RESUMO
PURPOSE: The purpose of this paper is to determine the significance of cyclic imide formation of an aspartic acid residue during storage on the pharmaceutical quality of a recombinant human glial cell line-derived neurotrophic factor (rhGDNF) formulation. METHODS: A combination of chromatography, peptide mapping, mass spectroscopy, and protein sequencing was used to purify and characterize the degradation product. Circular dichroism, 1,8-ANS and heparin binding, melting temperature determination, bioassays, and preclinical pharmacokinetic and toxicology testing were performed to examine its equivalence to native rhGDNF. RESULTS: The rhGDNF with cyclic imide at aspartic acid residue 96 showed identical activity, structure, pharmacokinetic profile, and toxicity profile to the native rhGDNF. CONCLUSIONS: Formation of cyclic imide at aspartic acid residue 96 does not affect the pharmaceutical quality of the rhGDNF formulation.
Assuntos
Proteínas de Drosophila , Imidas/química , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Animais , Ácido Aspártico/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Ciclização , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Gânglios Simpáticos/citologia , Gânglios Simpáticos/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Heparina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Mitógenos/farmacologia , Proteínas do Tecido Nervoso/farmacocinética , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Substância Negra/citologia , Substância Negra/efeitos dos fármacosRESUMO
Bcl10 was identified as a candidate gene responsible for low grade B cell lymphomas of mucosa-associated lymphoid tissue. Overexpression of Bcl10 in cultured cells was reported to promote apoptosis, however, the mechanism of regulation of apoptosis mediated by Bcl10 has not been demonstrated. In the present study, we analysed the apoptosis signaling pathway mediated by Bcl10, focusing on phosphorylation of Bcl10 and the dynamic interaction with its binding partners during apoptosis. Previously, we have demonstrated that Bcl10 potentially interacts with the other apoptosis regulator, TNF receptor associated factor-2 (TRAF2) and inhibitor of apoptosis proteins (cIAPs). The present results showed that the complex formation of these molecules was regulated by phosphorylation of Bcl10, that is, phosphorylation of Bcl10 resulted in binding of Bcl10 to cIAPs and the dissociation of it from TRAF2. Moreover, hyperphosphorylation of Bcl10 enhanced apoptosis, suggesting that changes in the binding partners of Bcl10 were correlated to the promotion of apoptosis as mediated by Bcl10. Indeed, the mutant which was deleted from the binding site of Bcl10 for cIAPs, could not induce apoptosis. These findings indicate that Bcl10 is a mediator of apoptosis signaling, by switching over binding to cIAPs from TRAF2 through the events of Bcl10 phosphorylation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Neoplasias/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Proteína 10 de Linfoma CCL de Células B , Linhagem Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias/química , Fosforilação , Estrutura Terciária de Proteína , Fator 2 Associado a Receptor de TNFRESUMO
When accumulation of a malfolded protein in the endoplastic reticulum (ER) is induced by various adverse conditions, such as hypoxia, glucose starvation, and perturbation of calcium homeostasis, cells respond to the stress by increasing transcription of genes encoding ER molecular chaperones, a process known as unfolded protein response. The signaling is initiated by IRE1s, ER stress sensors. Alternatively, excessive stress to the ER results in apoptosis. Caspase-12 is known to be essential for this ER stress-induced apoptosis. In this study, we analyzed the detailed regulatory mechanisms of IRE1s during ER stress. We identified c-Jun N-terminal inhibitory kinase (JIK) as a binding partner of IRE1alpha, and JIK was seen to modulate IRE1alpha-TRAF2 (tumor necrosis factor receptor-associated factor 2) complex formation and the resultant alteration to c-Jun N-terminal kinase signaling from IRE1s in response to ER stress. We also demonstrated that TRAF2 interacts with procaspase-12 and promotes the clustering of procaspase-12 and its activation by cleavage in response to ER stress. These results indicate that TRAF2 plays crucial roles not only in the signaling of the c-Jun N-terminal kinase pathway but also in activation of caspase-12 to transduce signals from IRE1s. Thus, we provide a missing link in the ER stress-induced apoptosis-signaling pathway, one which connects the stress sensor molecule IRE1 and the activation of caspase-12.
Assuntos
Caspases/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas/metabolismo , Estresse Fisiológico , Apoptose , Caspase 12 , Linhagem Celular , Dimerização , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Transfecção , Tunicamicina/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
In yeast, an endoplasmic reticulum (ER)-associated protein, Ire1p, is believed to initiate the unfolded protein response (UPR), that is responsible for protein folding in the ER under stressed conditions. Two mammalian homologs of Ire1p have been identified, Ire1 alpha and Ire1 beta. We have previously reported that familial Alzheimer's disease linked presenilin-1 variants downregulate the signaling pathway of the UPR by affecting the phosphorylation of Ire1 alpha. In the present study, we cloned the mouse homolog of Ire1 alpha for generating genetically modified mice. Ire1 alpha was ubiquitously expressed in all mouse tissues examined, and was expressed preferentially in neuronal cells in mouse brain. This led us to investigate the effects of the downregulation of the UPR on the survival of neuronal cells under conditions of ER stress. Morphological and biochemical studies using a dominant-negative form of mouse Ire1 alpha have revealed that cell death caused by ER stress can be attributed to apoptosis, and that the downregulation of the UPR enhances the apoptotic process in the mouse neuroblastoma cell line, Neuro2a. Our results indicate that genetically modified mice such as transgenic mice with a dominant-negative form of Ire1 alpha might provide further understanding of the pathogenic mechanisms of Alzheimer's disease and other neurodegenerative disorders.
Assuntos
Encéfalo/citologia , Proteínas de Membrana , Neuroblastoma , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Doença de Alzheimer/fisiopatologia , Animais , Antibacterianos/farmacologia , Apoptose/fisiologia , Encéfalo/enzimologia , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Clonagem Molecular , Grupo dos Citocromos c/metabolismo , DNA Complementar/isolamento & purificação , Retículo Endoplasmático/química , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Neurônios/citologia , Dobramento de Proteína , RNA Mensageiro/análise , Homologia de Sequência de Aminoácidos , Estresse Fisiológico/fisiopatologia , Células Tumorais Cultivadas , Tunicamicina/farmacologiaRESUMO
To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor kappaB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosis in vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas/fisiologia , Transdução de Sinais , Animais , Proteína 10 de Linfoma CCL de Células B , Sequência de Bases , Ativação Enzimática , Proteínas Inibidoras de Apoptose , MAP Quinase Quinase 4 , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas de Neoplasias/fisiologia , Fator 2 Associado a Receptor de TNF , Proteínas Virais/fisiologiaRESUMO
The compatibility of filgrastim with imipenem-cilastatin, ceftazidime, fluconazole, gentamicin, tobramycin, and amikacin was studied. Filgrastim 40 micrograms/mL or filgrastim 10 micrograms/mL (with human albumin) was combined with (1) imipenem-cilastatin 5 mg/mL (in terms of imipenem content), (2) ceftazidime 10 mg/mL (as the sodium salt), (3) fluconazole 2 mg/ mL, (4) gentamicin 1.6 mg/ mL (as the sulfate), (5) tobramycin 1.6 mg/mL (as the sulfate), or (6) amikacin 5 mg/ mL (as the sulfate). Equal volumes (5 mL) of the test-agent solutions were added in pairs to glass containers (simulating Y-site administration) in triplicate. Samples were analyzed for filgrastim activity, drug concentration, pH, and visible physical changes during storage at approximately 25 degrees C for up to four hours. Filgrastim activity was measured by the in vitro bioassay, and antimicrobial drug concentrations were measured by stability-indicating high-performance liquid chromatography or fluorescence polarization immunoassay. Filgrastim retained its activity, except for the combination of filgrastim at the lower concentration with gentamicin or at the higher concentration with imipenem-cilastatin. Antimicrobial drug concentrations did not change significantly during the study. No precipitation, color change, or haze was noted in any mixture. Changes in pH were negligible except for an increase in the mixture of filgrastim at either concentration with ceftazidime. In most cases, filgrastim retained its activity in the presence of a variety of antimicrobial drugs for up to four hours; in all cases, the antimicrobial drugs remained stable.