RESUMEN
Scaffold proteins Striatin and SG2NA assemble kinases and phosphatases into the signalling complexes called STRIPAK. Dysfunctional STRIPAKs cause cancer, cerebral cavernous malformations, etc. DJ-1, a sensor for oxidative stress, has long been associated with the Parkinson's disease, cancer, and immune disorders. SG2NA interacts with DJ-1 and Akt providing neuroprotection under oxidative stress. To dissect the role of SG2NA and DJ-1 in neuronal pathobiology, rat midbrain extracts were immunoprecipitated with SG2NA and sixty-three interacting proteins were identified. BN-PAGE followed by the LC-MS/MS showed 1030 comigrating proteins as the potential constituents of the multimeric complexes formed by SG2NA. Forty-three proteins were common between those identified by co-immunoprecipitation and the BN-PAGE. Co-immunoprecipitation with DJ-1 identified 179 interacting partners, of which forty-one also interact with SG2NA. Among those forty-one proteins immunoprecipitated with both SG2NA and DJ-1, thirty-nine comigrated with SG2NA in the BN-PAGE, and thus are bonafide constituents of the supramolecular assemblies comprising both DJ-1 and SG2NA. Among those thirty-nine proteins, seven are involved in mitochondrial oxidative phosphorylation. In rotenone-treated rats having Parkinson's like symptoms, the levels of both SG2NA and DJ-1 increased in the mitochondria; and the association of SG2NA with the electron transport complexes enhanced. In the hemi-Parkinson's model, where the rats were injected with 6-OHDA into the midbrain, the occupancy of SG2NA and DJ-1 in the mitochondrial complexes also increased. Our study thus reveals a new family of potential STRIPAK assemblies involving both SG2NA and DJ-1, with key roles in protecting midbrain from the oxidative stress.
Asunto(s)
Neoplasias , Enfermedad de Parkinson , Animales , Ratas , Cromatografía Liquida , Electrones , Mesencéfalo , Estrés Oxidativo , Espectrometría de Masas en TándemRESUMEN
Striatin and SG2NA are scaffold proteins that form signaling complexes called STRIPAK. It has been associated with developmental abnormalities, cancer, and several other diseases. Our earlier studies have shown that SG2NA forms a complex with the cancer-associated protein DJ-1 and the signaling kinase Akt, promoting cancer cell survival. In the present study, we used bioinformatics analyses to confirm the existence of two isoforms of human SG2NA, i.e., 78 and 87 kDas. In addition, several smaller isoforms like 35 kDa were also seen in western blot analyses of human cell lysates. The expression of these isoforms varies between different cancer cell lines of human origin. Also, the protein levels do not corroborate with its transcript levels, suggesting a complex regulation of its expression. In breast tumor tissues, the expression of the 35 and 78 kDa isoforms was higher as compared to the adjacent normal tissues, while the 87 kDa isoform was found in the breast tumor tissues only. With the progression of stages of breast cancer, while the expression of 78 kDa isoform decreased, 87 kDa became undetectable. In co-immunoprecipitation assays, the profile of the SG2NA interactome in breast tumors vis-à-vis adjacent normal breast tissues showed hundreds of common proteins. Also, some proteins were interacted with SG2NA in breast tumor tissues only. We conclude that SG2NA is involved in diverse cellular pathways and has roles in cellular reprogramming during tumorigenesis of the breast.
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Neoplasias de la Mama , Proteínas de Unión a Calmodulina , Autoantígenos/metabolismo , Neoplasias de la Mama/genética , Proteínas de Unión a Calmodulina/metabolismo , Femenino , Humanos , Isoformas de Proteínas/metabolismo , Transducción de SeñalRESUMEN
In cardiac muscle cells adrenergic agonists stimulate the generation of reactive oxygen species, followed by redox signaling. We postulated that the antagonists would attenuate such reactive oxygen species generation by the agonists. H9c2 cardiac myoblasts, neonatal rat cardiac myocytes, and HEK293 cells expressing ß1/ß2 adrenoceptors were stimulated with several agonists and antagonists. All the agonists and antagonists independently generated reactive oxygen species; but its generation was minimum whenever an agonists was added together with an antagonist. We monitored the Ca++ signaling in the treated cells and obtained similar results. In all treatment sets, superoxide and H2O2 were generated in the mitochondria and the cytosol respectively. NOX2 inhibitor gp91ds-tat blocked reactive oxygen species generation by both the agonists and the antagonists. The level of p47phox subunit of NOX2 rapidly increased upon treatment, and it translocated to the plasma membrane, confirming NOX2 activation. Inhibitor studies showed that the activation of NOX2 involves ERK, PI3K, and tyrosine kinases. Recombinant promoter-reporter assays showed that reactive oxygen species generated by both the agonists and antagonists modulated downstream gene expression. Mice injected with the ß-adrenergic agonist isoproterenol and fed with the antagonist metoprolol showed a robust induction of p47phox in the heart. We conclude that both the agonism and antagonism of adrenoceptors initiate redox signaling but when added together, they mutually counteract each other's effects. Our study thus highlights the importance of reactive oxygen species in adrenoceptor agonism and antagonism with relevance to the therapeutic use of the ß blockers.
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Especies Reactivas de Oxígeno , Agonistas Adrenérgicos , Animales , Células HEK293 , Humanos , Miocitos Cardíacos , RatasRESUMEN
Mitoapocynin is a triphenylphosphonium conjugated derivative of apocynin that specifically locates to the mitochondria. It has been developed as a mitochondrially targeted therapeutic antioxidant. We attempted to attenuate the mitochondrial ROS induced in H9c2 cardiac myoblast cells treated with norepinephrine. Mitoapocynin was a poor quencher of total ROS as detected by the fluoroprobe DCFH-DA. Using mitochondrial superoxide specific probe MitoSoxRed, we found that 5-10 µM mitoapocynin itself induces superoxide over and above that is generated by the norepinephrine treatment. A supposedly control molecule to mitoapocynin, the synthetic compound PhC11TPP, having the triphenylphosphonium group and a benzene moiety with C11 aliphatic chain spacer was also found to be a robust inducer of mitochondrial ROS. Subsequent assays with several cell lines viz., NIH3T3, HEK293, Neuro2A, MCF-7 and H9c2, showed that prolonged exposure to mitoapocynin induces cell death by apoptosis that can be partially prevented by the general antioxidant N-acetyl cysteine. Analyses of mitochondrial electron transport complexes by Blue Native Polyacrylamide gel electrophoresis showed that both mitoapocynin and PhC11TPP disrupt the mitochondrial Complex I and V, and in addition, PhC11TPP also damages the Complex IV. Our data thus highlights the limitations of the therapeutic use of mitoapocynin as an antioxidant.
Asunto(s)
Acetofenonas/farmacología , Apoptosis/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mioblastos Cardíacos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células HEK293 , Humanos , Células MCF-7 , Ratones , Células 3T3 NIHRESUMEN
SG2NA is a protein of the striatin family that organizes STRIPAK complexes. It has splice variants expressing differentially in tissues. Its 78 kDa isoform regulates cell cycle, maintains homeostasis in the endoplasmic reticulum, and prevents oxidative injuries. The 35 kDa variant is devoid of the signature WD-40 repeats in the carboxy terminal, and its function is unknown. We expressed it in NIH 3T3 cells that otherwise express 78 kDa variant only. These cells (35 EE) have altered morphology, faster rate of migration, and enhanced growth as measured by the MTT assay. Similar phenotypes were also seen in cells where the endogenous 78 kDa isoform was downregulated by siRNA (78 KD). Proteomic analyses showed that several cancer-associated proteins are modulated in both 35 EE and 78 KD cells. The 35 EE cells have diffused actin fibers, distinctive ultrastructure, reduced sialylation, and increased expression of MMP2 & 9. The 78 KD cells also had diffused actin fibers and an upregulated expression of MMP2. In both cells, markers epithelial to mesenchymal transition (EMT) viz, E- & N-cadherins, ß-catenin, slug, vimentin, and ZO-1 were modulated partially in tune with the EMT process. Since NIH 3T3 cells are mesenchymal, we also expressed 35 kDa SG2NA in MCF-7 cells of epithelial origin. In these cells (MCF-7-35), the actin fibers were also diffused and the modulation of the markers was more in tune with the EMT process. However, unlike in 35 EE cells, in MCF-7-35 cells, membrane sialylation rather increased. We infer that ectopic expression of 35 kDa and downregulation of 78 kDa SG2NAs partially induce transformed phenotypes.
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Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Sialiltransferasas/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/patología , Expresión Génica Ectópica , Transición Epitelial-Mesenquimal , Ratones , Células 3T3 NIH , Isoformas de Proteínas , Proteómica/métodosRESUMEN
SG2NA, a WD40 repeat protein of the Striatin subfamily, has four splicing and one messenger RNA edit variants. It is fast emerging as a scaffold for multimeric signaling complexes with roles in tissue development and disease. The green fluorescent protein (GFP)-tagged variants of SG2NA were ectopically expressed in NIH3T3 cells and their modulation by serum and GSK3ß-ERK signaling were monitored. The 87, 78, and 35 kDa variants showed a biphasic modulation by serum till 24 h but the 52 kDa variant remained largely unresponsive. Inhibition of phosphatases by okadaic acid increased the levels of the endogenous 78 kDa and the ectopically expressed GFP-tagged 87 and 78 kDa SG2NAs. Contrastingly, okadaic acid treatment reduced the level of GFP-tagged 35 kDa SG2NA, suggesting differential modes of their stability through phosphorylation-dephosphorylation. The inhibition of GSK3ß by LiCl showed a gradual decrease in the levels of 78 kDa. In the case of the other variants viz, GFP-tagged 35, 52, and 87 kDa, inhibition of GSK3ß caused an initial increase followed by a decrease with a subtle difference in kinetics and intensities. Similar results were also seen upon inhibition of GSK3ß by small interfering RNA. All the variants showed an increase followed by a decrease upon inhibition of extracellular-signal-regulated-kinase (ERK). These variants are localized in the plasma membrane, endoplasmic reticulum, mitochondria, and the nucleus with different propensities and no discernable subcellular distribution was seen upon stimulation by serum and the inhibition of phosphatases, GSK3ß, and ERK. Taken together, the variants of SG2NA are modulated by the kinase-phosphatase network in a similar but characteristic manner.
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Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Fracciones Subcelulares/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/genética , Proteínas de Unión a Calmodulina/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , Células 3T3 NIH , Fosforilación , Isoformas de Proteínas , Homología de Secuencia , Transducción de SeñalRESUMEN
Catestatin (CST) is a catecholamine release-inhibitory peptide secreted from the adrenergic neurons and the adrenal glands. It regulates the cardiovascular functions and it is associated with cardiovascular diseases. Though its mechanisms of actions are not known, there are evidences of cross-talk between the adrenergic and CST signaling. We hypothesized that CST moderates the adrenergic overdrive and studied its effects on norepinephrine-mediated hypertrophic responses in H9c2 cardiac myoblasts. CST alone regulated the expression of a number of fetal genes that are induced during hypertrophy. When cells were pre-treated CST, it blunted the modulation of those genes by norepinephrine. Norepinephrine (2 µM) treatment also increased cell size and enhanced the level of Troponin T in the sarcomere. These effects were attenuated by the treatment with CST. CST attenuated the immediate generation of ROS and the increase in glutathione peroxidase activity induced by norepinephrine treatment. Expression of fosB and AP-1 promoter-reporter constructs was used as the endpoint readout for the interaction between the CST and adrenergic signals at the gene level. It showed that CST largely attenuates the stimulatory effects of norepinephrine and other mitogenic signals through the modulation of the gene regulatory modules in a characteristic manner. Depending upon the dose, the signaling by CST appears to be disparate, and at 10-25 nM doses, it primarily moderated the signaling by the ß1/2-adrenoceptors. This study, for the first time, provides insights into the modulation of adrenergic signaling in the heart by CST.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Cromogranina A/farmacología , Mioblastos Cardíacos/metabolismo , Fragmentos de Péptidos/farmacología , Receptor de Adenosina A2B/metabolismo , Transducción de Señal/efectos de los fármacos , Cardiomegalia/metabolismo , Cardiomegalia/patología , Línea Celular , Humanos , Mioblastos Cardíacos/patologíaRESUMEN
Aqueous extract of the bark of Terminalia arjuna (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.
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RESUMEN
SG2NA was first discovered as nuclear autoantigen in lung and bladder cancer patient. It was named SG2NA as its expression increases during S to G2 phase of cell cycle. SG2NA/Striatin3 was classified as a member of Striatin family along with Straitin and Zinedin due to its structural and functional relatedness. At the molecular level, SG2NA is characterized by the presence of multiple protein-protein interaction domains viz., a caveolin binding motif, a coiled coil structure, Ca2+-calmodulin binding domain and a large WD-40 repeat domain in the same order from amino to the carboxyl termini. Analysis of secondary structures of 87 and 78 kDa SG2NA isoforms showed characteristic combinations of α-helix, ß-structure, ß-turns and random coil; suggesting of effective refolding after denaturation. This study for the first time establishes the structural differences between the two prevalent isoforms of SG2NA. Recently we observed that DJ-1 interacts with variants of SG2NA both in vitro and in vivo. The SG2NA isoforms purified from inclusion bodies showed the different secondary structure conformations, stability and interaction pattern for their interacting partners (DJ-1 and calmodulin) which imparts functional diversity of SG2NA. The SG2NA isoforms showed significant differential binding affinity to DJ-1 and Calmodulin.
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Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Calmodulina/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Autoantígenos/química , Autoantígenos/genética , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Dicroismo Circular , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de FluorescenciaRESUMEN
Oxidative stress is implicated in the pathogenesis of a plethora of cardiovascular diseases including interstitial fibrosis, contractile dysfunction, ischemia-reperfusion injury, and cardiac remodeling. However, antioxidant therapies targeting oxidative stress in the progression of those diseases have largely been unsuccessful. The current study evaluated the effects of a NADPH oxidase inhibitor, apocynin (Apo), on the production of reactive oxygen species and the development of pathological cardiac hypertrophy under sustained ß-adrenergic stimulation in male Wistar rats. As evident from the HW/BW ratio, HW/TL ratio, echocardiography, and histopathology, hypertrophic responses induced by isoproterenol (Iso; 5 mg/Kg body weight, subcutaneous) were blocked by Apo (10 mg/Kg body weight, intraperitoneal). Iso treatment increased the transcript levels of cybb and p22-phox, the two subunits of Nox. Iso treatment also caused a decrease in reduced glutathione level that was restored by Apo. Increase in mRNA levels of a number of markers of hypertrophy, viz., ANP, BNP, ß-MHC, and ACTA-1 by Iso was either partially or completely prevented by Apo. Activation of key signaling kinases such as PKA, Erk, and Akt by Iso was also prevented by Apo treatment. Our study thus provided hemodynamic, biochemical, and molecular evidences supporting the therapeutic value of Apo in ameliorating adrenergic stress-induced cardiac hypertrophy.
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Acetofenonas/farmacología , Agonistas Adrenérgicos beta/toxicidad , Cardiomegalia/inducido químicamente , Cardiomegalia/prevención & control , Isoproterenol/toxicidad , Animales , Biomarcadores/metabolismo , Peso Corporal , Cardiomegalia/diagnóstico por imagen , Ecocardiografía , Activación Enzimática , Glutatión/metabolismo , Corazón , Masculino , NADPH Oxidasa 2/genética , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Tamaño de los Órganos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3ß by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3ß. In cells depleted from SG2NA by shRNA, the levels of pGSK3ß and pERK are reduced, suggesting that these kinases and SG2NA regulate each other's expression.
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Autoantígenos/genética , Proteínas de Unión a Calmodulina/genética , Ciclo Celular/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Animales , Autoantígenos/química , Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Humanos , Ratones , Peso Molecular , Células 3T3 NIH , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARNRESUMEN
In recent years, NADPH oxidases (Noxes) have emerged as an important player in cardiovascular pathophysiology. Despite the growing evidences on the role of specific Nox isoforms, mechanisms of their activation, targets of reactive oxygen species (ROS) generated, and their downstream effects are poorly understood as yet. In this study, we treated H9c2 cardiac myoblasts with norepinephrine (NE, 2 µM), inducing ROS generation that was inhibited by Nox2-specific peptide inhibitor gp91ds-tat. Organelle-specific hydrogen peroxide-sensitive probe HyPer showed that the site of ROS generation is primarily in the cytosol, to some extent in the endoplasmic reticulum (ER) but not the mitochondria. Modulation of mRNAs of marker genes of cardiac hypertrophy i.e. induction in ANP and ß-MHC, and reduction in α-MHC by NE treatment was prevented by specific inhibition of Nox2 by gp91ds-tat. Induction of ANP and ß-MHC at the protein level were also attenuated by the inhibition of Nox2. Induction of c-Jun and FosB, the two members of the transcription factor family AP-1, were also blocked by the inhibition of Nox2 by gp91ds-tat. Induction of promoter-reporter constructs harboring multiple AP-1 elements and the upstream of FosB and ANP genes by NE were also blocked by the inhibition of Nox2 by gp91ds-tat and a dominant negative mutant of p22phox, a constituent of Nox2 that prevents its activation. This study for the first time establishes the significant role of Nox2 in mediating the NE-induced pathological adrenergic signaling in cardiac myoblasts.
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Cardiomegalia/metabolismo , Mioblastos Cardíacos/metabolismo , NADPH Oxidasa 2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Adrenérgicos/metabolismo , Transducción de Señal , Animales , Cardiomegalia/genética , Cardiomegalia/patología , Línea Celular , Ratones , Mioblastos Cardíacos/patología , NADPH Oxidasa 2/genética , Receptores Adrenérgicos/genéticaRESUMEN
SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.
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Autoantígenos/genética , Proteínas de Unión a Calmodulina/genética , Estrés del Retículo Endoplásmico/genética , Retículo Endoplásmico/genética , Homeostasis , Animales , Autoantígenos/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclina D1/genética , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Células 3T3 NIH , Isoformas de Proteínas/genética , Transducción de Señal/genética , Tapsigargina/farmacología , Tunicamicina/farmacología , Repeticiones WD40/genéticaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Aqueous bark extract of Terminalia arjuna (TA) has been in use as an ethnomedicine for cardiovascular ailments in the Indian subcontinent for centuries. Studies using hemodynamic, ROS scavenging and anti-inflammatory parameters in animal models have shown its anti-atherogenic, hypotensive, inotropic, anti-inflammatory effects. However, details analysis on its effects on established molecular and cell biological markers are a prerequisite for its wider acceptance to the medical community. AIMS OF THE STUDY: To test the efficacy of TA extract in ameliorating cardiac hypertrophy induced by ISO in rats. METHODS: Cardiac hypertrophy was induced by ISO (5mg/kg/day s.c. for 14 days) in rats and a standardized aqueous extract of TA stem bark was orally administered by gavage. Total RNA and protein were isolated from control, ISO, ISO plus TA and TA treated rat hearts and analyzed for the transcripts for the markers of hypertrophy, signaling kinases, transcription factors and total protein profile. RESULTS: TA extract reversed the induction of fetal genes like ß-myosin heavy chain, skeletal α-actin and brain natriuretic peptide in hypertrophic rat hearts. While ISO slightly increased the level of phospho-ERK, TA repressed it to about one third of the base line level. Survival kinase Akt, ER stress marker Grp78 and epigenetic regulator HDAC5 were augmented by ISO and TA restored them by various extents. ISO administration moderately increased the transcription factor NFκB binding activity, while coadministration of TA further increased it. AP-1 binding activity was largely unchanged by ISO treatment but it was upregulated when administered along with TA. MEF2D binding activity was increased by ISO and TA restored it to the baseline level. Global proteomic analysis revealed that TA treatment restored a subset of proteins up- and down-regulated in the hypertrophied hearts. Amongst those restored by TA were purinergic receptor X, myosin light chain 3, tropomyosin, and kininogen; suggesting a nodal role of TA in modulating cardiac function. CONCLUSIONS: This study for the first time reveals that TA partially or completely restores the marker mRNAs, signaling kinases, transcription factors and total protein profile in rat heart, thereby demonstrating its efficacy in preventing ISO-induced cardiac hypertrophy.
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Cardiomegalia/tratamiento farmacológico , Isoproterenol/farmacología , Extractos Vegetales/uso terapéutico , Proteómica , Terminalia , Animales , Cardiomegalia/inducido químicamente , Masculino , Fitoterapia , Corteza de la Planta , Ratas , Ratas WistarRESUMEN
Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses.
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Apoptosis/efectos de los fármacos , Norepinefrina/toxicidad , Especies Reactivas de Oxígeno/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Catalasa/metabolismo , Línea Celular , Daño del ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Ensayo de Inmunoadsorción Enzimática , Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo/metabolismo , Microscopía Fluorescente , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/metabolismo , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
SG2NA in association with striatin and zinedin forms a striatin family of WD-40 repeat proteins. This family of proteins functions as scaffold in different signal transduction pathways. They also act as a regulatory subunit of protein phosphatase 2A. We have shown that SG2NA which evolved first in the metazoan evolution among the striatin family members expresses different isoforms generated out of alternative splicing. We have also shown that SG2NA protects cells from oxidative stress by recruiting DJ-1 and Akt to mitochondria and membrane in the post-mitotic neuronal cells. DJ-1 is both cancer and Parkinson's disease related protein. In the present study we have shown that SG2NA protects DJ-1 from proteasomal degradation in cancer cells. Hence, downregulation of SG2NA reduces DJ-1/Akt colocalization in cancer cells resulting in the reduction of anchorage dependent and independent growth. Thus SG2NA enhances cancer cell survival. Reactive oxygen species enhances SG2NA, DJ-1 and Akt trimerization. Removal of the reactive oxygen species by N-acetyl-cysteine thus reduces cancer cell growth.
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Autoantígenos/fisiología , Proteínas de Unión a Calmodulina/fisiología , Supervivencia Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Peroxirredoxinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Activación Enzimática , Humanos , Ratones , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Proteína Desglicasa DJ-1 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
SG2NA belongs to a three member Striatin subfamily of WD-40 repeat superfamily. It has multiple protein-protein interaction domains that are involved in the assembly of supra-molecular signaling complexes. Earlier we had demonstrated that there are at least five variants of SG2NA, generated by alternative splicing. We now demonstrate that a 52kDa novel variant is generated by the editing of the transcript for the 82kDa isoform. The 52kDa protein is abundant in mouse tissues but it is barely present in immortalized cells, suggesting its role in cell differentiation. Besides splicing and editing, expression of SG2NAs in tissues is also regulated by differential polyadenylation and mRNA/protein stability. Further, the longer UTR is seen only in the brain mRNA from 1month old mouse and 8-10day old chick embryo. Like alternative splicing, differential polyadenylation of Sg2na transcripts is also conserved in evolution. Taken together, these results suggest a highly versatile and dynamic mode of regulation of SG2NA with potential implications in tissue development.
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Autoantígenos/genética , Autoantígenos/metabolismo , Encéfalo/metabolismo , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/metabolismo , Animales , Encéfalo/embriología , Células CHO , Línea Celular Tumoral , Embrión de Pollo , Cricetulus , Embrión de Mamíferos/metabolismo , Masculino , Ratones , Especificidad de Órganos , Poliadenilación , Estabilidad Proteica , Edición de ARN , Empalme del ARNRESUMEN
SG2NA is a WD-40 repeat protein with multiple protein-protein interaction domains of unknown functions. We demonstrate that it associates with the antioxidant protein DJ-1 and the survival kinase Akt. The C-terminal WD-40 repeat domain of SG2NA is required for its interaction with Akt, while DJ-1 binds it further upstream. No interaction between DJ-1 and Akt occurs in the absence of SG2NA. SG2NA, DJ-1, and Akt colocalize in mitochondria and plasma membrane. Their association is enhanced by increasing levels of reactive oxygen species up to a threshold level but falters thereafter with further increase in oxidants. Mutants of DJ-1 found in patients with familial parkinsonism are not recruited by SG2NA, suggesting its role in neuroprotection. Cells depleted of SG2NA are susceptible, while those overexpressing it are resistant to apoptosis induced by oxidative stress. Our study thus unravels a novel pathway of recruitment of Akt and DJ-1 that provides protection against oxidative stress, especially in neurons.