RESUMO
Antibodies are among the most important tools for protein detection but, prior to their usage, proper validation of their appropriateness for given applications is required. The utility of an antibody depends on its sensitivity and specificity. We studied these two aspects in a panel of commercial antibodies against Shb, a platform protein involved in receptor tyrosine kinase signalling, but the function of which is still incompletely understood. Several of the antibodies showed shortcomings or were not acceptable for detection of the endogenous protein. The few that could detect Shb were doing so in either western blotting or immunoprecipitation experiments but a given antibody could not work in both applications. This article provides a resource for the available molecular tools that can be used in future research on Shb.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Anticorpos/imunologia , Proteínas Proto-Oncogênicas/análise , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Western Blotting , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Proto-Oncogênicas/imunologiaRESUMO
BACKGROUND: Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast. CONCLUSIONS/SIGNIFICANCE: The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.
Assuntos
Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Mitocôndrias/enzimologia , Éteres Fosfolipídicos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Deleção de Genes , Humanos , Leishmaniose/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Resultado do TratamentoRESUMO
The nuclear factor κB (NF-κB) transcription factor is a master regulator of inflammation. Short-term NF-κB activation is generally beneficial. However, sustained NF-κB might be detrimental, directly causing apoptosis of cells or leading to a persistent damaging inflammatory response. NF-κB activity in stressed cells needs therefore to be controlled for homeostasis maintenance. In mildly stressed cells, caspase-3 cleaves p120 RasGAP, also known as RASA1, into an N-terminal fragment, which we call fragment N. We show here that this fragment is a potent NF-κB inhibitor. Fragment N decreases the transcriptional activity of NF-κB by promoting its export from the nucleus. Cells unable to generate fragment N displayed increased NF-κB activation upon stress. Knock-in mice expressing an uncleavable p120 RasGAP mutant showed exaggerated NF-κB activation when their epidermis was treated with anthralin, a drug used for the treatment of psoriasis. Our study provides biochemical and genetic evidence of the importance of the caspase-3-p120-RasGAP stress-sensing module in the control of stress-induced NF-κB activation.
Assuntos
Caspase 3/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/química , Ratos , Estresse Fisiológico/fisiologia , Proteína p120 Ativadora de GTPase/químicaRESUMO
Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.
Assuntos
Caspase 3/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Caspase 3/genética , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Microinjeções , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Ovário/citologia , Ovário/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Cultura Primária de Células , Estrutura Terciária de Proteína , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Xenopus laevis , Proteína p120 Ativadora de GTPase/genéticaRESUMO
The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.
Assuntos
Antineoplásicos/farmacologia , Microdomínios da Membrana/metabolismo , Éteres Fosfolipídicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Antineoplásicos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Endocitose , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Microdomínios da Membrana/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Éteres Fosfolipídicos/metabolismo , Transporte Proteico , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Proteínas de Transporte de Nucleotídeos/metabolismo , Éteres Fosfolipídicos/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Membranas Intracelulares/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Transporte Proteico , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Ubiquitinação/efeitos dos fármacosRESUMO
Caspases are a family of proteases that participate in the progression and execution of the apoptotic program. However, regulation of the caspase activation and their substrates has not yet been fully elucidated. Here we explore the effect of the ectopic expression of the human initiator caspases-8 and -10 in Saccharomyces cerevisiae. Our results showed that the expression of human CASP10 and CASP8 triggers certain apoptotic markers such as a massive production of reactive oxygen species (ROS), chromatin condensation and phosphatidylserine externalization, finally leading to cell death. In response to hydroxyurea (HU), yeast cells expressing caspase-10 did not reduce the replication of DNA and escaped to the intra-S checkpoint of the cell cycle. In addition, caspase-10 expression induced yeast vacuolization and a vacuole-associated phenotype resembling autophagy. Other intracellular alterations such as disorganization of the actin cytoskeleton, cell wall damage, and aberrations within the endoplasmic reticulum lumen were also associated with caspase-10 expression. Furthermore, caspase-induced cell death was completely dependent on the proteolytic activation of the enzyme but, in contrast, was not dependent on either of the endogenous yeast apoptotic proteins Aif1 and Mca1 or the mitochondria.