RESUMO
Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.
Assuntos
Estruturas do Núcleo Celular/ultraestrutura , Proteínas HMGB/metabolismo , Trypanosoma cruzi , Pontos de Checagem do Ciclo Celular , Nucléolo Celular , Citocinese , Proteínas HMGB/farmacologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Trypanosoma cruzi/ultraestrutura , VirulênciaRESUMO
Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman-Bodian-Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S-80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.
Assuntos
Proteínas Nucleares/fisiologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Linhagem Celular , Estruturas do Núcleo Celular/química , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Poli A-U/metabolismo , Poli U/metabolismo , Polirribossomos/química , RNA/química , RNA/metabolismo , Precursores de RNA/química , RNA Ribossômico/químicaRESUMO
Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Amiloide/metabolismo , Animais , Compartimento Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Dactinomicina/farmacologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Peptídeos/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
Lamin A/C is the most studied nucleoskeletal constituent. Lamin A/C expression indicates cell differentiation and is also a structural component of nuclear speckles, which are involved in gene expression regulation. Hypertonicity has been reported to induce renal epithelial cell differentiation and expression of TonEBP (NFAT5), a transcriptional activator of hypertonicity-induced gene transcription. In this paper, we investigate the effect of hypertonicity on lamin A/C expression in MDCK cells and the involvement of TonEBP. Hypertonicity increased lamin A/C expression and its distribution to nucleoplasm with speckled pattern. Microscopy showed codistribution of TonEBP and lamin A/C in nucleoplasmic speckles, and immunoprecipitation demonstrated their interaction. TonEBP silencing caused lamin A/C redistribution from nucleoplasmic speckles to the nuclear rim, followed by lamin decrease, thus showing that hypertonicity induces lamin A/C speckles through a TonEBP-dependent mechanism. We suggest that lamin A/C speckles could serve TonEBP as scaffold thus favoring its role in hypertonicity.
Assuntos
Estruturas do Núcleo Celular/efeitos dos fármacos , Estruturas do Núcleo Celular/metabolismo , Soluções Hipertônicas/farmacologia , Lamina Tipo A/biossíntese , Lamina Tipo A/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Cães , Inativação Gênica/efeitos dos fármacos , Imunoprecipitação , Transporte Proteico/efeitos dos fármacosRESUMO
BACKGROUND: The close apposition of multivalents with the XY body has been repeatedly described in heterozygous carriers of chromosomal rearrangements. Because in many of these carriers spermatogenesis is deeply disturbed at the spermatocyte level, the association of autosomal chromatin with the XY body may impair the spermatocyte life. METHODS: Testicular biopsies from three men carriers of three different chromosomal rearrangements have been analysed by electron microscopy (EM) and immunolocalization of meiotic proteins. RESULTS: There is an ordered transition from isolated multivalents at early pachytene to XY body association in late pachytene, as shown in a carrier of a rob t(13;14) translocation by EM and in a reciprocal translocation t(9;14) carrier by immunofluorescence. The non-synapsed ends of the quadrivalent show BRCA1 located on the axes and the variant histone gamma-H2AX located on the chromatin. The area covered by gamma-H2AX increases with the association of the asynaptic ends with the XY body in the t(9;14) carrier, and the area covered with gamma-H2AX in the t(Y;15) carrier is larger than that of the XY body of controls. CONCLUSIONS: The affinity between the inactive XY body and asynaptic regions of multivalents is given a material basis, and transcriptional inactivation is probably shared by these two chromatin types.
Assuntos
Azoospermia/genética , Estruturas do Núcleo Celular/genética , Cromatina/genética , Cromatina/ultraestrutura , Histonas/genética , Oligospermia/genética , Espermatócitos/ultraestrutura , Translocação Genética/genética , Adulto , Azoospermia/patologia , Proteína BRCA1/genética , Biópsia , Proteínas de Ciclo Celular , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 9/genética , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Proteínas de Ligação a DNA , Humanos , Masculino , Proteínas Nucleares/genética , Oligospermia/patologia , Testículo/patologiaRESUMO
This study allowed the characterization of the tambaqui Colossoma macropomum testes structural organization, emphasizing Sertoli and interstitial cells and analyzing morphometrically the Sertoli cell nucleus diameter and the interstitial tissue area during the reproductive cycle. Fragments of tambaqui testes were collected in the following reproductive cycle stages: immature, resting, maturation I and II, mature, and regression, and were histologically processed. The Sertoli cells were found at the periphery of the cysts of germinative lineage cells and the nuclei were shown to be smaller as these cells developed. The interstitial cells were better observed between the seminiferous lobules next to vessels in the interstitial tissue of maturing testes.
Assuntos
Peixes/anatomia & histologia , Células Intersticiais do Testículo/ultraestrutura , Reprodução/fisiologia , Células de Sertoli/ultraestrutura , Animais , Estruturas do Núcleo Celular , Peixes/fisiologia , Masculino , Testículo/citologiaRESUMO
Endopeptidase 24.15 (EP24.15) and 24.16 (EP24.16) are closely related metalloendopeptidases implicated in the metabolism of several neuropeptides and widely expressed in mammalian brain. To gain insight into the functional role of these two enzymes in the central nervous system, we examined their cellular and subcellular distribution in rat brain by using electron microscopic immunogold labeling. In all areas examined, EP24.15 and EP24.16 immunoreactivity were observed in selective subpopulations of neuronal and glial cells. Subcellular localization of EP24.15 in neurons revealed that this enzyme was predominantly concentrated in the nucleus, whereas EP24.16 was almost exclusively cytoplasmic. The amount of EP24.15 found in the nucleus was inversely correlated with that found in the cytoplasm, suggesting that the enzyme could be mobilized from one compartment to the other. Within the cytoplasm, EP24.15 and EP24.16 immunoreactivity showed comparable distributional patterns. Both enzymes were detected throughout perikarya and dendrites, as well as within axons and axon terminals. In all neuronal compartments, EP24.15 and EP24.16 showed a major association with membranes of neurosecretory elements, including Golgi cisternae, tubulovesicular organelles, synaptic vesicles, and endosomes. However, whereas EP24.15 always faced the cytoplasmic face of the membranes, EP24.16 was observed on both cytoplasmic and luminal sides, suggesting that the latter was more likely to contribute to the processing of peptides or to the degradation of internalized ligands. Taken together, the present results suggest that EP24.15 could play a major role in the hydrolysis of intranuclear substrates, whereas EP24.16 would be predominantly involved in the processing and inactivation of signaling peptides.