RESUMEN

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.

Asunto(s)

Isquemia Encefálica/metabolismo , Región CA1 Hipocampal/metabolismo , Lactoilglutatión Liasa/genética , Neuronas/metabolismo , Tioléster Hidrolasas/genética , Animales , Apoptosis , Isquemia Encefálica/patología , Región CA1 Hipocampal/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Fragmentación del ADN , Expresión Génica , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Gerbillinae , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismo , Peróxido de Hidrógeno/farmacología , Lactoilglutatión Liasa/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo , Piruvaldehído/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tioléster Hidrolasas/metabolismo

RESUMEN

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.

Asunto(s)

Cisteamina/análogos & derivados , Neuronas Dopaminérgicas/efectos de los fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Sustancia Negra/efectos de los fármacos , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisteamina/metabolismo , Fragmentación del ADN/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Escherichia coli , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/metabolismo , Péptidos/genética , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Prueba de Desempeño de Rotación con Aceleración Constante , Sustancia Negra/metabolismo , Sustancia Negra/patología

RESUMEN

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.

Asunto(s)

Agrocybe/química , Diabetes Mellitus Experimental/prevención & control , Polisacáridos/farmacología , Animales , Glucemia/metabolismo , Western Blotting , Secuencia de Carbohidratos , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitroprusiato/farmacología , Polisacáridos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estreptozocina

RESUMEN

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].

Asunto(s)

Proteínas Quinasas Activadas por AMP/genética , Ciclooxigenasa 2/metabolismo , Cisteamina/análogos & derivados , Lipopolisacáridos/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Péptidos/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Línea Celular Tumoral , Cisteamina/metabolismo , Ratones , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factores de Tiempo , Transducción Genética

RESUMEN

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.

Asunto(s)

Productos del Gen tat , Hidrolasas , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Proteínas Recombinantes de Fusión , Animales , Línea Celular , Supervivencia Celular , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/metabolismo , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Macrófagos/citología , Ratones , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

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.

Asunto(s)

Esclerosis Amiotrófica Lateral/enzimología , Citoprotección , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas Mutantes/metabolismo , Neuronas/citología , Superóxido Dismutasa/metabolismo , Transducción Genética , Astrocitos/citología , Astrocitos/metabolismo , Muerte Celular , Supervivencia Celular , Cisteamina/análogos & derivados , Cisteamina/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Neuronas/metabolismo , Estrés Oxidativo , Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Superóxido Dismutasa/química , Superóxido Dismutasa-1

RESUMEN

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.

Asunto(s)

Forma BB de la Creatina-Quinasa/genética , Transducción Genética/métodos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Clonación Molecular , Forma BB de la Creatina-Quinasa/aislamiento & purificación , Humanos , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/aislamiento & purificación

RESUMEN

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).

Asunto(s)

Encéfalo/enzimología , Cisteamina/análogos & derivados , Péptidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Western Blotting , Cisteamina/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Células PC12 , Péptidos/genética , Monoéster Fosfórico Hidrolasas/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección

RESUMEN

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.

Asunto(s)

Esclerosis Amiotrófica Lateral , Astrocitos/fisiología , Cisteamina/análogos & derivados , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Superóxido Dismutasa/metabolismo , Transducción Genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Astrocitos/citología , Supervivencia Celular , Células Cultivadas , Cisteamina/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Estrés Oxidativo , Péptidos/genética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa-1

RESUMEN

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.

Asunto(s)

Ciclooxigenasa 2/metabolismo , Cisteamina/análogos & derivados , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Fosfohidrolasa PTEN/genética , Péptidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Línea Celular , Activación Enzimática , Humanos , Ratones , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Recombinantes de Fusión/genética , Transducción de Señal