RESUMO
Amyloid-ß peptide 1-42 (Aß(1-42)), the predominant form in senile plaques, plays important roles in the pathogenesis of Alzheimer's disease. Because Aß(1-42) has aggregation-prone nature, it has been difficult to produce in a soluble state in bacterial expression systems. In this study, we modified our expression system to increase the soluble fraction of Aß(1-42) in Escherichia coli (E. coli) cells. The expression level and solubility of recombinant Aß(1-42) induced at the low temperature (16°C) is highly increased compared to that induced at 37°C. To optimize expression temperature, the coding region of Aß(1-42) was constructed in a pCold vector, pCold-TF, which has a hexahistidine-tagged trigger factor (TF). Recombinant Aß(1-42) was expressed primarily as a soluble protein using pCold vector system and purified with a nickel-chelating resin. When the toxic effect of recombinant Aß(1-42) examined on human neuroblastoma SH-SY5Y cells, the purified Aß(1-42) induced cell toxicity on SH-SY5Y cells. In conclusion, the system developed in this study will provide a useful method for the production of aggregation prone-peptide such as Aß(1-42).
Assuntos
Peptídeos beta-Amiloides/biossíntese , Temperatura Baixa , Vetores Genéticos/genética , Fragmentos de Peptídeos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Humanos , Neuroblastoma/patologia , Fases de Leitura Aberta , Fragmentos de Peptídeos/genética , Proteólise , Proteínas Recombinantes de Fusão/genética , Solubilidade , Transformação GenéticaRESUMO
Reactive oxygen species (ROS) have been implicated in the pathogenesis of ischemic brain injury. Sensitive to apoptosis gene (SAG) is a RING-finger protein that exhibits antioxidant activity against a variety of redox reagents. However, the protective effect of SAG in brain ischemic injury is unclear. Here, we investigated the protective effects of a Tat-SAG fusion protein against cell death and ischemic insult. When Tat-SAG fusion protein was added to the culture medium of astrocytes, it rapidly entered the cells and protected them against oxidative stress-induced cell death. Immunohistochemical analysis revealed that, when Tat-SAG fusion protein was intraperitoneally injected into gerbils, wild-type Tat-SAG prevented neuronal cell death in the CA1 region of the hippocampus in response to transient forebrain ischemia. In addition, wild-type Tat-SAG fusion protein decreased lipid peroxidation in the brain compared with mutant Tat-SAG- or vehicle-treated animals. Our results demonstrate that Tat-SAG fusion protein is a tool for the treatment of ischemic insult and it can be used in protein therapy for various disorders related to ROS, including stroke.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Produtos do Gene tat/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Citoproteção , Produtos do Gene tat/genética , Gerbillinae , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Isquemia/genética , Isquemia/metabolismo , Isquemia/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Estresse Oxidativo , Engenharia de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Transdução Genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.
Assuntos
Creatina Quinase Forma BB/genética , Transdução Genética/métodos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Clonagem Molecular , Creatina Quinase Forma BB/isolamento & purificação , Humanos , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Produtos do Gene tat do Vírus da Imunodeficiência Humana/isolamento & purificaçãoRESUMO
Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).
Assuntos
Encéfalo/enzimologia , Cisteamina/análogos & derivados , Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Western Blotting , Cisteamina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Células PC12 , Peptídeos/genética , Monoéster Fosfórico Hidrolases/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.
Assuntos
Antioxidantes/metabolismo , Sinais de Localização Nuclear/genética , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase/metabolismo , Transdução Genética , Western Blotting , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Imunofluorescência , Células HeLa , Herbicidas/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Sinais de Localização Nuclear/metabolismo , Estresse Oxidativo , Paraquat/farmacologia , Fragmentos de Peptídeos/farmacologia , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Frações Subcelulares , Superóxido Dismutase/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the selective death of motor neurons. Mutations in the SOD1 gene are responsible for a familial form of ALS (FALS). Although many studies suggest that mutant SOD1 proteins are cytotoxic, the mechanism is not fully understood. To investigate the role of mutant SOD1 in FALS, human SOD1 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce in-frame PEP-1-SOD fusion proteins (wild type and mutants). The expressed and purified PEP-1-SOD fusion proteins were efficiently transduced into neuronal cells. Neurones harboring the A4V, G93A, G85R, and D90A mutants of PEP-1-SOD were more vulnerable to oxidative stress induced by paraquat than those harboring wild-type proteins. Moreover, neurones harboring the mutant SOD proteins had lower heat shock protein (Hsp) expression levels than those harboring wild-type SOD. The effects of the transduced SOD1 fusion proteins may provide an explanation for the association of SOD1 with FALS, and Hsps could be candidate agents for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica , Astrócitos/fisiologia , Cisteamina/análogos & derivados , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase/metabolismo , Transdução Genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Astrócitos/citologia , Sobrevivência Celular , Células Cultivadas , Cisteamina/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Estresse Oxidativo , Peptídeos/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappaB induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.