RESUMEN

Galleria mellonella is a lepidopteran whose larval stage has shown the ability to degrade polystyrene (PS), one of the most recalcitrant plastics to biodegradation. In the present study, we fed G. mellonella larvae with PS for 54 days and determined candidate enzymes for its degradation. We first confirmed the biodegradation of PS by Fourier transform infrared spectroscopy- Attenuated total reflectance (FTIR-ATR) and then identified candidate enzymes in the larval gut by proteomic analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Two of these proteins have structural similarities to the styrene-degrading enzymes described so far. In addition, potential hydrolases, isomerases, dehydrogenases, and oxidases were identified that show little similarity to the bacterial enzymes that degrade styrene. However, their response to a diet based solely on polystyrene makes them interesting candidates as a potential new group of polystyrene-metabolizing enzymes in eukaryotes.

Asunto(s)

Mariposas Nocturnas , Poliestirenos , Animales , Poliestirenos/metabolismo , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Mariposas Nocturnas/microbiología , Larva/metabolismo , Biodegradación Ambiental

RESUMEN

Rheb is a small GTPase member of the Ras superfamily and an activator of mTORC1, a protein complex master regulator of cell metabolism, growth, and proliferation. Rheb/mTORC1 pathway is hyperactivated in proliferative diseases, such as Tuberous Sclerosis Complex syndrome and cancer. Therefore, targeting Rheb-dependent signaling is a rational strategy for developing new drug therapies. Rheb activates mTORC1 in the cytosolic surface of lysosomal membranes. Rheb's farnesylation allows its anchorage on membranes, while its proper localization depends on the prenyl-binding chaperone PDEδ. Recently, the use of PDEδ inhibitors has been proposed as anticancer agents because they interrupted KRas signaling leading to antiproliferative effects in KRas-dependent pancreatic cancer cells. However, the effect of PDEδ inhibition on the Rheb/mTORC1 pathway has been poorly investigated. Here, we evaluated the impact of a new PDEδ inhibitor, called Deltasonamide 1, in Tsc2-null MEFs, a Rheb-dependent overactivated mTORC1 cell line. By using a yeast two-hybrid assay, we first validated that Deltasonamide 1 disrupts Rheb-PDEδ interaction. Accordingly, we found that Deltasonamide 1 reduces mTORC1 targets activation. In addition, our results showed that Deltasonamide 1 has antiproliferative and cytotoxic effects on Tsc2-null MEFs but has less effect on Tsc2-wild type MEFs viability. This work proposes the pharmacological PDEδ inhibition as a new approach to target the abnormal Rheb/mTORC1 activation in Tuberous Sclerosis Complex cells.

RESUMEN

SALL2 is a poorly characterized transcription factor that belongs to the Spalt-like family involved in development. Mutations on SALL2 have been associated with ocular coloboma and cancer. In cancers, SALL2 is deregulated and is proposed as a tumor suppressor in ovarian cancer. SALL2 has been implicated in stemness, cell death, proliferation, and quiescence. However, mechanisms underlying roles of SALL2 related to cancer remain largely unknown. Here, we investigated the role of SALL2 in cell proliferation using mouse embryo fibroblasts (MEFs) derived from Sall2-/- mice. Compared to Sall2+/+ MEFs, Sall2-/- MEFs exhibit enhanced cell proliferation and faster postmitotic progression through G1 and S phases. Accordingly, Sall2-/- MEFs exhibit higher mRNA and protein levels of cyclins D1 and E1. Chromatin immunoprecipitation and promoter reporter assays showed that SALL2 binds and represses CCND1 and CCNE1 promoters, identifying a novel mechanism by which SALL2 may control cell cycle. In addition, the analysis of tissues from Sall2+/+ and Sall2-/- mice confirmed the inverse correlation between expression of SALL2 and G1-S cyclins. Consistent with an antiproliferative function of SALL2, immortalized Sall2-/- MEFs showed enhanced growth rate, foci formation, and anchorage-independent growth, confirming tumor suppressor properties for SALL2. Finally, cancer data analyses show negative correlations between SALL2 and G1-S cyclins' mRNA levels in several cancers. Altogether, our results demonstrated that SALL2 is a negative regulator of cell proliferation, an effect mediated in part by repression of G1-S cyclins' expression. Our results have implications for the understanding and significance of SALL2 role under physiological and pathological conditions.

Asunto(s)

Ciclo Celular/genética , Ciclina D1/genética , Ciclina E/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas Represoras/metabolismo , Animales , Proliferación Celular , Transformación Celular Neoplásica/patología , Ciclina D1/metabolismo , Ciclina E/metabolismo , Proteínas de Unión al ADN , Fibroblastos/metabolismo , Fase G1 , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Ratones Noqueados , Modelos Biológicos , Fenotipo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase S , Factores de Transcripción , Transcripción Genética

RESUMEN

Tuberous sclerosis complex (TSC) disease results from inactivation of the TSC1 or TSC2 gene, and is characterized by benign tumors in several organs. Because TSC tumorigenesis correlates with hyperactivation of mTORC1, current therapies focus on mTORC1 inhibition with rapamycin or its analogs. Rapamycin-induced tumor shrinkage has been reported, but tumor recurrence occurs on withdrawal from rapamycin. Autophagy has been associated with development of TSC tumors and with tumor cell survival during rapamycin treatment. mTORC1 and AMPK directly inhibit and activate autophagy, respectively. AMPK is hyperactivated in TSC cells and tumors, and drives cytoplasmic sequestration of the cell-cycle inhibitor p27KIP (p27). Whether AMPK and p27 are involved in rapamycin-induced autophagy and survival of TSC cells remain unexplored. Here, we show that inhibition of AMPK by compound C or by shRNA-mediated depletion of LKB1 reduces activation of autophagy by rapamycin in Tsc2-null cells. Similarly, shRNA-mediated depletion of p27 inhibited rapamycin-induced autophagy. In support of p27 lying downstream of AMPK on the activation of autophagy in Tsc2-null cells, a p27 mutant that preferentially localizes in the cytosol recovered the effect of rapamycin on autophagy in both p27- and LKB1-depleted cells, but a nuclear p27 mutant was inactive. Finally, we show that p27-dependent activation of autophagy is involved in Tsc2-null cell survival under rapamycin treatment. These results indicate that an AMPK/p27 axis is promoting a survival mechanism that could explain in part the relapse of TSC tumors treated with rapamycin, exposing new avenues for designing more efficient treatments for TSC patients.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/efectos de los fármacos , Sirolimus/farmacología , Proteínas Supresoras de Tumor/deficiencia , Animales , Antibióticos Antineoplásicos/farmacología , Autofagia/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Immunoblotting , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Interferencia de ARN , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética

RESUMEN

RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5'UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.

Asunto(s)

Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Transcripción Genética , Regiones no Traducidas 5' , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Mutación , Motivos de Nucleótidos , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero , Alineación de Secuencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Activación Transcripcional

RESUMEN

In amphibians, sperm histone transition post-fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin-like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin-like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post-fertilization and this phosphorylation is dependent on CDK-cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin-like activity in sea urchins participating during male pronucleus formation post-fecundation.

Asunto(s)

Ensamble y Desensamble de Cromatina/fisiología , Nucleoplasminas/metabolismo , Erizos de Mar/metabolismo , Espermatozoides/metabolismo , Animales , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Histonas/metabolismo , Cinetina/farmacología , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Roscovitina , Erizos de Mar/citología , Espermatozoides/citología

RESUMEN

Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

Asunto(s)

Catepsina L/metabolismo , Endopeptidasas/metabolismo , Histonas/metabolismo , Erizos de Mar/embriología , Erizos de Mar/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catepsina L/análisis , Catepsina L/genética , ADN Complementario/genética , Endopeptidasas/análisis , Endopeptidasas/genética , Fertilización , Regulación del Desarrollo de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , Erizos de Mar/citología , Erizos de Mar/genética , Alineación de Secuencia , Espermatozoides/metabolismo

RESUMEN

This study evaluated the condition factor, gonadosomatic, and hepatosomatic indexes, occurrence of plasmatic vitellogenin (Vg), and frequency of spermatogenic maturity stages in male Chilean flounders, Paralichthys adspersus, caught at three different coastal sites off the Bio-Bio region, central Chile, during 1 year. The Vg was detected by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Western blot analyses using an antibody against Chilean flounder Vg. The spermatogenic maturity stages were analyzed by histological gonadic diagnostic. The prevalence of plasmatic Vg induction in male fish differed significantly among sites. The flounders sampled from the Itata area were the most affected. Evaluations of biometric data, plasmatic Vg induction, and spermatogenic maturity stages of the flounder showed the following: (1) lower gonadosomatic index, (2) greater hepatosomatic index, (3) greater prevalence of plasmatic Vg, and (4) delayed development of the gonad. The results suggest that estrogenic endocrine-disruption compounds are introduced into the marine environment, negatively affecting the fish studied. The relevance of this report is discussed in relation to estrogenic compounds introduced by industrial and municipal wastewater effluents in the areas studied.

Asunto(s)

Disruptores Endocrinos/toxicidad , Lenguado , Reproducción/efectos de los fármacos , Vitelogeninas/biosíntesis , Contaminantes Químicos del Agua/toxicidad , Animales , Western Blotting/métodos , Chile , Electroforesis en Gel de Poliacrilamida/métodos , Disruptores Endocrinos/análisis , Monitoreo del Ambiente/métodos , Estrógenos , Gónadas/efectos de los fármacos , Gónadas/crecimiento & desarrollo , Masculino , Contaminantes Químicos del Agua/análisis

RESUMEN

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell-cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH-protease) participates in male chromatin remodeling and in cell-cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38-42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco-2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non-proliferative cells.

Asunto(s)

Células CACO-2/enzimología , Catepsina L , Proteasas de Cisteína/análisis , Células HeLa/enzimología , Erizos de Mar/enzimología , Transporte Activo de Núcleo Celular , Animales , Ciclo Celular , Clonación Molecular , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Femenino , Humanos , Masculino , Proteínas Nucleares/análisis , Homología de Secuencia , Huso Acromático/metabolismo

RESUMEN

We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17beta-estradiol. The Vg was detected by SDS-PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.

Asunto(s)

Biomarcadores/metabolismo , Disruptores Endocrinos/análisis , Monitoreo del Ambiente/métodos , Peces Planos/metabolismo , Vitelogeninas/aislamiento & purificación , Vitelogeninas/metabolismo , Contaminantes Químicos del Agua/análisis , Animales , Western Blotting , Chile , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Masculino , Espectrometría de Masas , Océano Pacífico , Especificidad de la Especie

RESUMEN

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.

Asunto(s)

Catepsinas/antagonistas & inhibidores , División Celular/fisiología , Cromosomas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Mitosis/fisiología , Erizos de Mar , Animales , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Replicación del ADN , Masculino , Erizos de Mar/embriología , Erizos de Mar/genética

RESUMEN

We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.

Asunto(s)

Ensamble y Desensamble de Cromatina/fisiología , Histonas/metabolismo , Meiosis , Nucleosomas/ultraestructura , Espermatozoides/fisiología , Animales , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Cisteína Endopeptidasas/farmacología , Cisteína Endopeptidasas/fisiología , Femenino , Fertilización , Histonas/efectos de los fármacos , Masculino , Meiosis/fisiología , Nucleosomas/química , Nucleosomas/efectos de los fármacos , Erizos de Mar , Espermatozoides/citología , Espermatozoides/metabolismo , Cigoto/química , Cigoto/ultraestructura

RESUMEN

Previously we have identified a cysteine-protease involved in male chromatin remodeling which segregates into the nuclei of the two blastomeres at the first cleavage division. Here we have investigated the fate of this protease during early embryogenesis by immunodetecting this protein with antibodies elicited against its N-terminal sequence. As shown in this report, the major 60 kDa active form of this protease was found to be present in the extracts of chromosomal proteins obtained from all developmental stages analyzed. In morula and gastrula the 70 kDa inactive precursor, which corresponds to the major form of the zymogen found in unfertilized eggs, was detected. In plutei larvas, the major 60 kDa form of this enzyme was found together with a higher molecular weight precursor (90 kDa) which is consistent with the less abundant zymogen primarily detected in unfertilized eggs. As reported here, either the active protease or its zymogens were visualized in most of the embryonic territories indicating that this enzyme lacks a specific pattern of spatial-temporal developmental segregation. Taken together our results indicate that this protease persists in the embryo and is ubiquitously distributed up to larval stages of development, either as an active enzyme and/or as an inactive precursor. These results suggest that this enzyme may display yet unknown functions during embryonic development that complement its role in male chromatin remodeling after fertilization.

Asunto(s)

Núcleo Celular/enzimología , Ensamble y Desensamble de Cromatina/fisiología , Cisteína Endopeptidasas/inmunología , Fertilización , Erizos de Mar/embriología , Animales , Anticuerpos/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Embrión no Mamífero , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/fisiología , Masculino , Factores de Tiempo , Distribución Tisular

RESUMEN

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development.

Asunto(s)

Ciclo Celular/inmunología , Ensamble y Desensamble de Cromatina/fisiología , Cisteína Endopeptidasas/inmunología , Inhibidores de Cisteína Proteinasa/inmunología , Erizos de Mar/embriología , Animales , Anticuerpos/farmacología , Ciclo Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Replicación del ADN/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/fisiología , Fertilización/fisiología , Inmunoglobulinas/efectos de los fármacos , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Microinyecciones/métodos , Mitosis/efectos de los fármacos , Cigoto/citología

RESUMEN

Male pronucleus formation involves sperm nucleus decondensation and sperm chromatin remodeling. In sea urchins, male pronucleus decondensation was shown to be modulated by protein kinase C and a cdc2-like kinase sensitive to olomoucine in vitro assays. It was further demonstrated that olomoucine blocks SpH2B and SpH1 phosphorylation. These phosphorylations were postulated to participate in the initial steps of male chromatin remodeling during male pronucleus formation. At final steps of male chromatin remodeling, all sperm histones (SpH) disappear from male chromatin and are subsequently degraded by a cysteine protease. As a result of this remodeling, the SpH are replaced by maternal histone variants (CS). To define if sperm nucleus decondensation is coupled with sperm chromatin remodeling, we have followed the loss of SpH in zygotes treated with olomoucine. SpH degradation was followed with anti-SpH antibodies that had no cross-reactivity with CS histone variants. We found that olomoucine blocks SpH1 and SpH2B phosphorylation and inhibits male pronucleus decondensation in vivo. Interestingly, the normal schedule of SpH degradation remains unaltered in the presence of olomoucine. Taken together these results, it was concluded that male nucleus decondensation is uncoupled from the degradation of SpH associated to male chromatin remodeling. From these results, it also emerges that the phosphorylation of SpH2B and SpH1 is not required for the degradation of the SpH that is concurrent to male chromatin remodeling.

Asunto(s)

Núcleo Celular , Ensamble y Desensamble de Cromatina , Erizos de Mar/citología , Erizos de Mar/genética , Animales , Fertilización , Histonas/metabolismo , Masculino , Fosforilación , Erizos de Mar/embriología , Espermatozoides/metabolismo

RESUMEN

We postulated an essential role for a cysteine-protease in sea urchins sperm histones degradation which follows fertilization. We now report the purification of this enzyme, the determination of its N-terminal amino acid sequence and the localization of the protein with antibodies generated against this amino-terminal peptide. The immunofluorescence data confirmed the presence of this enzyme in the nucleus of unfertilized eggs. After fertilization labeling is observed both in female and male pronuclei suggesting a rapid recruitment of the enzyme to the male pronuclei. Interestingly, we have found that this cysteine-protease persists in the nucleus of the zygotes during S phase of the cell cycle and co-localizes with alpha-tubulin that organizes the mitotic spindle during the initial embryonic cell division.

Asunto(s)

Ensamble y Desensamble de Cromatina/fisiología , Cisteína Endopeptidasas/fisiología , Fertilización/fisiología , Mitosis/fisiología , Erizos de Mar , Tubulina (Proteína)/metabolismo , Animales , Núcleo Celular/enzimología , Cisteína Endopeptidasas/metabolismo , Femenino , Immunoblotting , Inmunohistoquímica , Masculino , Óvulo/metabolismo , Fase S , Erizos de Mar/embriología , Distribución Tisular , Cigoto/citología , Cigoto/enzimología , Cigoto/metabolismo

RESUMEN

Transcriptional activation of specific genes is initiated after fertilization by the interaction of specific transcription factors with its cognate sequences in the chromatin context, thereby leading to a concerted and coordinated program which determines early development. Remodeling of the sperm chromatin after fertilization is a fundamental event for transcriptional activation and expression of the paternally inherited genome. The transitions in chromosomal proteins, as well as the mechanisms that participate in these transitions, have been investigated only to a limited extent as compared to the signal transduction patterns that govern egg activation or the dynamics and structural changes accompanying sperm nuclear membrane dissociation-association following insemination. In this review, we will discuss the remodeling of sperm chromatin that follows fertilization. We will emphasize the transitions of chromosomal proteins, as well as the post-translational modifications associated with these transitions. The molecular mechanisms that may be participating in these events will also be analyzed. We will further discuss the mechanisms that govern chromatin remodeling and the role of specific transcription factors in the control of the transcriptional program during sea urchin early development.

Asunto(s)

Núcleo Celular/metabolismo , Cromatina/metabolismo , Erizos de Mar/genética , Activación Transcripcional , Animales , Núcleo Celular/genética , Cromatina/genética , Metilación de ADN , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Modelos Biológicos , Erizos de Mar/embriología

RESUMEN

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants.

Asunto(s)

Histonas/biosíntesis , Erizos de Mar/fisiología , Animales , Blástula/metabolismo , Western Blotting , Bromodesoxiuridina , ADN/análisis , ADN/biosíntesis , Fertilización , Variación Genética , Histonas/análisis , Histonas/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Microscopía Fluorescente , Factores de Tiempo