RESUMEN
Capsaicin is widely used as a food additive and as an analgesic agent. Besides its well-known role in nociception, which is mediated by vanilloid receptor 1 specifically expressed in dorsal root ganglion neurons, capsaicin has also been considered as a potential anticancer agent, as it inhibits cell proliferation and induces apoptosis in various types of cancer cells. Here we identified a new molecular target of capsaicin from human myeloid leukemia cells. We show that capsaicin binds to prohibitin (PHB) 2, which is normally localized to the inner mitochondrial membrane, and induces its translocation to the nucleus. PHB2 is implicated in the maintenance of mitochondrial morphology and the control of apoptosis. We also provide evidence suggesting that capsaicin causes apoptosis directly through the mitochondria and that PHB2 contributes to capsaicin-induced apoptosis at multiple levels. This work will serve as an important foundation for further understanding of anticancer activity of capsaicin.
Asunto(s)
Analgésicos/metabolismo , Capsaicina/metabolismo , Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Proteínas Represoras/metabolismo , Transporte Activo de Núcleo Celular , Translocador 2 del Nucleótido Adenina/metabolismo , Analgésicos/farmacología , Apoptosis , Capsaicina/farmacología , Citocromos c/metabolismo , Humanos , Membranas Mitocondriales/metabolismo , ProhibitinasRESUMEN
We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.
Asunto(s)
Aequorina/análisis , Separación Inmunomagnética/métodos , Mediciones Luminiscentes/métodos , Metotrexato/análisis , Mapeo de Interacción de Proteínas/métodos , Tetrahidrofolato Deshidrogenasa/análisis , Sistemas de Liberación de Medicamentos/métodos , Metotrexato/química , Unión Proteica , Sensibilidad y Especificidad , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.
Asunto(s)
Dietilhexil Ftalato/análogos & derivados , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Glucogenólisis/efectos de los fármacos , Testículo/efectos de los fármacos , Análisis de Varianza , Animales , Células COS , Chlorocebus aethiops , Dietilhexil Ftalato/metabolismo , Dietilhexil Ftalato/toxicidad , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Cinética , Hígado/metabolismo , Masculino , Metabolómica , Unión Proteica , Ratas , Ratas Wistar , Espermatozoides/metabolismo , Testículo/metabolismoRESUMEN
Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX) must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT), the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.
Asunto(s)
Mitocondrias Hepáticas/enzimología , Translocasas Mitocondriales de ADP y ATP/metabolismo , Animales , Hemo/química , Hemo/metabolismo , Hemoproteínas/metabolismo , Cinética , Translocasas Mitocondriales de ADP y ATP/química , Modelos Moleculares , Peso Molecular , Conformación Proteica , Protoporfirinas/metabolismo , RatasRESUMEN
Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-beta-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.