RESUMEN
Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Erythrina/química , Neoplasias Gástricas/tratamiento farmacológico , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Reelin is a secreted protein that plays versatile roles in neuronal development and function. The strength of Reelin signaling is regulated by proteolytic processing, but its importance in vivo is not yet fully understood. Here, we generated Reelin knock-in (PA-DV KI) mice in which the key cleavage site of Reelin was abolished by mutation. As expected, the cleavage of Reelin was severely abrogated in the cerebral cortex and hippocampus of PA-DV KI mice. The amount of Dab1, whose degradation is induced by Reelin signaling, decreased in these tissues, indicating that the signaling strength of Reelin was augmented. The brains of PA-DV KI mice were largely structurally normal, but unexpectedly, the hippocampal layer was disturbed. This phenotype was ameliorated in hemizygote PA-DV KI mice, indicating that excess Reelin signaling is detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice had more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Animales , Encéfalo/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Sustitución del Gen , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteolisis , Proteína ReelinaRESUMEN
Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.
Asunto(s)
Proteínas ADAMTS/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Proteínas del Tejido Nervioso/genética , Procolágeno N-Endopeptidasa/genética , Serina Endopeptidasas/genética , Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismo , Proteína Reelina , Serina Endopeptidasas/metabolismoRESUMEN
Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.
Asunto(s)
Proteínas ADAMTS/metabolismo , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/química , Corteza Cerebral/citología , Proteínas de la Matriz Extracelular/química , Proteínas del Tejido Nervioso/química , Procolágeno N-Endopeptidasa/metabolismo , Serina Endopeptidasas/química , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas/citología , Neuronas/metabolismo , Proteolisis , Proteína ReelinaRESUMEN
Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.
Asunto(s)
Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas ADAMTS/genética , Animales , Cerebelo/crecimiento & desarrollo , Corteza Cerebral/crecimiento & desarrollo , Femenino , Células HEK293 , Hipocampo/crecimiento & desarrollo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteolisis , Proteína ReelinaRESUMEN
Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.
Asunto(s)
Proteínas ADAMTS/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas ADAMTS/antagonistas & inhibidores , Proteínas ADAMTS/genética , Enfermedad de Alzheimer , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.
Asunto(s)
Benzofuranos/química , Erythrina/química , Células HL-60/química , Isoflavonas/química , Leucemia/tratamiento farmacológico , Apoptosis , Humanos , Leucemia/patologíaRESUMEN
TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.