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1.
PLoS Pathog ; 16(9): e1008842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898178

RESUMO

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.


Assuntos
Adenosina Desaminase/metabolismo , Apoptose , Proteína DEAD-box 58/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Replicação Viral , Células A549 , Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Células HEK293 , Humanos , Influenza Humana/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Ligação a RNA/genética
2.
Int J Occup Environ Med ; 11(3): 140-147, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32683426

RESUMO

BACKGROUND: The major portion of lead in the body resides in skeletal system. The bone turnover affects the release of lead into the circulation from bones. The bone turnover biomarkers (BTM) in lead-battery workers with long-term exposure to lead have not been explored yet. OBJECTIVE: To evaluate the BTM (formation and resorption) in lead-battery workers with long-term exposure to lead in lead-battery manufacturing plant. METHODS: 176 male lead-exposed workers and 80 matched comparison group were studied. All participants were examined for blood lead levels (BLLs), bone formation biomarkers- serum osteocalcin (OC), alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP)-and bone resorption biomarkers-serum pyridinoline (PYD), deoxypyridinoline (DPYD), tartarate-resistant acid phosphatase-5b (TRACP-5b), and urinary hydroxyproline (UHYP). RESULTS: We found a significantly higher bone formation biomarkers such as BALP (p=0.007) and bone resorption biomarkers, eg, PYD (p=0.048), TRCAP-5b (p=0.001), and UHYP (p=0.001) in lead-exposed workers. A significant (p=0.041) negative correlation (ρ ­0.128) was noted between BLLs and OC. A significant positive correlation was noted between BLLs and TRACP-5b (ρ 0.176, p=0.005) and UHYP (ρ 0.258, p=0.004). Serum OC (p=0.040) and UHYP (p=0.015) levels changed significantly with BLL level. Bone resorption biomarkers levels- PYD, TRACP-5b, and BALP-were higher among those with higher BLLs levels. The duration of exposure was significantly associated with BALP (p=0.037), DPYD (p=0.016), TRACP-5b (p=0.001), and UHYP (p=0.002) levels. CONCLUSION: Long-term lead exposure affects the bone turnover.


Assuntos
Biomarcadores/sangue , Remodelação Óssea/fisiologia , Fontes de Energia Elétrica , Chumbo/toxicidade , Exposição Ocupacional/análise , Fosfatase Ácida/sangue , Fosfatase Ácida/metabolismo , Adulto , Fosfatase Alcalina/sangue , Biomarcadores/análise , Reabsorção Óssea/sangue , Estudos de Casos e Controles , Estudos Transversais , Fontes de Energia Elétrica/efeitos adversos , Humanos , Isoenzimas/sangue , Isoenzimas/metabolismo , Chumbo/química , Intoxicação por Chumbo/sangue , Intoxicação por Chumbo/diagnóstico , Masculino , Instalações Industriais e de Manufatura , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/estatística & dados numéricos , Osteocalcina/sangue , Local de Trabalho
3.
PLoS One ; 15(7): e0235747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658903

RESUMO

Despite development of markers for identification of cancer stem cells, the mechanism underlying the survival and division of cancer stem cells in breast cancer remains unclear. Here we report that PKCλ expression was enriched in basal-like breast cancer, among breast cancer subtypes, and was correlated with ALDH1A3 expression (p = 0.016, χ2-test). Late stage breast cancer patients expressing PKCλhigh and ALDH1A3high had poorer disease-specific survival than those expressing PKCλlow and ALDH1A3low (p = 0.018, log rank test for Kaplan-Meier survival curves: hazard ratio 2.58, 95% CI 1.24-5.37, p = 0.011, multivariate Cox regression analysis). Functional inhibition of PKCλ through siRNA-mediated knockdown or CRISPR-Cas9-mediated knockout in ALDH1high MDA-MB 157 and MDA-MB 468 basal-like breast cancer cells led to increases in the numbers of trypan blue-positive and active-caspase 3-positive cells, as well as suppression of tumor-sphere formation and cell migration. Furthermore, the amount of CASP3 and PARP mRNA and the level of cleaved caspase-3 protein were enhanced in PKCλ-deficient ALDH1high cells. An Apoptosis inhibitor (z-VAD-FMK) suppressed the enhancement of cell death as well as the levels of cleaved caspase-3 protein in PKCλ deficient ALDH1high cells. It also altered the asymmetric/symmetric distribution ratio of ALDH1A3 protein. In addition, PKCλ knockdown led to increases in cellular ROS levels in ALDH1high cells. These results suggest that PKCλ is essential for cancer cell survival and migration, tumorigenesis, the asymmetric distribution of ALDH1A3 protein among cancer cells, and the maintenance of low ROS levels in ALDH1-positive breast cancer stem cells. This makes it a key contributor to the poorer prognosis seen in late-stage breast cancer patients.


Assuntos
Aldeído Oxirredutases/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/mortalidade , Regulação Neoplásica da Expressão Gênica , Isoenzimas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteína Quinase C/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
4.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640193

RESUMO

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Xenoenxertos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neuroglia/metabolismo , Neuroglia/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
5.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629995

RESUMO

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Desiminases de Arginina em Proteínas/metabolismo , Betacoronavirus/isolamento & purificação , Estudos de Casos e Controles , Linhagem Celular , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Bases de Dados Factuais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vesículas Extracelulares/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Desiminases de Arginina em Proteínas/genética , RNA Mensageiro/metabolismo
6.
Anticancer Res ; 40(7): 3857-3863, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620625

RESUMO

BACKGROUND: The aim of this study was to define the alterations in the activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in normal and cancerous lung cells. MATERIALS AND METHODS: Lung tissues were taken from 36 patients during surgical resection of cancer. The activities of tested enzymes were measured by spectrofluorometric method (ADH I, ADH II, total ALDH) and photometric method (ADH III, ADH IV, total ADH). RESULTS: The activities of class II and III ADH were significantly lower in lung cancer cells compared to histologically normal lung tissue. CONCLUSION: Reduced activity of isoenzyme class II ADH may affect disorders in retinoic acid biosynthesis, leading to its deficit. Lower ADH III activity may result in depletion of glutathione, and in initiation of oxidative stress, leading to cancer progression. These data suggest that alterations in ADH isoenzyme activities can contribute to carcinogenesis in human lungs.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Neoplasias Pulmonares/enzimologia , Adenocarcinoma de Pulmão/enzimologia , Carcinoma de Células Escamosas/enzimologia , Feminino , Humanos , Isoenzimas/metabolismo , Masculino , Fatores Sexuais
7.
Proc Natl Acad Sci U S A ; 117(25): 14280-14291, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513715

RESUMO

In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.


Assuntos
Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Proteína Quinase C/metabolismo , Trofoblastos/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , PPAR gama/metabolismo , Placenta/citologia , Placentação/fisiologia , Gravidez , Proteína Quinase C/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Trofoblastos/citologia
8.
Nat Commun ; 11(1): 2738, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483131

RESUMO

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.


Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Metaloproteínas/metabolismo , Staphylococcus aureus/enzimologia , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Evolução Molecular , Ferro/química , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Manganês/química , Metaloproteínas/química , Metaloproteínas/genética , Mutação , Oxirredução , Filogenia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/genética , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxidos/metabolismo
9.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
10.
Am J Physiol Heart Circ Physiol ; 319(1): H144-H158, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32442021

RESUMO

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.


Assuntos
Artérias/metabolismo , Canais de Cálcio Tipo L/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Potenciais de Ação , Androsterona/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Pressão Sanguínea , Bovinos , Cavéolas/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Vasoconstrição
11.
Clin Sci (Lond) ; 134(10): 1167-1180, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32458968

RESUMO

In the present study, we evaluated the metabolic effects of green tea polyphenols (GTPs) in high-fat diet (HFD) fed Zucker fatty (ZF) rats, in particular the effects of GTP on skeletal muscle insulin sensitivity. Body weight, visceral fat, glucose tolerance, lipid profiles and whole-body insulin sensitivity were measured in HFD-fed ZF rats after 8-week-treatment with GTP (200 mg/kg of body weight) or saline (5 ml/kg of body weight). Zucker lean rats were studied as controls. Ex vivo insulin-mediated muscle glucose uptake was assessed. Immunoblotting was used to evaluate the expression of key insulin signalling proteins in skeletal muscle. GTP treatment attenuated weight gain (P<0.05) and visceral fat accumulation (27.6%, P<0.05), and significantly reduced fasting serum glucose (P<0.05) and insulin (P<0.01) levels. Homoeostasis model assessment of insulin resistance (HOMA-IR), a measure of insulin resistance, was lower (P<0.01) in GTP-treated animals compared with ZF controls. Moreover, insulin-stimulated glucose uptake by isolated soleus muscle was increased (P<0.05) in GTP-ZF rats compared with ZF-controls. GTP treatment attenuated the accumulation of ectopic lipids (triacyl- and diacyl-glycerols), enhanced the expression and translocation of glucose transporter-4, and decreased pSer612IRS-1 and increased pSer473Akt2 expression in skeletal muscle. These molecular changes were also associated with significantly decreased activation of the inhibitory (muscle-specific) protein kinase (PKC) isoform, PKC-θ. Taken together, the present study has shown that regular ingestion of GTP exerts a number of favourable metabolic and molecular effects in an established animal model of obesity and insulin resistance. The benefits of GTP are mediated in part by inhibiting PKC-θ and improving muscle insulin sensitivity.


Assuntos
Resistência à Insulina , Insulina/metabolismo , Músculo Esquelético/metabolismo , Polifenóis/farmacologia , Transdução de Sinais , Chá/química , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/metabolismo , Gordura Intra-Abdominal/efeitos dos fármacos , Gordura Intra-Abdominal/metabolismo , Isoenzimas/metabolismo , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos Zucker
12.
Nat Cell Biol ; 22(4): 389-400, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231305

RESUMO

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Receptores de Netrina/genética , Netrina-1/genética , Receptores de Superfície Celular/genética , Via de Sinalização Wnt/genética , Animais , Linhagem Celular , Embrião de Mamíferos , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Netrina/metabolismo , Netrina-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Receptores de Superfície Celular/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
13.
Gene ; 749: 144708, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32334022

RESUMO

The kallikrein-related peptidase 15 (KLK15) gene is a member of the largest cluster of serine proteases in the human genome. Exhibiting trypsin-like activity, KLK15 is most likely involved in the activation of prostate-specific antigen (PSA; also known as KLK3), an established biomarker for the diagnosis and screening of prostate cancer. High mRNA expression levels of KLK15 have already been reported in ovarian and prostate cancer, in contrast with breast cancer, where KLK15 has been proposed as a biomarker of favorable prognosis. In this study, we exploited the next-generation sequencing (NGS) technology along with 3' rapid amplification of cDNA ends (3' RACE) to discover alternative KLK15 splice variants. Extensive computational analysis of the obtained NGS data revealed the existence of novel splice junctions, thus supporting the existence of novel KLK15 transcripts. Six novel KLK15 splice variants were identified and verified by Sanger sequencing. Two of them (KLK15 v.11 and v.12) contain an open reading frame and are hence predicted to encode two novel KLK15 protein isoforms. Expression analysis of each KLK15 splice variant in sixteen cDNA pools from malignant cell lines and in normal cell lines (HEK293, HaCaT, and BJ cells) revealed very different expression profiles of particular KLK15 transcripts. Moreover, the new KLK15 splice variants were shown to be expressed in breast, ovarian, prostate, urinary bladder, colon, and renal tissue specimens. Due to the prominent clinical value of KLK15 mRNA expression, the novel KLK15 transcripts appear as candidate cancer biomarkers for diagnostic and/or prognostic purposes and, therefore, merit further investigation.


Assuntos
Processamento Alternativo , Calicreínas/genética , Linhagem Celular , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Calicreínas/metabolismo
14.
Mutat Res ; 819-820: 111690, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32120136

RESUMO

The serine/threonine kinase AKT, also known as protein kinase B (PKB), is the major substrate to phosphoinositide 3-kinase (PI3K) and consists of three paralogs: AKT1 (PKBα), AKT2 (PKBß) and AKT3 (PKBγ). The PI3K/AKT pathway is normally activated by binding of ligands to membrane-bound receptor tyrosine kinases (RTKs) as well as downstream to G-protein coupled receptors and integrin-linked kinase. Through multiple downstream substrates, activated AKT controls a wide variety of cellular functions including cell proliferation, survival, metabolism, and angiogenesis in both normal and malignant cells. In human cancers, the PI3K/AKT pathway is most frequently hyperactivated due to mutations and/or overexpression of upstream components. Aberrant expression of RTKs, gain of function mutations in PIK3CA, RAS, PDPK1, and AKT itself, as well as loss of function mutation in AKT phosphatases are genetic lesions that confer hyperactivation of AKT. Activated AKT stimulates DNA repair, e.g. double strand break repair after radiotherapy. Likewise, AKT attenuates chemotherapy-induced apoptosis. These observations suggest that a crucial link exists between AKT and DNA damage. Thus, AKT could be a major predictive marker of conventional cancer therapy, molecularly targeted therapy, and immunotherapy for solid tumors. In this review, we summarize the current understanding by which activated AKT mediates resistance to cancer treatment modalities, i.e. radiotherapy, chemotherapy, and RTK targeted therapy. Next, the effect of AKT on response of tumor cells to RTK targeted strategies will be discussed. Finally, we will provide a brief summary on the clinical trials of AKT inhibitors in combination with radiochemotherapy, RTK targeted therapy, and immunotherapy.


Assuntos
DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Raios gama/uso terapêutico , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Resultado do Tratamento , Proteínas ras/genética , Proteínas ras/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-32126452

RESUMO

Abscisic acid (ABA) triggers and regulates, while ethylene modulates autumn leaf senescence. The expression profiles of genes encoding ABA route components and the ACC oxidase isozymes were investigated in Populus tremula during the early and moderate stages of autumn leaf senescence. The targets of interest were Ptre-HAB1-like genes (Ptre-HAB1, Ptre-HAB3a and Ptre-HAB3b), the subclass 3 of Ptre-SnRK2s genes (Ptre-SnRK2.6a, Ptre-SnRK2.6b and Ptre-SnRK2.6b) and Ptre-RbohD1, Ptre-RbohF1, and Ptre-RbohF2 genes encoding the poplar components, which are counterparts of the ABA route key regulators or the counterparts of its secondary messengers, such as Homology to ABA-insensitive 1 (HAB1), Sucrose non-fermenting 1-related protein kinases 2 (SnRK2s) or Respiratory burst oxidase D and Respiratory burst oxidase F (RbohD and RbohF, respectively) in Arabidopsis, and Ptre-ACO3, Ptre-ACO5, and Ptre-ACO6 genes encoding ACC oxidase isozymes involved in ethylene biosynthesis. The fold change in their expression levels enabled to distinguish the distinct expression patterns for the following pairs of genes: Ptre-HAB3a and Ptre-SnRK2.6a, Ptre-HAB3b and Ptre-SnRK2.2, and Ptre-HAB1 and Ptre-SnRK2.6b, where each pair involves the genes encoding the negative and positive regulators of ABA route, respectively. Among the investigated genes, the fold change of expression was the highest for Ptre-ACO3, Ptre-ACO6, and Ptre-SnRK2.6b genes during both the studied stages, and additionally for Ptre-HAB1 and Ptre-RbohD1 genes during the moderate stage. In contrast, the Ptre-RbohF1 and Ptre-RbohF2 genes exhibited only the transient upregulation at the early stage of senescence. In an in vitro study, the ability of protein kinases Ptre-SnRK2.6a and Ptre-SnRK2.6b to phosphorylate the N-terminal regions of Ptre-RbohD1 and Ptre-RbohF2 was studied; the activity of Ptre-SnRK2.6b against the studied Ptre-Rbohs was noticeably lower than that exhibited by Ptre-SnRK2.6a. It seems that despite the high similarity of their polypeptides, Ptre-SnRK2.6a and Ptre-SnRK2.6b may play different biological roles; nonetheless, it requires in vivo confirmation. Surprisingly, the highest protein kinase activity against the Ptre-Rbohs was detected in the heterologous reaction with AT-SnRK2.6/OST1 which suggests that the discussed interactions are evolutionary conserved.


Assuntos
Aminoácido Oxirredutases/genética , Populus/genética , Transdução de Sinais/genética , Transcriptoma , Ácido Abscísico , Aminoácido Oxirredutases/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Populus/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(14): 7729-7738, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213584

RESUMO

Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments.


Assuntos
Diglicerídeos/química , Lipídeos/química , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Sobrevivência Celular , Isoenzimas/metabolismo , Cinética , Luz , Modelos Biológicos , Proteína Quinase C/metabolismo , Transdução de Sinais
17.
Nat Commun ; 11(1): 1308, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161261

RESUMO

Cancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes.


Assuntos
Antineoplásicos/farmacologia , Arilamina N-Acetiltransferase/genética , Neoplasias Colorretais/tratamento farmacológico , Perda de Heterozigosidade , Inibidores de Proteínas Quinases/farmacologia , Alelos , Animais , Antineoplásicos/uso terapêutico , Arilamina N-Acetiltransferase/metabolismo , Efeito Espectador/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Polimorfismo Genético , Inibidores de Proteínas Quinases/uso terapêutico , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Chem Biol ; 16(4): 391-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042197

RESUMO

Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor-hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes.


Assuntos
Fosfolipase D/ultraestrutura , Sequência de Aminoácidos , Domínio Catalítico , Desenho de Fármacos , Humanos , Isoenzimas/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade
19.
PLoS One ; 15(2): e0228747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092058

RESUMO

Alliinase is the key enzyme in allicin biosynthesis pathway. In the current study, the identification and sequencing of alliinase genes along with determination of allicin contents were reported for Allium species with a novel report for Iranian endemic species. The presence of different isoforms in the Allium being discovered for the first time. In bulbs tissue, the highest allicin concentration was in Allium sativum, A. umbilicatum, and A. fistolosum (1.185%, 0.367%, and 0.34%, respectively), followed by A. spititatum (0.072%), A. lenkoranicum (0.055%), A. atroviolaseum (0.36%), A. rubellum (0.041%), and A. stamineum (0.007%). The highest allicin content in the leaves and roots were in A. sativum (0.13%), and A. stamineum (0.195%), respectively. The ORFs length ranged from 1416 in A. sativum (iso-alliinase2; ISA2) to 1523 bp in A. sativum (alliinase); the identity with A. sativum (alliinase) varies from 95% to 68% for A. ampeloprasum, and A. sativum (iso-alliinase1, ISA1) respectively. These data suggested that both ISA1 and ISA2 had a high expression in the roots and bulbs compared to A. sativum as the control in all species. Note that ISA1 and ISA2 were not expressed in the leaves. The results showed that isoforms expression patterns among different tissues in Allium species were variable. The presence of various isoforms is a possible explanation for the difference between the species in terms of obtained results, especially the amount of allicin.


Assuntos
Allium/genética , Allium/metabolismo , Cisteína/metabolismo , Regulação da Expressão Gênica de Plantas , Liases/genética , Ácidos Sulfínicos/metabolismo , Sequência de Aminoácidos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Liases/química , Liases/metabolismo
20.
Nat Cell Biol ; 22(2): 167-174, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029896

RESUMO

Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.


Assuntos
Adenocarcinoma/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transaminases/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Transaminases/metabolismo
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