However, 30 min into recovery Ser293-PDH-E1α phosphorylation reached pre-exercise values while Ser300-PDH-E1α was still reduced by 44%. Thirty minutes after the sprint Ser293-PDH-E1α phosphorylation was greater with antioxidants, resulting in 74% higher muscle lactate concentration. Changes in Ser293 and Ser300-PDH-E1α phosphorylation from pre to immediately after the sprints were linearly related after placebo (r = 0.74, P < 0.001; n = 18, but not after antioxidants ingestion (r = 0.35, P = 0.15. In summary, lactate accumulation during sprint exercise in severe acute hypoxia is not caused by a reduced activation of the PDH. The ingestion of antioxidants is associated with increased PDH re-phosphorylation and slower elimination of muscle lactate during the recovery period. Ser293 re-phosphorylates at a faster rate than Ser300-PDH-E1α during the recovery period, suggesting slightly different regulatory mechanisms.

Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice.

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Carr, Michael I; Roderick, Justine E; Zhang, Hong; Woda, Bruce A; Kelliher, Michelle A; Jones, Stephen N

2016-12-27

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2 S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2 Y393F ) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2 Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2 Y393F/S394A mice and Mdm2 S394A mice display similar phenotypes.

Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis.

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Chen, Yanmei; Hoehenwarter, Wolfgang

2015-12-01

Salinity and oxidative stress are major factors affecting and limiting the productivity of agricultural crops. The molecular and biochemical processes governing the plant response to abiotic stress have often been researched in a reductionist manner. Here, we report a systemic approach combining metabolic labeling and phosphoproteomics to capture early signaling events with quantitative metabolome analysis and enzyme activity assays to determine the effects of salt and oxidative stress on plant physiology. K(+) and Na(+) transporters showed coordinated changes in their phosphorylation pattern, indicating the importance of dynamic ion homeostasis for adaptation to salt stress. Unique phosphorylation sites were found for Arabidopsis (Arabidopsis thaliana) SNF1 kinase homolog10 and 11, indicating their central roles in the stress-regulated responses. Seven Sucrose Non-fermenting1-Related Protein Kinase2 kinases showed varying levels of phosphorylation at multiple serine/threonine residues in their kinase domain upon stress, showing temporally distinct modulation of the various isoforms. Salinity and oxidative stress also lead to changes in protein phosphorylation of proteins central to photosynthesis, in particular the kinase State Transition Protein7 required for state transition and light-harvesting II complex proteins. Furthermore, stress-induced changes of the phosphorylation of enzymes of central metabolism were observed. The phosphorylation patterns of these proteins were concurrent with changes in enzyme activity. This was reflected by altered levels of metabolites, such as the sugars sucrose and fructose, glycolysis intermediates, and amino acids. Together, our study provides evidence for a link between early signaling in the salt and oxidative stress response that regulates the state transition of photosynthesis and the rearrangement of primary metabolism. © 2015 American Society of Plant Biologists. All Rights Reserved.

Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.

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Vichaiwong, Kanokwan; Purohit, Suneet; An, Ding; Toyoda, Taro; Jessen, Niels; Hirshman, Michael F; Goodyear, Laurie J

2010-10-15

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.

Evidence of oxidative stress and mitochondrial dysfunction in spinocerebellar ataxia type 2 (SCA2) patient fibroblasts

DEFF Research Database (Denmark)

Cornelius, Nanna; Wardman, Jonathan H; Hargreaves, Iain P

2017-01-01

Spinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disorder caused by a CAG repeat expansion in the ataxin-2 gene. We show increased oxidative stress, abnormalities in the antioxidant system, changes in complexes involved in oxidative phosphorylation and changes in mitochondrial mor...

Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

International Nuclear Information System (INIS)

Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

2017-01-01

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

Raptor is phosphorylated by cdc2 during mitosis.

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Dana M Gwinn

2010-02-01

Full Text Available The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1. As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct

Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

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Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

2012-01-01

Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

Tyrosine phosphorylation of Kv1.5 is upregulated in intrauterine growth retardation rats with exaggerated pulmonary hypertension

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L.C. Fu

2017-09-01

Full Text Available Intrauterine growth retardation (IUGR is associated with the development of adult-onset diseases, including pulmonary hypertension. However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular dysfunction later in life is not fully understood. Here, we investigated the role of tyrosine phosphorylation of voltage-gated potassium channel 1.5 (Kv1.5 in this prenatal event that results in exaggerated adult vascular dysfunction. A rat model of chronic hypoxia (2 weeks of hypoxia at 12 weeks old following IUGR was used to investigate the physiological and structural effect of intrauterine malnutrition on the pulmonary artery by evaluating pulmonary artery systolic pressure and vascular diameter in male rats. Kv1.5 expression and tyrosine phosphorylation in pulmonary artery smooth muscle cells (PASMCs were determined. We found that IUGR increased mean pulmonary artery pressure and resulted in thicker pulmonary artery smooth muscle layer in 14-week-old rats after 2 weeks of hypoxia, while no difference was observed in normoxia groups. In the PASMCs of IUGR-hypoxia rats, Kv1.5 mRNA and protein expression decreased while that of tyrosine-phosphorylated Kv1.5 significantly increased. These results demonstrate that IUGR leads to exaggerated chronic hypoxia pulmonary arterial hypertension (CH-PAH in association with decreased Kv1.5 expression in PASMCs. This phenomenon may be mediated by increased tyrosine phosphorylation of Kv1.5 in PASMCs and it provides new insight into the prevention and treatment of IUGR-related CH-PAH.

Deciphering the role of the signal- and Sty1 kinase-dependent phosphorylation of the stress-responsive transcription factor Atf1 on gene activation.

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Salat-Canela, Clàudia; Paulo, Esther; Sánchez-Mir, Laura; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2017-08-18

Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast ( Schizosaccharomyces pombe ), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1 itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that, in cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA but, rather, establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells by promoting transcription initiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

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Slavka eBekesova

2015-05-01

Full Text Available The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+ used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™ offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

PKCδ-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

International Nuclear Information System (INIS)

Greene, Michael W.; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

2006-01-01

The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCδ on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCδ-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCδ catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1

A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction

International Nuclear Information System (INIS)

Corvera, S.; Folander, K.; Clairmont, K.B.; Czech, M.P.

1988-01-01

Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32 P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of ∼ 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at ∼ 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated ∼ 3-fold in this clathrin-enriched subfraction. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction

Carbon monoxide alleviates ethanol-induced oxidative damage and inflammatory stress through activating p38 MAPK pathway

International Nuclear Information System (INIS)

Li, Yanyan; Gao, Chao; Shi, Yanru; Tang, Yuhan; Liu, Liang; Xiong, Ting; Du, Min; Xing, Mingyou; Liu, Liegang; Yao, Ping

2013-01-01

Stress-inducible protein heme oxygenase-1(HO-1) is well-appreciative to counteract oxidative damage and inflammatory stress involving the pathogenesis of alcoholic liver diseases (ALD). The potential role and signaling pathways of HO-1 metabolite carbon monoxide (CO), however, still remained unclear. To explore the precise mechanisms, ethanol-dosed adult male Balb/c mice (5.0 g/kg.bw.) or ethanol-incubated primary rat hepatocytes (100 mmol/L) were pretreated by tricarbonyldichlororuthenium (II) dimmer (CORM-2, 8 mg/kg for mice or 20 μmol/L for hepatocytes), as well as other pharmacological reagents. Our data showed that CO released from HO-1 induction by quercetin prevented ethanol-derived oxidative injury, which was abolished by CO scavenger hemoglobin. The protection was mimicked by CORM-2 with the attenuation of GSH depletion, SOD inactivation, MDA overproduction, and the leakage of AST, ALT or LDH in serum and culture medium induced by ethanol. Moreover, CORM-2 injection or incubation stimulated p38 phosphorylation and suppressed abnormal Tnfa and IL-6, accompanying the alleviation of redox imbalance induced by ethanol and aggravated by inflammatory factors. The protective role of CORM-2 was abolished by SB203580 (p38 inhibitor) but not by PD98059 (ERK inhibitor) or SP600125 (JNK inhibitor). Thus, HO-1 released CO prevented ethanol-elicited hepatic oxidative damage and inflammatory stress through activating p38 MAPK pathway, suggesting a potential therapeutic role of gaseous signal molecule on ALD induced by naturally occurring phytochemicals. - Highlights: • CO alleviated ethanol-derived liver oxidative and inflammatory stress in mice. • CO eased ethanol and inflammatory factor-induced oxidative damage in hepatocytes. • The p38 MAPK is a key signaling mechanism for the protective function of CO in ALD

Carbon monoxide alleviates ethanol-induced oxidative damage and inflammatory stress through activating p38 MAPK pathway

Energy Technology Data Exchange (ETDEWEB)

Li, Yanyan; Gao, Chao; Shi, Yanru; Tang, Yuhan; Liu, Liang; Xiong, Ting; Du, Min [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Xing, Mingyou [Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Liu, Liegang [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Yao, Ping, E-mail: yaoping@mails.tjmu.edu.cn [Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Ministry of Education Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China); Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030 (China)

2013-11-15

Stress-inducible protein heme oxygenase-1(HO-1) is well-appreciative to counteract oxidative damage and inflammatory stress involving the pathogenesis of alcoholic liver diseases (ALD). The potential role and signaling pathways of HO-1 metabolite carbon monoxide (CO), however, still remained unclear. To explore the precise mechanisms, ethanol-dosed adult male Balb/c mice (5.0 g/kg.bw.) or ethanol-incubated primary rat hepatocytes (100 mmol/L) were pretreated by tricarbonyldichlororuthenium (II) dimmer (CORM-2, 8 mg/kg for mice or 20 μmol/L for hepatocytes), as well as other pharmacological reagents. Our data showed that CO released from HO-1 induction by quercetin prevented ethanol-derived oxidative injury, which was abolished by CO scavenger hemoglobin. The protection was mimicked by CORM-2 with the attenuation of GSH depletion, SOD inactivation, MDA overproduction, and the leakage of AST, ALT or LDH in serum and culture medium induced by ethanol. Moreover, CORM-2 injection or incubation stimulated p38 phosphorylation and suppressed abnormal Tnfa and IL-6, accompanying the alleviation of redox imbalance induced by ethanol and aggravated by inflammatory factors. The protective role of CORM-2 was abolished by SB203580 (p38 inhibitor) but not by PD98059 (ERK inhibitor) or SP600125 (JNK inhibitor). Thus, HO-1 released CO prevented ethanol-elicited hepatic oxidative damage and inflammatory stress through activating p38 MAPK pathway, suggesting a potential therapeutic role of gaseous signal molecule on ALD induced by naturally occurring phytochemicals. - Highlights: • CO alleviated ethanol-derived liver oxidative and inflammatory stress in mice. • CO eased ethanol and inflammatory factor-induced oxidative damage in hepatocytes. • The p38 MAPK is a key signaling mechanism for the protective function of CO in ALD.

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Macek, B

2006-01-01

for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

Science.gov (United States)

Thériault, Jimmy R; Lambert, Herman; Chávez-Zobel, Aura T; Charest, Gabriel; Lavigne, Pierre; Landry, Jacques

2004-05-28

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

Science.gov (United States)

Verma, Rakesh; Venkatareddy, Madhusudan; Kalinowski, Anne; Patel, Sanjeevkumar R.; Salant, David J.

2015-01-01

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury. PMID:26644409

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

Science.gov (United States)

Copeland, O'Neal; Sadayappan, Sakthivel; Messer, Andrew E; Steinen, Ger J M; van der Velden, Jolanda; Marston, Steven B

2010-12-01

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the

Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

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Bakthisaran, Raman; Akula, Kranthi Kiran; Tangirala, Ramakrishna; Rao, Ch Mohan

2016-01-01

αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin

PhosphoRice: a meta-predictor of rice-specific phosphorylation sites

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Que Shufu

2012-02-01

Full Text Available Abstract Background As a result of the growing body of protein phosphorylation sites data, the number of phosphoprotein databases is constantly increasing, and dozens of tools are available for predicting protein phosphorylation sites to achieve fast automatic results. However, none of the existing tools has been developed to predict protein phosphorylation sites in rice. Results In this paper, the phosphorylation site predictors, NetPhos 2.0, NetPhosK, Kinasephos, Scansite, Disphos and Predphosphos, were integrated to construct meta-predictors of rice-specific phosphorylation sites using several methods, including unweighted voting, unreduced weighted voting, reduced unweighted voting and weighted voting strategies. PhosphoRice, the meta-predictor produced by using weighted voting strategy with parameters selected by restricted grid search and conditional random search, performed the best at predicting phosphorylation sites in rice. Its Matthew's Correlation Coefficient (MCC and Accuracy (ACC reached to 0.474 and 73.8%, respectively. Compared to the best individual element predictor (Disphos_default, PhosphoRice archieved a significant increase in MCC of 0.071 (P Conclusions PhosphoRice is a powerful tool for predicting unidentified phosphorylation sites in rice. Compared to the existing methods, we found that our tool showed greater robustness in ACC and MCC. PhosphoRice is available to the public at http://bioinformatics.fafu.edu.cn/PhosphoRice.

The Effect of Nitric Oxide Synthetase Inhibitor (L-NAME on Prevention of Morphine Dependence in Rats

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A. Rafati

2004-10-01

Full Text Available Prevention of dependency to morphine or delaying to it and decreasing of tendency to morphine craving and also decreasing in morphine induced hyperalgesia(tolerance were the aims of this study. Nitric oxide is one of the neurotransmitters, which involves in the Dopamine reuptake in striatum. Dopamine is one of the most important neurotransmitters in reward system in central nervous system and it has a critical role in morphine addiction and dependency, tendency and tolerance to it, so in this study we survied the role of L- NAME as a nitric oxide synthetase (NOS inhibitor on the prevention of morphine addiction in rats. In this study we evaluated behavioral changes such as morphine craving by self - administration as a criterion for tendency, dependency by observation of withdrawal syndrom signs (e.g Jumping, wet dog shaking and also responses to nociceptive condenced bim of light by using tail flick analgesia metric device in sham (consuming tap water, control (consuming increasing doses of morphine sulfate solution from 0.1mg/ml up to 0.4mg/ml and test (treated with 45 mg/kg of L- NAME 30 minutes before consuming of morphine sulfate solution per day groups. The results showed that pretreatment with L- NAME in test group lead to a significant decline in tendency to morphine craving, withdrawal signs and also a significant reversal of morphine induced hyperalgesia. We concluded that L- NAME is a potent agent in the prevention of morphine addiction.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

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Callender, Tracy L; Laureau, Raphaelle; Wan, Lihong; Chen, Xiangyu; Sandhu, Rima; Laljee, Saif; Zhou, Sai; Suhandynata, Ray T; Prugar, Evelyn; Gaines, William A; Kwon, YoungHo; Börner, G Valentin; Nicolas, Alain; Neiman, Aaron M; Hollingsworth, Nancy M

2016-08-01

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

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Tracy L Callender

2016-08-01

Full Text Available During meiosis, programmed double strand breaks (DSBs are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i phosphorylation of Rad54 by Mek1 and (ii binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

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Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

2015-05-08

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication.

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Sui, Jiangdong; Lin, Yu-Fen; Xu, Kangling; Lee, Kyung-Jong; Wang, Dong; Chen, Benjamin P C

2015-07-13

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Physical inactivity and muscle oxidative capacity in humans

DEFF Research Database (Denmark)

Gram, Martin; Dahl, Rannvá; Dela, Flemming

2014-01-01

Physical inactivity is associated with a high prevalence of type 2 diabetes and is an independent predictor of mortality. It is possible that the detrimental effects of physical inactivity are mediated through a lack of adequate muscle oxidative capacity. This short review will cover the present...... literature on the effects of different models of inactivity on muscle oxidative capacity in humans. Effects of physical inactivity include decreased mitochondrial content, decreased activity of oxidative enzymes, changes in markers of oxidative stress and a decreased expression of genes and contents...... of proteins related to oxidative phosphorylation. With such a substantial down-regulation, it is likely that a range of adenosine triphosphate (ATP)-dependent pathways such as calcium signalling, respiratory capacity and apoptosis are affected by physical inactivity. However, this has not been investigated...

Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

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Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

1987-01-01

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

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Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-04-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

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Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

DEFF Research Database (Denmark)

Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

2011-01-01

Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

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Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

2017-01-06

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

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Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

2018-01-05

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

Antioxidant supplements for preventing gastrointestinal cancers

DEFF Research Database (Denmark)

Bjelakovic, G; Nikolova, D; Simonetti, R G

2004-01-01

Oxidative stress may cause gastrointestinal cancers. The evidence on whether antioxidant supplements are effective in preventing gastrointestinal cancers is contradictory.......Oxidative stress may cause gastrointestinal cancers. The evidence on whether antioxidant supplements are effective in preventing gastrointestinal cancers is contradictory....

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

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Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Physical basis of the effect of hemoglobin on the 31P NMR chemical shifts of various phosphoryl compounds

International Nuclear Information System (INIS)

Kirk, K.; Kuchel, P.W.

1988-01-01

The marked difference between the intra- and extracellular 31 P NMR chemical shifts of various phosphoryl compounds when added to a red cell suspension may be largely understood in terms of the effects of hemoglobin on the 31 P NMR chemical shifts. The presence of [oxy- or (carbonmonoxy)-] hemoglobin inside the red cell causes the bulk magnetic susceptibility of the cell cytoplasm to be significantly less than that of the external solution. This difference is sufficient to account for the difference in the intra- and extracellular chemical shifts of the two phosphate esters trimethyl phosphate and triethyl phosphate. However, in the case of the compounds dimethyl methylphosphonate, diethyl methylphosphonate, and trimethylphosphine oxide as well as the hypophosphite, phenylphosphinate, and diphenylphosphinate ions, hemoglobin exerts an additional, much larger, effect, causing the 31 P NMR resonances to shift to lower frequency in a manner that cannot be accounted for in terms of magnetic susceptibility. Lysozyme is a protein structurally unrelated to hemoglobin and was shown to cause similar shifts to lower frequency of the resonances of these six compounds; this suggests that the mechanism may involve a property of proteins in general and not a specific property of hemoglobin. The effect of different solvents on the chemical shifts of the eight phosphoryl compounds provided an insight into the possible physical basis of the effect. It is proposed that, in addition to magnetic susceptibility effects, hemoglobin exerts its influence on phosphoryl chemical shifts by disrupting the hydrogen bonding of the phosphoryl group to solvent water

Inhibition of peptide aggregation by means of enzymatic phosphorylation

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Kristin Folmert

2016-11-01

Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

[Acetylcholine activation of alpha-ketoglutarate oxidation in liver mitochondria].

Science.gov (United States)

Shostakovskaia, I V; Doliba, N M; Gordiĭ, S K; Babskiĭ, A M; Kondrashova, M N

1986-01-01

Activation of alpha-ketoglutarate oxidation in the rat liver mitochondria takes place 15 and 30 min after intraperitoneal injection of acetyl choline. This mediator in doses of 25, 50 and 100 micrograms per 100 g of body weight causes a pronounced stimulation of phosphorylation respiration rate and calcium capacity of mitochondria with alpha-ketoglutarate oxidation. Acetyl choline is found to have a moderate inhibitory action on oxidation of lower (physiological) concentrations of succinate. Its stimulating action on alpha-ketoglutarate oxidation is associated with activation of M-cholinoreceptors; atropine, a choline-blocker, removes completely this effect. It is supposed that alpha-ketoglutarate and succinate are included into the composition of two reciprocal hormonal-substrate nucleotide systems.

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

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Andaleeb Sajid

2011-03-01

Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

The Traditional Herbal Medicine, Dangkwisoo-San, Prevents Cerebral Ischemic Injury through Nitric Oxide-Dependent Mechanisms

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Ji Hyun Kim

2011-01-01

Full Text Available Dangkwisoo-San (DS is an herbal extract that is widely used in traditional Korean medicine to treat traumatic ecchymosis and pain by promoting blood circulation and relieving blood stasis. However, the effect of DS in cerebrovascular disease has not been examined experimentally. The protective effects of DS on focal ischemic brain were investigated in a mouse model. DS stimulated nitric oxide (NO production in human brain microvascular endothelial cells (HBMECs. DS (10–300 μg/mL produced a concentration-dependent relaxation in mouse aorta, which was significantly attenuated by the nitric oxide synthase (NOS inhibitor L-NAME, suggesting that DS causes vasodilation via a NO-dependent mechanism. DS increased resting cerebral blood flow (CBF, although it caused mild hypotension. To investigate the effect of DS on the acute cerebral injury, C57/BL6J mice received 90 min of middle cerebral artery occlusion followed by 22.5 h of reperfusion. DS administered 3 days before arterial occlusion significantly reduced cerebral infarct size by 53.7% compared with vehicle treatment. However, DS did not reduce brain infarction in mice treated with the relatively specific endothelial NOS (eNOS inhibitor, N5-(1-iminoethyl-L-ornithine, suggesting that the neuroprotective effect of DS is primarily endothelium-dependent. This correlated with increased phosphorylation of eNOS in the brains of DS-treated mice. DS acutely improves CBF in eNOS-dependent vasodilation and reduces infarct size in focal cerebral ischemia. These data provide causal evidence that DS is cerebroprotective via the eNOS-dependent production of NO, which ameliorates blood circulation.

Tat-PRAS40 prevent hippocampal HT-22 cell death and oxidative stress induced animal brain ischemic insults.

Science.gov (United States)

Shin, Min Jea; Kim, Dae Won; Jo, Hyo Sang; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Yeo, Eun Ji; Choi, Yeon Joo; Kim, Ji An; Hwang, Jung Soon; Sohn, Eun Jeong; Jeong, Ji-Heon; Kim, Duk-Soo; Kwon, Hyeok Yil; Cho, Yong-Jun; Lee, Keunwook; Han, Kyu Hyung; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

2016-08-01

Proline rich Akt substrate (PRAS40) is a component of mammalian target of rapamycin complex 1 (mTORC1) and is known to play an important role against reactive oxygen species-induced cell death. However, the precise function of PRAS40 in ischemia remains unclear. Thus, we investigated whether Tat-PRAS40, a cell-permeable fusion protein, has a protective function against oxidative stress-induced hippocampal neuronal (HT-22) cell death in an animal model of ischemia. We showed that Tat-PRAS40 transduced into HT-22 cells, and significantly protected against cell death by reducing the levels of H2O2 and derived reactive species, and DNA fragmentation as well as via the regulation of Bcl-2, Bax, and caspase 3 expression levels in H2O2 treated cells. Also, we showed that transduced Tat-PARS40 protein markedly increased phosphorylated RRAS40 expression levels and 14-3-3σ complex via the Akt signaling pathway. In an animal ischemia model, Tat-PRAS40 effectively transduced into the hippocampus in animal brain and significantly protected against neuronal cell death in the CA1 region. We showed that Tat-PRAS40 protein effectively transduced into hippocampal neuronal cells and markedly protected against neuronal cell damage. Therefore, we suggest that Tat-PRAS40 protein may be used as a therapeutic protein for ischemia and oxidative stress-induced brain disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

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Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

2017-06-01

Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

The role of oxidative stress in EBV lytic reactivation, radioresistance and the potential preventive and therapeutic implications.

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Hu, Jianmin; Li, Hongde; Luo, Xiangjian; Li, Yueshuo; Bode, Ann; Cao, Ya

2017-11-01

Epstein-Barr virus (EBV) is an important cancer causing virus. Cancer associated with EBV account for approximately 1.5% of all cancers, and represent 1.8% of all cancer deaths worldwide. EBV reactivation plays an important role in the development of EBV-related diseases and is closely related with patients' survival and clinical stages of EBV-related cancers. The therapy regarding to EBV-related cancers is very urgent, especially in endemic areas. Generating oxidative stress is a critical mechanism by which host cells defend against infection by virus. In addition, ROS-mediated oxidative stress plays a significant but paradoxical role acting as a "double-edged sword" to regulate cellular response to radiation, which is the main therapy strategy for EBV-related cancers, especially nasopharyngeal carcinoma. Therefore, in this review we primarily discuss the possible interplay among the oxidative stress, EBV lytic reactivation and radioresistance. Understanding the role of oxidative stress in EBV lytic reactivation and radioresistance will assist in the development of effective strategies for prevention and treatment of EBV-related cancers. © 2017 UICC.

Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

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Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

2011-01-01

Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

The 5-lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines.

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Loftus, J P; Cavatorta, D; Bushey, J J; Levine, C B; Sevier, C S; Wakshlag, J J

2016-06-01

The 5-lipoxygenase (5-LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase-3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5-LOX inhibitor regardless of cellular 5-LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5-LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide-3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response. © 2014 John Wiley & Sons Ltd.

Glutamine prevents oxidative stress in a model of portal hypertension.

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Zabot, Gilmara Pandolfo; Carvalhal, Gustavo Franco; Marroni, Norma Possa; Licks, Francielli; Hartmann, Renata Minuzzo; da Silva, Vinícius Duval; Fillmann, Henrique Sarubbi

2017-07-07

To evaluate the protective effects of glutamine in a model of portal hypertension (PH) induced by partial portal vein ligation (PPVL). Male Wistar rats were housed in a controlled environment and were allowed access to food and water ad libitum . Twenty-four male Wistar rats were divided into four experimental groups: (1) control group (SO) - rats underwent exploratory laparotomy; (2) control + glutamine group (SO + G) - rats were subjected to laparotomy and were treated intraperitoneally with glutamine; (3) portal hypertension group (PPVL) - rats were subjected to PPVL; and (4) PPVL + glutamine group (PPVL + G) - rats were treated intraperitoneally with glutamine for seven days. Local injuries were determined by evaluating intestinal segments for oxidative stress using lipid peroxidation and the activities of glutathione peroxidase (GPx), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) after PPVL. Lipid peroxidation of the membrane was increased in the animals subjected to PH ( P 0.05). The activity of the antioxidant enzyme GTx was decreased in the gut of animals subjected to PH compared with that in the control group of animals not subjected to PH ( P 0.05). At least 10 random, non-overlapping images of each histological slide with 200 × magnification (44 pixel = 1 μm) were captured. The sum means of all areas, of each group were calculated. The mean areas of eNOS staining for both of the control groups were similar. The PPVL group showed the largest area of staining for eNOS. The PPVL + G group had the second highest amount of staining, but the mean value was much lower than that of the PPVL group ( P < 0.01). For iNOS, the control (SO) and control + G (SO + G) groups showed similar areas of staining. The PPVL group contained the largest area of iNOS staining, followed by the PPVL + G group; however, this area was significantly smaller than that of the group that underwent PH without glutamine ( P < 0.01). Treatment with

Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

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Zhang, Menghuan; Li, Hong; He, Ying; Sun, Han; Xia, Li; Wang, Lishun; Sun, Bo; Ma, Liangxiao; Zhang, Guoqing; Li, Jing; Li, Yixue; Xie, Lu

2015-07-02

Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

International Nuclear Information System (INIS)

Haycock, J.W.; Browning, M.D.; Greengard, P.

1988-01-01

Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

Inhibitory effects of myricitrin on oxidative stress-induced endothelial damage and early atherosclerosis in ApoE −/− mice

Energy Technology Data Exchange (ETDEWEB)

Sun, Gui-bo; Qin, Meng [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 100193, Beijing (China); Ye, Jing-xue [Jilin Agricultural University, No. 2888, Xincheng Street, Changchun, 130118 Jilin (China); Pan, Rui-le; Meng, Xiang-bao; Wang, Min; Luo, Yun; Li, Zong-yang [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 100193, Beijing (China); Wang, Hong-wei, E-mail: hwang@nju.edu.cn [Center for Translational Medicine and Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu 210093 (China); Sun, Xiao-bo, E-mail: sunsubmit@163.com [Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, 100193, Beijing (China)

2013-08-15

Atherosclerosis (AS) is a state of heightened oxidative stress characterized by lipid and protein oxidation in vascular walls. Oxidative stress-induced vascular endothelial cell (VEC) injury is a major factor in the pathogenesis of AS. Myricitrin, a natural flavonoid isolated from the root bark of Myrica cerifera, was recently found to have a strong antioxidative effect. However, its use for treating cardiovascular diseases, especially AS is still unreported. Consequently, we evaluated the cytoprotective effect of myricitrin on AS by assessing oxidative stress-induced VEC damage. The in vivo study using an ApoE −/− mouse model of AS demonstrated that myricitrin treatment protects against VEC damage and inhibits early AS plaque formation. This effect is associated with the antioxidative effect of myricitrin, as observed in a hydrogen peroxide (H{sub 2}O{sub 2})-induced rat model of artery endothelial injury and primary cultured human VECs. Myricitrin treatment also prevents and attenuates H{sub 2}O{sub 2}-induced endothelial injury. Further investigation of the cytoprotective effects of myricitrin demonstrated that myricitrin exerts its function by scavenging for reactive oxygen species, as well as reducing lipid peroxidation, blocking NO release, and maintaining mitochondrial transmembrane potential. Myricitrin treatment also significantly decreased H{sub 2}O{sub 2}-induced apoptosis in VECs, which was associated with significant inhibition of p53 gene expression, activation of caspase-3 and the MAPK signaling pathway, and alteration of the patterns of pro-apoptotic and anti-apoptotic gene expression. The resulting significantly increased bcl-2/bax ratio indicates that myricitrin may prevent the apoptosis induced by oxidative stress injury. - Highlights: • Myricitrin prevents early atherosclerosis in ApoE−/− mice. • Myricitrin protects endothelial cell from H{sub 2}O{sub 2} induced injury in rat and HUVECs. • Myricitrin enhanced NO release and up

Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

International Nuclear Information System (INIS)

Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji

2007-01-01

Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS

c-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells

Directory of Open Access Journals (Sweden)

Diana M. Dunn

2015-08-01

Full Text Available The ability of Heat Shock Protein 90 (Hsp90 to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1, promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

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Yen-Hsing Li

2015-07-01

Full Text Available The Hedgehog (Hh pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

Bad phosphorylation as a target of inhibition in oncology.

Science.gov (United States)

Bui, Ngoc-Linh-Chi; Pandey, Vijay; Zhu, Tao; Ma, Lan; Basappa; Lobie, Peter E

2018-02-28

Bcl-2 agonist of cell death (BAD) is a BH3-only member of the Bcl-2 family which possesses important regulatory function in apoptosis. BAD has also been shown to possess many non-apoptotic functions closely linked to cancer including regulation of glycolysis, autophagy, cell cycle progression and immune system development. Interestingly, BAD can be either pro-apoptotic or pro-survival depending on the phosphorylation state of three specific serine residues (human S75, S99 and S118). Expression of BAD and BAD phosphorylation patterns have been shown to influence tumor initiation and progression and play a predictive role in disease prognosis, drug response and chemosensitivity in various cancers. This review aims to summarize the current evidence on the functional role of BAD phosphorylation in human cancer and evaluate the potential utility of modulating BAD phosphorylation in cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Polyphenol-Rich Blackcurrant Juice Prevents Endothelial Dysfunction in the Mesenteric Artery of Cirrhotic Rats with Portal Hypertension: Role of Oxidative Stress and the Angiotensin System.

Science.gov (United States)

Rashid, Sherzad; Idris-Khodja, Noureddine; Auger, Cyril; Kevers, Claire; Pincemail, Joël; Alhosin, Mahmoud; Boehm, Nelly; Oswald-Mammosser, Monique; Schini-Kerth, Valérie B

2018-04-01

Chronic liver diseases with portal hypertension are characterized by a progressive vasodilatation, endothelial dysfunction, and NADPH oxidase-derived vascular oxidative stress, which have been suggested to involve the angiotensin system. This study evaluated the possibility that oral intake of polyphenol-rich blackcurrant juice (PRBJ), a rich natural source of antioxidants, prevents endothelial dysfunction in a rat model of cirrhosis induced by chronic bile duct ligation (CBDL), and, if so, determined the underlying mechanism. Male Wistar rats received either control drinking water or water containing 60 mg/kg gallic acid equivalents of PRBJ for 3 weeks before undergoing surgery with CBDL or sham surgery. After 4 weeks, vascular reactivity was assessed in mesenteric artery rings using organ chambers. Both the acetylcholine-induced nitric oxide (NO)- and endothelium-dependent hyperpolarization (EDH)-mediated relaxations in mesenteric artery rings were significantly reduced in CBDL rats compared to sham rats. An increased level of oxidative stress and expression of NADPH oxidase subunits, COX-2, NOS, and of the vascular angiotensin system are observed in arterial sections in the CBDL group. Chronic intake of PRBJ prevented the CBDL-induced impaired EDH-mediated relaxation, oxidative stress, and expression of the different target proteins in the arterial wall. In addition, PRBJ prevented the CBDL-induced increase in the plasma level of proinflammatory cytokines (interleukin [IL]-1α, monocyte chemotactic protein 1, and tumor necrosis factor α) and the decrease of the anti-inflammatory cytokine, IL-4. Altogether, these observations indicate that regular ingestion of PRBJ prevents the CBDL-induced endothelial dysfunction in the mesenteric artery most likely by normalizing the level of vascular oxidative stress and the angiotensin system.

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

Directory of Open Access Journals (Sweden)

Jens T Stieler

Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

Retosiban Prevents Stretch-Induced Human Myometrial Contractility and Delays Labor in Cynomolgus Monkeys.

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Aye, Irving L M H; Moraitis, Alexandros A; Stanislaus, Dinesh; Charnock-Jones, D Stephen; Smith, Gordon C S

2018-03-01

Stretch of the myometrium promotes its contractility and is believed to contribute to the control of parturition at term and to the increased risk of preterm birth in multiple pregnancies. To determine the effects of the putative oxytocin receptor (OTR) inverse agonist retosiban on (1) the contractility of human myometrial explants and (2) labor in nonhuman primates. Human myometrial biopsies were obtained at planned term cesarean, and explants were exposed to stretch in the presence and absence of a range of drugs, including retosiban. The in vivo effects of retosiban were determined in cynomolgus monkeys. Prolonged mechanical stretch promoted myometrial extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Moreover, stretch-induced stimulation of myometrial contractility was prevented by ERK1/2 inhibitors. Retosiban (10 nM) prevented stretch-induced stimulation of myometrial contractility and phosphorylation of ERK1/2. Moreover, the inhibitory effect of retosiban on stretch-induced ERK1/2 phosphorylation was prevented by coincubation with a 100-fold excess of a peptide OTR antagonist, atosiban. Compared with vehicle-treated cynomolgus monkeys, treatment with oral retosiban (100 to 150 days of gestational age) reduced the risk of spontaneous delivery (hazard ratio = 0.07, 95% confidence interval 0.01 to 0.60, P = 0.015). The OTR acts as a uterine mechanosensor, whereby stretch increases myometrial contractility through agonist-free activation of the OTR. Retosiban prevents this through inverse agonism of the OTR and, in vivo, reduced the likelihood of spontaneous labor in nonhuman primates. We hypothesize that retosiban may be an effective preventative treatment of preterm birth in high-risk multiple pregnancies, an area of unmet clinical need.

Solid polymer electrolyte from phosphorylated chitosan

Energy Technology Data Exchange (ETDEWEB)

Fauzi, Iqbal, E-mail: arcana@chem.itb.ac.id; Arcana, I Made, E-mail: arcana@chem.itb.ac.id [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)

2014-03-24

Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

Energy Technology Data Exchange (ETDEWEB)

Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

2016-10-07

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

Phosphorylation of proteins in Clostridium thermohydrosulfuricum

International Nuclear Information System (INIS)

Londesborough, J.

1986-01-01

Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (γ- 32 P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10μM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 μM brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32 P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro

Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.