RESUMO
The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1ß, P2α and P2ß, which form two heterodimers, P1α/P2ß and P1ß/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1ß-P2ß heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1ß/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2ß-P0-P1ß/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.
Assuntos
Subunidades Proteicas/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Dimerização , Mutação , Fenótipo , Polirribossomos/metabolismo , Subunidades Proteicas/genética , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In this study, we found an imbalance between stress-mediated and survival signaling and elevated apoptotic markers in retinal pigment epithelium (RPE) from diabetic patients. Since fenofibric acid (FA) treatment reduces the progression of diabetic retinopathy (DR), we investigated the effect of hyperglycemia and hypoxia, two components of the diabetic milieu, on stress, apoptosis, and survival pathways in ARPE-19 cells (immortalized human RPE cell line) and whether FA is able to prevent the deleterious effects induced by these conditions. ARPE-19 cells cultured in high-glucose (HG) medium or under hypoxia (1% oxygen)-induced phosphorylation of the stress-activated kinases JNK and p38 MAPK. This effect was increased by the combination of both conditions. Likewise, hyperglycemia and hypoxia triggered the phosphorylation of the endoplasmic reticulum (ER) stress markers PERK and eIF2α and the induction of the pro-apoptotic transcription factor CHOP. Under these experimental conditions, reactive oxygen species (ROS) were elevated and the integrity of tight junctions was disrupted. Conversely, ARPE-19 cells treated with FA were protected against these deleterious effects induced by hyperglycemia and hypoxia. FA increased insulin-like growth factor I receptor (IGF-IR)-mediated survival signaling in cells cultured under hyperglycemia and hypoxia, thereby suppressing caspase-3 activation and down-regulation of BclxL. Moreover, FA increased LC3-II, an autophagy marker. In conclusion, our results demonstrated that FA elicits a dual protective effect in RPE by down-regulation of stress-mediated signaling and induction of autophagy and survival pathways. These molecular mechanisms could be involved in the beneficial effects of fenofibrate reported in clinical trials.
Assuntos
Retinopatia Diabética/metabolismo , Fenofibrato/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Idoso , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Biomarcadores/metabolismo , Glicemia/metabolismo , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Estudos de Casos e Controles , Caspase 3/metabolismo , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/patologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Permeabilidade , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptor IGF Tipo 1/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fatores de Tempo , Fator de Transcrição CHOP/metabolismo , Proteína bcl-X/metabolismo , eIF-2 Quinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of metabolism and cell growth by its ability to dephosphorylate tyrosine kinase receptors and modulate the intensity of their signaling cascades. Because liver regeneration involves tyrosine phosphorylation-mediated signaling, we investigated the role of PTP1B in this process by performing partial hepatectomy in wild-type (PTP1B(+/+)) and PTP1B-deficient (PTP1B(-/-)) mice. The expression of PCNA and cyclins D1 and E (cell proliferation markers) was enhanced in PTP1B(-/-) regenerating livers, in parallel with 5'-bromo-2'-deoxyuridine incorporation. Phosphorylation of JNK1/2 and STAT3, early triggers of hepatic regeneration in response to TNF-α and IL-6, was accelerated in PTP1B(-/-) mice compared with PTP1B(+/+) mice. These phosphorylations were increased in PTP1B(-/-) hepatocytes or by silencing PTP1B in wild-type cells and decreased further after the addition of recombinant PTP1B. Enhanced EGF- and HGF receptor-mediated signaling was observed in regenerating livers lacking PTP1B and in EGF- or HGF-stimulated PTP1B(-/-) hepatocytes. Moreover, PTP1B(-/-) mice displayed a more rapid increase in intrahepatic lipid accumulation than PTP1B(+/+) control mice. Late responses to partial hepatectomy revealed additional divergences because stress-mediated signaling was attenuated at 24 to 96 hours in PTP1B(-/-) mice compared with PTP1B(+/+) mice. Finally, PTP1B deficiency also improves hepatic regeneration in mice fed a high-fat diet. These results suggest that pharmacological inhibition of PTP1B would improve liver regeneration in patients with acute or chronic liver injury.
Assuntos
Regeneração Hepática , Fígado/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Alanina Transaminase/metabolismo , Animais , Apoptose , Proliferação de Células , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Interleucina-6/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. METHODS: We performed major histocompatibility complex mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2) donors and BALB/c (H-2) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (quantitative real-time reverse-transcriptase polymerase chain reaction). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. RESULTS: We noted significantly greater intimal hyperplasia in HET versus WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-кB activation, gauged by higher levels of IkBα, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET versus WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFNγ and Granzyme B CD4 T cells and natural killer cells, and lower number of FoxP3 regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. CONCLUSIONS: A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings.
Assuntos
Aorta/transplante , Arteriosclerose/etiologia , Haploinsuficiência , Complicações Pós-Operatórias/etiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Arteriosclerose/genética , Arteriosclerose/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Complicações Pós-Operatórias/genética , Complicações Pós-Operatórias/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/biossíntese , Túnica Íntima/patologiaRESUMO
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the tyrosine kinase growth factor signaling pathway, which is involved in major physiological mechanisms such as liver regeneration. We investigate early hepatic metabolic events produced by partial hepatectomy (PHx) for PTP1B deficient (PTP1B KO) and wild type (WT) mice using proton nuclear magnetic resonance spectroscopy. Metabolic response of the two genotypes produced 24 h upon PHx is compared using magic angle spinning high-resolution nuclear magnetic resonance ((1)H-HR-MAS-NMR) on intact liver tissues. In addition, genotype-associated metabolic profile changes were monitored during the first 48 h after PHx using high-resolution nuclear magnetic resonance ((1)H-HR-NMR) on liver extracts. A marked increase of lipid-related signals in regenerating livers was observed after 24 h PHx in either intact tissues or liver extracts studies. In spite of this common initial metabolic response, results obtained 48 h after PHx on liver extracts indicate a genotype-differential metabolic pattern. This metabolic pattern resulted in line with well known regenerative features such as more sustained cell proliferation, a better management of lipids as energy fuel and lessened liver injury for PTP1B KO mice as compared to WT. Taken together, these findings suggest the metabolic basis to the pivotal role of PTP1B in liver regeneration.
Assuntos
Hepatectomia , Regeneração Hepática , Metaboloma , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Acetonitrilas/química , Animais , Clorofórmio/química , Técnicas de Inativação de Genes , Fígado/citologia , Fígado/metabolismo , Fígado/cirurgia , Espectroscopia de Ressonância Magnética , Masculino , Metanol/química , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Fatores de Tempo , Água/químicaRESUMO
PURPOSE: Mice with deletion of insulin receptor substrate (IRS) 2 develop type 2 diabetes and photoreceptor degeneration. Loss of protein tyrosine phosphatase 1B (PTP1B) in diabetic IRS2(-/-) mice restores insulin sensitivity and normalizes glucose homeostasis. Since insulin-like growth factor (IGF)-IR promotes survival of photoreceptors and is a substrate of PTP1B, we investigated IGF-IR-mediated survival signaling and visual function in PTP1B(-/-) and double mutant IRS2(-/-)/PTP1B(-/-) mice. METHODS: IGF-IR-mediated Akt signaling was evaluated in IGF-I-stimulated retinal explants. Histologic and electroretinogram analysis was performed in wild-type (WT), IRS2(-/-), PTP1B(-/-), and the double mutant IRS2(-/-)/PTP1B(-/-) mice. RESULTS: IGF-I stimulated the tyrosine phosphorylation of its receptor and Akt activation in retinal explants of WT mice. In PTP1B(-/-) retinal explants, these responses were enhanced. Conversely, in retinas from IRS2(-/-) mice, expression of PTP1B was increased, coincident with decreased IGF-I-mediated Akt serine 473 phosphorylation. PTP1B deletion in IRS2(-/-) mice also enhanced IGF-IR tyrosine phosphorylation but, unexpectedly, did not rescue Akt activation in response to IGF-I. One potential explanation is that PTEN was increased in retinas of IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice. Histologic evaluation revealed alterations in various structures of the retina in IRS2(-/-) and IRS2(-/-)/PTP1B(-/-) mice, specifically in the outer nuclear layer (ONL) and retinal outer segments (ROS). Electroretinogram (ERG) analysis confirmed that PTP1B deficiency did not restore visual function in IRS2(-/-) mice. CONCLUSIONS: Although loss of PTP1B enhances tyrosine phosphorylation of the IGF-IR in retinal explants of IRS2(-/-) mice, Akt activation remains defective owing to elevated PTEN levels and, thus, structural and functional visual defects persist in this model.
Assuntos
Proteínas Substratos do Receptor de Insulina/deficiência , Fator de Crescimento Insulin-Like I/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Doenças Retinianas/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Insulin resistance and type 2 diabetes correlate with impaired leptin and insulin signaling. Insulin receptor substrate-2 deficient (IRS2(-/-)) mice are an accepted model for the exploration of alterations in these signaling pathways and their relationship with diabetes; however, disturbances in hypothalamic signaling and the effect on neuropeptides controlling food intake remain unclear. Our aim was to analyze how leptin and insulin signaling may differentially affect the expression of hypothalamic neuropeptides regulating food intake and hypothalamic inflammation in diabetic (D) and nondiabetic (ND) IRS2(-/-) mice. We analyzed the activation of leptin and insulin targets by Western blotting and their association by immunoprecipitation, as well as the mRNA levels of neuropeptide Y (NPY), proopiomelanocortin, and inflammatory markers by real-time PCR and colocalization of forkhead box protein O1 (FOXO1) and NPY by double immunohistochemistry in the hypothalamus. Serum leptin and insulin levels and hypothalamic Janus kinase 2 and signal transducer and activator of transcription factor 3 activation were increased in ND IRS2(-/-) mice. IRS1 levels and its association with Janus kinase 2 and p85 and protein kinase B activation were increased in ND IRS2(-/-). Increased FOXO1 positively correlated with NPY mRNA levels in D IRS2(-/-) mice, with FOXO1 showing mainly nuclear localization in D IRS2(-/-) and cytoplasmic in ND IRS2(-/-) mice. D IRS2(-/-) mice exhibited higher hypothalamic inflammation markers than ND IRS2(-/-) mice. In conclusion, differential activation of these pathways and changes in the expression of NPY and inflammation may exert a protective effect against hypothalamic deregulation of appetite, suggesting that manipulation of these targets could be of interest in the treatment of insulin resistance and type 2 diabetes.
Assuntos
Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Regulação da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Neuropeptídeo Y/biossíntese , Pró-Opiomelanocortina/biossíntese , Animais , Glicemia/metabolismo , Hipotálamo/metabolismo , Imuno-Histoquímica/métodos , Inflamação , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos BiológicosRESUMO
Insulin is an inducer of brown fat adipogenesis through the activation of a signalling network that involves positive/negative modulators. Given the importance of brown adipose tissue (BAT) for basal thermogenic energy expenditure, we investigated the role of PTP1B in the acquisition of terminal differentiated phenotype and in the apoptotic responses of brown adipocytes. Immortalized brown preadipocytes lacking (PTP1B(-/-)) or expressing (PTP1B(+/+)) PTP1B have been generated. PTP1B deficiency accelerated a full program of brown adipogenesis including induction of transcription factors, coactivators, adipogenic markers and signalling molecules. Fully differentiated PTP1B(-/-) brown adipocytes were resistant to tumor necrosis factor (TNFalpha)-induced apoptosis as these cells were protected against caspase-8 activation, FLIP degradation, Bid cleavage and caspase-3 activation compared to wild-type controls. These events were recovered by PTP1B rescue. Survival signalling including phosphorylation of IRS-1 and Akt/PKB and BclxL expression were decreased in TNFalpha-treated PTP1B(-/-) cells but not in the wild-type. Similarly, PTP1B(-/-) brown adipocytes were protected against resveratrol-induced apoptosis. Phosphorylation of Akt/PKB and Foxo1 phosphorylation/acetylation decreased exclusively in resveratrol-treated wild-type cells, leading to nuclear localization of Foxo1 and up-regulation of Bim. Thus, PTP1B inhibition could be of benefit against obesity by counteracting TNFalpha-induced brown fat atrophy, and combined with resveratrol might improve low-grade inflammation.