RESUMO
Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H2O2-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H2O2-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H2O2-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.
Assuntos
Isquemia Encefálica/metabolismo , Região CA1 Hipocampal/metabolismo , Lactoilglutationa Liase/genética , Neurônios/metabolismo , Tioléster Hidrolases/genética , Animais , Apoptose , Isquemia Encefálica/patologia , Região CA1 Hipocampal/patologia , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Fragmentação do DNA , Expressão Gênica , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Gerbillinae , Produtos Finais de Glicação Avançada/genética , Produtos Finais de Glicação Avançada/metabolismo , Peróxido de Hidrogênio/farmacologia , Lactoilglutationa Liase/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estresse Oxidativo , Aldeído Pirúvico/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tioléster Hidrolases/metabolismoRESUMO
Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP(+) ) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP(+) -induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.
Assuntos
Cisteamina/análogos & derivados , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Doença de Parkinson Secundária/tratamento farmacológico , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Substância Negra/efeitos dos fármacos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisteamina/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Escherichia coli , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/metabolismo , Peptídeos/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-Rod , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
The aim of this study was to investigate the preventive effect of Agrocybe chaxingu polysaccharide on streptozocin (STZ)-induced pancreatic beta-cells destruction. Agrocybe chaxingu polysaccharide markedly reduced nitric oxide (NO) production and iNOS expression levels in RINm5F cells in a dose-dependent manner. In addition, Agrocybe chaxingu polysaccharide significantly inhibited iNOS expression and blood glucose levels in STZ-induced diabetic mice. Moreover, immunohistochemical analysis revealed that it enhanced pancreatic beta-cells resistance to destruction by STZ. These results suggest that Agrocybe chaxingu polysaccharide may have value as a therapeutic agent against diabetes mellitus.
Assuntos
Agrocybe/química , Diabetes Mellitus Experimental/prevenção & controle , Polissacarídeos/farmacologia , Animais , Glicemia/metabolismo , Western Blotting , Sequência de Carboidratos , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitroprussiato/farmacologia , Polissacarídeos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , EstreptozocinaRESUMO
AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that plays a central role in cellular metabolic stress. Modulation of nitric oxide (NO) and cyclooxygenase-2 (COX-2) is considered a promising approach for the treatment of inflammation and neuronal diseases. In this study, the AMPK gene was fused in-frame with PEP-1 peptide in a bacterial expression vector to produce a PEP-1-AMPK fusion protein. Expressed and purified PEP-1-AMPK fusion proteins were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose-dependent manner. Furthermore, transduced PEP-1-AMPK fusion protein markedly inhibited LPS-induced iNOS and COX-2 expression. These results suggest that the PEP-1-AMPK fusion protein can be used for the protein therapy of COX-2 and NO-related disorders such as inflammation and neuronal diseases. [BMB reports 2010; 43(1): 40-45].
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Ciclo-Oxigenase 2/metabolismo , Cisteamina/análogos & derivados , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Peptídeos/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular Tumoral , Cisteamina/metabolismo , Camundongos , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de Tempo , Transdução GenéticaRESUMO
Arginine deiminase (ADI), an arginine-degrading enzyme, has anti-proliferative and anti-tumor activities and is capable of inhibiting the production of nitric oxide (NO). Modulation of nitric oxide (NO) production is considered a promising approach for the treatment of various diseases including cancer, inflammation and neuronal disorders. In this study, an ADI gene was fused with an HIV-1 Tat peptide in a bacterial expression vector to produce an genetic in-frame Tat-ADI fusion protein. When added exogenously to the culture media, the expressed and purified Tat-ADI fusion proteins were efficiently transduced into macrophage Raw 264.7 cells in a time- and dose-dependent manner. Furthermore, transduced Tat-ADI fusion proteins markedly increased cell viability in cells treated with lipopolysaccharide (LPS). This increase in viability was mediated by an inhibition of NO production. These results suggest that this Tat-ADI fusion protein can be used in protein therapies of NO-related disorders such as cancer, inflammation and neuronal diseases.
Assuntos
Produtos do Gene tat , Hidrolases , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Proteínas Recombinantes de Fusão , Animais , Linhagem Celular , Sobrevivência Celular , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , HIV-1/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Macrófagos/citologia , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Familial Amyotrophic lateral sclerosis (FALS) is a progressive neurodegenetative disorder induced by mutations of the SOD1 gene. Heat shock protein 27 (HSP27) is well-defined as a stress-inducible protein, however the its role in ALS protection has not yet been established. To investigate the role HSP27 may have in SOD1 mutant-mediated apoptosis, human SOD1 or HSP27 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame fusion protein, which was then transduced into cells. We found the purified PEP-1-HSP27 fusion proteins can be transduced efficiently into neuronal cells and protect against cell death by enhancing mutant SOD1 activity. Moreover, transduced PEP-1-HSP27 efficiently prevents protein aggregation produced by oxidative stress. These results suggest that transduced HSP27 fusion protein may be explored as a potential therapeutic agent for FALS patients.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Citoproteção , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas Mutantes/metabolismo , Neurônios/citologia , Superóxido Dismutase/metabolismo , Transdução Genética , Astrócitos/citologia , Astrócitos/metabolismo , Morte Celular , Sobrevivência Celular , Cisteamina/análogos & derivados , Cisteamina/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Neurônios/metabolismo , Estresse Oxidativo , Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação , Superóxido Dismutase/química , Superóxido Dismutase-1RESUMO
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
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.