RESUMO
The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.
Assuntos
Ácidos Graxos , Insulina , Miocárdio , Oxirredução , Complexo Piruvato Desidrogenase , Animais , Insulina/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Camundongos , Miocárdio/metabolismo , Miocárdio/enzimologia , Ácidos Graxos/metabolismo , Acetil-CoA Carboxilase/metabolismo , Acetil-CoA Carboxilase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/genética , Malonil Coenzima A/metabolismo , Masculino , Camundongos Knockout , Glucose/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.
Assuntos
Diacilglicerol O-Aciltransferase , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Diacilglicerol O-Aciltransferase/metabolismo , Retículo Endoplasmático/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismoRESUMO
Acetyl-CoA carboxylase (ACC) promotes prandial liver metabolism by producing malonyl-CoA, a substrate for de novo lipogenesis and an inhibitor of CPT-1-mediated fat oxidation. We report that inhibition of ACC also produces unexpected secondary effects on metabolism. Liver-specific double ACC1/2 knockout (LDKO) or pharmacologic inhibition of ACC increased anaplerosis, tricarboxylic acid (TCA) cycle intermediates, and gluconeogenesis by activating hepatic CPT-1 and pyruvate carboxylase flux in the fed state. Fasting should have marginalized the role of ACC, but LDKO mice maintained elevated TCA cycle intermediates and preserved glycemia during fasting. These effects were accompanied by a compensatory induction of proteolysis and increased amino acid supply for gluconeogenesis, which was offset by increased protein synthesis during feeding. Such adaptations may be related to Nrf2 activity, which was induced by ACC inhibition and correlated with fasting amino acids. The findings reveal unexpected roles for malonyl-CoA synthesis in liver and provide insight into the broader effects of pharmacologic ACC inhibition.
Assuntos
Acetil-CoA Carboxilase , Aminoácidos , Gluconeogênese , Fígado , Malonil Coenzima A , Camundongos Knockout , Oxirredução , Animais , Malonil Coenzima A/metabolismo , Fígado/metabolismo , Acetil-CoA Carboxilase/metabolismo , Camundongos , Aminoácidos/metabolismo , Masculino , Piruvato Carboxilase/metabolismo , Ciclo do Ácido Cítrico , Ácido Pirúvico/metabolismo , Camundongos Endogâmicos C57BL , Jejum/metabolismo , Carnitina O-Palmitoiltransferase/metabolismoRESUMO
Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (> 5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (< 1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by > 50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Ácidos Graxos/biossíntese , Proteínas Associadas aos Microtúbulos/metabolismo , Multimerização Proteica , Animais , Citosol/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Fosforilação , Ligação ProteicaRESUMO
Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 Å and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.
Assuntos
Ácidos Graxos/biossíntese , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Ácidos Graxos/química , Ácidos Graxos/genética , Feminino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/genéticaRESUMO
Transglutaminase 2 (TGase2) is a ubiquitously expressed enzyme that catalyzes irreversible post-translational modification of protein, forming cross-linked protein aggregates. We previously reported that intracellular TGase2 is activated by oxidative stress. To elucidate the functional role of TGase2 activation in cells under the oxidatively stressed condition, we identified the mediator that activates TGase2. In this study, we showed that low levels of oxidative stress trigger the release of TGFbeta, which subsequently activates TGase2 through the nuclear translocation of Smad3. Analysis of substrate proteins reveals that TGase2-mediated protein modification results in a decrease of protein solubility and a collapse of intermediate filament network, which leads to aggregation of proteins. We confirm these results using lens tissues from TGase2-deficient mice. Among several antioxidants tried, only N-acetylcysteine effectively inhibits TGFbeta-mediated activation of TGase2. These results indicate that TGFbeta mediates oxidative stress-induced protein aggregation through activation of TGase2 and suggest that the formation of protein aggregation may not be a passive process of self-assembly of oxidatively damaged proteins but may be an active cellular response to oxidative stress. Therefore, TGFbeta-TGase2 pathway may have implications for both the pathogenesis of age-related degenerative diseases and the development of pharmaceutics.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Estresse Oxidativo/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Transglutaminases/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Proteínas de Ligação ao GTP/efeitos dos fármacos , Humanos , Cristalino/efeitos dos fármacos , Cristalino/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Proteína 2 Glutamina gama-Glutamiltransferase , Transfecção , Transglutaminases/efeitos dos fármacosRESUMO
Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/sangue , Fígado Gorduroso/tratamento farmacológico , Triglicerídeos/sangue , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Lipoproteínas VLDL/genética , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Knockout , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/genéticaRESUMO
High-risk human papillomavirus (HPV) E7 is a major oncoprotein that plays a crucial role in the development of cervical cancer. A previous study showed that transglutaminase (TGase) 2 catalyzes the incorporation of polyamines into HPV 18 E7 protein, and thereby diminishes its ability to bind Rb. Therefore, TGase 2 activity may be implicated in a suppressive function of host against HPV-induced carcinogenesis. To better understand the nature of polyamination of HPV 18 E7, we investigated the Rb binding of E7 polyaminated in vitro with different type of polyamines. The incorporation of spermine diminished the Rb binding of E7 more profoundly compared with that of spermidine, suggesting that either the additional positive charge or a steric effect or both may have altered the chemical or structural properties of the protein. In addition, the treatment of either spermidine or spermine in cultured cell system reduced the ability of E7 to inactivate Rb with a TGase activity-dependent manner. Spermine was more effective in inhibiting E7 activity than spermidine. These results may provide the basis for future investigation aiming at delineating the significance of polyamine metabolism on HPV E7 functions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Oncogênicas Virais/fisiologia , Poliaminas/farmacologia , Proteína do Retinoblastoma/metabolismo , Transglutaminases/metabolismo , Aminas/química , Biotinilação , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Fatores de Transcrição E2F/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes Reguladores , Genes do Retinoblastoma , Glutationa Transferase/metabolismo , Humanos , Imuno-Histoquímica , Modelos Químicos , Proteínas Oncogênicas Virais/metabolismo , Poliaminas/química , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/química , Espermidina/química , Espermina/química , Ativação Transcricional , TransfecçãoRESUMO
Filaggrin is expressed in the cornified layer of epidermis and known to be one of the antigenic targets in rheumatoid arthritis. Although the citrulline residue in filaggrin is thought to be an antigenic determinant recognized by autoantibodies, the diagnostic sensitivity of synthetic citrullinated peptide is variable. To investigate the implication of anti-filaggrin antibodies recognizing uncitrullinated filaggrin in rheumatoid arthritis, we assayed antibody titers using unmodified recombinant filaggrin in the sera from 73 patients with rheumatoid arthritis, 150 patients with other connective tissue diseases and 70 normal controls. We also performed the correlation analysis between antibody titers and the clinical variables in patients with rheumatoid arthritis. Titers of IgG anti-filaggrin antibodies were significantly higher in rheumatoid arthritis patients compared to normal controls (P=0.02), but not in patients with osteoarthritis, ankylosing spondylitis or systemic lupus erythematosus. IgG anti-filaggrin antibodies were more frequently found in patients with rheumatoid arthritis compared to normal controls (12.3% vs 1.4% respectively, P=0.04). An anti-filaggrin antibody titer was correlated with visual analogue scale of pain, tender joint count, Ritchie articular index or C-reactive protein, but not with anti-nuclear antibody or rheumatoid factor. These results suggest that anti-filaggrin antibody recognizes the uncitrullinated filaggrin as an antigen and its titer correlates with clinical parameters, explaining the variable sensitivity of anti-filaggrin antibody test.
Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Citrulina/análise , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/imunologia , Sequência de Aminoácidos , Artrite Reumatoide/sangue , Estudos de Casos e Controles , Proteínas Filagrinas , Humanos , Proteínas de Filamentos Intermediários/isolamento & purificação , Dados de Sequência MolecularRESUMO
Polyamine incorporation or cross-linking of proteins, post-translational modifications mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an artificial polyamine derivative. When measured in vitro, BP showed a significantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives different results with different substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required.
Assuntos
Biotina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Poliaminas/metabolismo , Transglutaminases/metabolismo , Aminas/química , Aminas/metabolismo , Biotina/química , Cálcio/metabolismo , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Humanos , Poliaminas/química , Proteína 2 Glutamina gama-Glutamiltransferase , Reprodutibilidade dos Testes , Espermina/síntese química , Espermina/química , Espermina/metabolismo , Transglutaminases/genéticaRESUMO
Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.
Assuntos
Proteínas de Ligação a DNA , Imunoquímica/métodos , Proteínas Oncogênicas Virais/análise , Papillomaviridae/imunologia , Extratos Celulares/química , Extratos Celulares/imunologia , Linhagem Celular , Humanos , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/química , Proteínas E7 de PapillomavirusRESUMO
The treatment of cystamine, a transglutaminase(TGase) inhibitor, has beneficial effects in several diseases including CAG-expansion disorders and cataract. We compared the inhibition characteristics of cystamine with those of cysteamine, a reduced form of cystamine expect-ed to be present inside cells. Cystamine is a more potent inhibitor for TGase than cysteamine with different kinetics pattern in a non-reducing condition. By contrast, under reducing conditions, the inhibitory effect of cystamine was comparable with that of cysteamine. How-ever, cystamine inhibited intracellular TGase activity more strongly than cysteamine despite of cytoplasmic reducing environment, suggest-ing that cystamine itself inhibits in situ TGase activity by forming mixed disulfides.
Assuntos
Cistamina/farmacologia , Cisteamina/farmacologia , Inibidores Enzimáticos/farmacologia , Transglutaminases/antagonistas & inibidores , Linhagem Celular Tumoral , HumanosRESUMO
The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFß or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Ativadores de Enzimas/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Animais , Cafeína/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/farmacologia , Ativação Enzimática , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Líquido Intracelular/enzimologia , Camundongos , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais/efeitos dos fármacos , Transglutaminases/genéticaRESUMO
Transglutaminase 4 (TG4) is a member of the enzyme family that catalyzes the calcium-dependent post-translational modification of proteins via cross-linking, polyamination, or deamidation. TG4 exhibits prostate-specific expression pattern and plays a crucial role in the formation of the copulatory plug in rodents. However, the physiological function(s) of human TG4 remains speculative. Human TG4 has been postulated to participate in the maturation process of sperm by modifying its cell surface, which results in suppression of sperm antigenicity in the female genital tract. To better understand the pathophysiological role of TG4 in prostate tissue, we generated monoclonal antibodies (MAb) against human TG4 in mice by repeated injections with the recombinant human TG4. Western blot analysis demonstrated that the selected MAbs react specifically with TG4, but not with other isoenzymes of the TG family. Immunocytochemical and immunohistochemical analyses showed that specific staining is observed with the cells overexpressing TG4 and with the paraffin-embedded prostate tissue specimens obtained from the benign prostate hyperplasia and prostate cancer patients, respectively. Our results indicate that these MAbs are suitable for detecting TG4 in the cultured cells or prostate tissues for investigating the biological functions of human TG4.
Assuntos
Anticorpos Monoclonais/imunologia , Isoenzimas/imunologia , Neoplasias da Próstata/enzimologia , Transglutaminases/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Próstata/enzimologia , Próstata/patologia , Neoplasias da Próstata/imunologia , Transfecção , Transglutaminases/genética , Transglutaminases/isolamento & purificaçãoRESUMO
An abrupt increase of intracellular Ca(2+) is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca(2+) triggers apoptotic cell death through mitochondrial swelling and activation of Ca(2+)-dependent enzymes. Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca(2+)-overload. Overexpression of TG2 inhibited the A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca(2+)-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca(2+)-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.
Assuntos
Apoptose , Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Transglutaminases/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Calcimicina/farmacologia , Caspases/metabolismo , Morte Celular , Sobrevivência Celular , Citocromos c/metabolismo , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Ionóforos/farmacologia , Mitocôndrias/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Proteína X Associada a bcl-2/genéticaRESUMO
Transglutaminase 4 is a member of enzyme family that catalyzes calcium-dependent posttranslational modification of proteins. Although transglutaminase 4 has been shown to have prostate-restricted expression pattern, little is known about the biological function of transglutaminase 4 in human. To gain insight into its role in prostate, we analyzed the expression status of human transglutaminase 4 in benign prostate hyperplasia (BPH) and prostate cancer (PCa). Unexpectedly, RT-PCR and nucleotide sequence analysis showed four alternative splicing variants of transglutaminase 4: transglutaminase 4-L, -M (-M1 and -M2) and -S. The difference between transglutaminase 4-M1 and -M2 is attributed to splicing sites, but not nucleotide size. The deduced amino acid sequences showed that transglutaminase 4-L, -M1 and -M2 have correct open reading frames, whereas transglutaminase 4-S has a truncated reading frame. RT-PCR analysis of clinical samples revealed that transglutaminase 4-M and -S were detected in all tested prostate tissue (80 BPH and 48 PCa). Interestingly, transglutaminase 4-L was found in 56% of BPH (45 out of 80) and only in 15% of PCa (7 out of 48). However, transglutaminase 4-L expression did not correlate with serum prostate-specific antigen (PSA) level, prostate volumes or PSA densities. These results will provide a clue to future investigation aiming at delineating physiological and pathological roles of human transglutaminase 4.
Assuntos
Processamento Alternativo/genética , Hiperplasia Prostática/enzimologia , Hiperplasia Prostática/genética , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Transglutaminases/genética , Idoso , Sequência de Aminoácidos , Sequência de Bases , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Dados de Sequência Molecular , Saccharomyces cerevisiae , Transglutaminases/química , Transglutaminases/metabolismoRESUMO
Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the transamidation reaction. There is conflicting evidence on the role of TG2 in apoptosis. In this report, we show that TG2 increases in response to low level of oxidative stress, whereas TG2 diminishes under high stress conditions. Monitoring TG2 expression, activity and calcium concentration in cells treated with A23187 revealed that the initial rise of calcium activates TG2 but subsequent calcium-overload induces the degradation of TG2 via calcium-mediated polyubiquitination. These results indicate that the role of TG2 in apoptosis depends on the level of calcium influx triggered by oxidative stress.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Estresse Oxidativo , Transglutaminases/metabolismo , Adenoviridae/genética , Antibacterianos/farmacologia , Apoptose , Calcimicina/farmacologia , Morte Celular , Linhagem Celular , Meios de Cultura Livres de Soro , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Rim/citologia , Leupeptinas/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de Tempo , UbiquitinaçãoRESUMO
Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/fisiologia , Transglutaminases/metabolismo , Células 3T3 , Animais , Eritrócitos/enzimologia , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/enzimologia , Proteína 2 Glutamina gama-Glutamiltransferase , Fatores de Tempo , Transfecção , Transglutaminases/isolamento & purificação , Transglutaminases/fisiologiaRESUMO
Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.