RESUMO
Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t(½) < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t(½) = 16 min) to the extracellular space (t(½) = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn(113), a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.
Assuntos
Retículo Endoplasmático/enzimologia , Dobramento de Proteína , Multimerização Proteica/fisiologia , Ribonuclease Pancreático/metabolismo , Animais , Células CHO , Bovinos , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Estabilidade Enzimática/genética , Pancrelipase/metabolismo , Biossíntese de Proteínas/fisiologia , Transporte Proteico/fisiologia , Ribonuclease Pancreático/genéticaRESUMO
The C-terminal domain, Cp, of the Semliki Forest virus capsid protein, known for its rapid, efficient and chaperone-independent folding, was used to measure bulk fluid flow in the secretory pathway of Chinese hamster ovary cells. Being small, nonglycosylated, soluble and cytoplasmic in origin, Cp was not likely to interact with lectins, cargo receptors and retention factors. Using pulse-chase analysis, we observed that translocation into the endoplasmic reticulum resulted in rapid and efficient folding and transport of the newly synthesized Cp protein to the extracellular medium. The first Cp molecules were secreted 15 min after synthesis, which is the fastest transport of a protein so far recorded in mammalian cells. The rate constant of secretion was 1.2% per min, which amounts to an estimated bulk flow rate of about 155 coat protein II (COPII) vesicles per second. Transport was independent of expression level, and blocked by CI-976, brefeldin A and ATP depletion indicating that it depended on COPII vesicle formation, and followed the classical secretory pathway. In polarized Madin-Darby canine kidney cells, the secretion rate was similar but occurred mainly apically. The results demonstrated that fluid flow in the secretory pathway is fast, and can therefore play a significant role in the secretion of soluble secretory products.
Assuntos
Vírus da Floresta de Semliki/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Capsídeo , Cricetinae , Cricetulus , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Transporte Proteico , Proteínas Virais/químicaRESUMO
Pharmacological blockade of NMDA receptor function induces apoptotic neurodegeneration in the developing rat brain. However, the use of NMDA receptor antagonists as anesthetics and sedatives represents a difficult-to-avoid clinical practice in pediatrics. This warrants the search for adjunctive neuroprotective measures that will prevent or ameliorate neurotoxicity of NMDA receptor antagonists. The NMDA receptor antagonist MK801 triggered apoptosis in the neonatal rat forebrain, most notably in cortex and thalamus. MK801 exposure reduced mRNA levels of erythropoietin (EPO) and the EPO receptor, suggesting that loss of endogenous EPO activity may contribute to MK801-induced apoptosis. Coadministration of recombinant EPO (rEPO) conferred 50% neuroprotection, partially restored MK801-induced reduction of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, and prevented decreased phosphorylation levels of extracellular signal-regulated protein kinase-1/2 (ERK1/2) and Akt. These observations indicate that rEPO partly rescues newborn rats from MK801-mediated brain damage by enhancing neurotrophin-associated signaling pathways.
Assuntos
Encéfalo/efeitos dos fármacos , Eritropoetina/farmacologia , Antagonistas de Aminoácidos Excitatórios/toxicidade , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Proteínas Serina-Treonina Quinases , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/genética , Maleato de Dizocilpina/antagonistas & inibidores , Maleato de Dizocilpina/toxicidade , Eritropoetina/deficiência , Eritropoetina/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Fatores de Crescimento Neural/genética , Fármacos Neuroprotetores/uso terapêutico , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores da Eritropoetina/efeitos dos fármacos , Receptores da Eritropoetina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The developing rodent brain is vulnerable to pharmacological blockade of N-methyl-d-aspartate (NMDA) receptors which can lead to severe and disseminated apoptotic neurodegeneration. Here, we show that systemic administration of the NMDA receptor antagonist MK801 to 7-day-old rats leads to impaired activity of extracellular signal-regulated kinase 1/2 (ERK1/2) and reduces levels of phosphorylated cAMP-responsive element binding protein (CREB) in brain regions which display severe apoptotic neurodegeneration. Impaired ERK1/2 and CREB activity were temporally paralleled by sustained depletion of neurotrophin expression, particularly brain-derived neurotrophic factor (BDNF). BDNF supplementation fully prevented MK801-induced neurotoxicity in immature neuronal cultures and transgenic constitutive activation of Ras was associated with marked protection against MK801-induced apoptotic neuronal death. These data indicate that uncoupling of NMDA receptors from the ERK1/2-CREB signaling pathway in vivo results in massive apoptotic deletion of neurons in the developing rodent brain.
Assuntos
Apoptose/fisiologia , Giro do Cíngulo/crescimento & desenvolvimento , Giro do Cíngulo/patologia , Degeneração Neural/patologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Fatores Etários , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Maleato de Dizocilpina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Expressão Gênica/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Degeneração Neural/induzido quimicamente , Fatores de Crescimento Neural/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Substantial neurologic morbidity occurs in survivors of premature birth. Premature infants are exposed to partial oxygen pressures that are fourfold higher compared to intrauterine conditions, even if no supplemental oxygen is administered. Here we report that short exposures to nonphysiologic oxygen levels can trigger apoptotic neurodegeneration in the brains of infant rodents. Vulnerability to oxygen neurotoxicity is confined to the first 2 weeks of life, a period characterized by rapid growth, which in humans expands from the sixth month of pregnancy to the third year of life. Oxygen caused oxidative stress, decreased expression of neurotrophins, and inactivation of survival signaling proteins Ras, extracellular signal-regulated kinase (ERK 1/2), and protein kinase B (Akt). The synRas-transgenic mice overexpressing constitutively activated Ras and phosphorylated kinases ERK1/2 in the brain were protected against oxygen neurotoxicity. Our findings reveal a mechanism that could potentially damage the developing brain of human premature neonates.