RESUMO
The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects.
Assuntos
Espectrometria de Massas/métodos , Células-Tronco Mesenquimais/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Oxigênio/farmacologia , Reprodutibilidade dos TestesRESUMO
In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Linhagem Celular Tumoral , Cromatografia de Afinidade , Cromatografia Líquida , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Espectrometria de Massas , Proteômica/métodos , Espectrometria de Massas em Tandem , Células U937RESUMO
Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.
Assuntos
Espaço Intracelular/enzimologia , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Western Blotting , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Humanos , Interferon gama/farmacologia , Espaço Intracelular/metabolismo , Cinética , Leucemia/metabolismo , Leucemia/patologia , Espectrometria de Massas/métodos , Microscopia Confocal , Microssomos/enzimologia , Microssomos/metabolismo , Complexos Multienzimáticos/química , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Células U937RESUMO
The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Linhagem Celular , Homeostase , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peso Molecular , Complexo de Endopeptidases do Proteassoma/química , Mapas de Interação de Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , ProteômicaRESUMO
We have identified a novel evolutionarily conserved protein motif - designated the THAP domain - that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-Xaa(2-4)-Cys-Xaa(35-50)-Cys-Xaa(2)-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Transposases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The physiologic function of the secondary lymphoid organs to recruit large numbers of naïve lymphocytes increases the probability that antigens encounter their rare, sometimes unique, specific T lymphocytes and initiate a specific immune response. In peripheral lymph nodes (LNs), this recruitment is a multistep process, initiated predominantly within the high endothelial venules (HEVs), beginning with rolling and chemokine-dependent firm adhesion of the lymphocytes on the venular endothelium surface. We report here that, in C57BL/6 mice, the recruitment of naïve lymphocytes is impaired in LNs draining a B16 melanoma tumor. Intravital microscopy analysis of the tumor-draining LNs revealed that this effect is associated with an important defect in lymphocyte adhesion in the HEVs and a progressive decrease in the expression of the LN chemokine CCL21. In parallel with these effects, the tumor up-regulated, essentially through a P-selectin-dependent mechanism, the rolling and sticking of circulating polymorphonuclear cells within the LN low-order venules where few rolling and sticking events are usually observed. These effects of the tumor were independent of the presence of metastasis into the LN and occurred as long as the tumor developed. Together, these results indicate that the tumor proximity disturbs the LN physiology by modifying the molecular, spatial, and cellular rules that usually control leukocyte-endothelium interactions into the peripheral LNs. In addition, they emphasize a new role for the low-order venules of the peripheral LNs, which compared with the HEVs, seem to be the preferential port of entry for cells linked to inflammatory processes.
Assuntos
Endotélio Linfático/citologia , Endotélio Linfático/imunologia , Leucócitos/metabolismo , Linfonodos/imunologia , Linfócitos/fisiologia , Melanoma Experimental/imunologia , Animais , Apresentação de Antígeno/imunologia , Adesão Celular/imunologia , Quimiocina CCL21 , Quimiocinas CC/metabolismo , Feminino , Selectina L/metabolismo , Leucócitos/imunologia , Metástase Linfática/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Selectina-P/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Promyelocytic leukemia (PML) nuclear bodies (PML NBs) are discrete subnuclear domains organized by the promyelocytic leukemia protein PML, a tumor suppressor essential for multiple apoptotic pathways. We have recently described a novel family of cellular factors, the THAP proteins, characterized by the presence at their amino-terminus of an evolutionary conserved putative DNA-binding motif, designated THAP domain. Here, we report that THAP1 is a novel nuclear proapoptotic factor associated with PML NBs, which potentiates both serum withdrawal- and TNF alpha-induced apoptosis, and interacts with prostate-apoptosis-response-4 (Par-4), a well characterized proapoptotic factor, previously linked to prostate cancer and neurodegenerative diseases. We show that endogenous Par-4 colocalizes with ectopic THAP1 within PML NBs in primary endothelial cells and fibroblasts. In addition, we found that Par-4 is a component of PML NBs in blood vessels, a major site of PML expression in vivo. Finally, we investigated the role of the THAP domain in THAP1 activities and found that this putative DNA-binding domain is not required for Par-4 binding and localization within PML NBs, but is essential for THAP1 proapoptotic activity. Together, our results provide an unexpected link between a nuclear factor of the THAP family, the proapoptotic protein Par-4 and PML nuclear bodies.
Assuntos
Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Epitélio/metabolismo , Fibroblastos/metabolismo , Humanos , Proteína da Leucemia Promielocítica , Estrutura Terciária de Proteína , Proteínas Supressoras de TumorRESUMO
Lymphocyte emigration from the blood into most secondary lymphoid organs and chronically inflamed tissues occurs at the level of high endothelial venules (HEV). A unique characteristic of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that sulfate uptake into HEVEC is mediated by two distinct functional classes of sulfate transporters: Na+-coupled transporters and sulfate/anion exchangers. Here, we report the molecular characterization from human HEVEC of SLC26A11, a novel member of the SLC26 sulfate/anion exchanger family. Functional expression studies in COS-7 and Sf9 insect cells revealed that SLC26A11 is targeted to the cell membrane and exhibits Na+-independent sulfate transport activity, sensitive to the anion exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Northern blot analysis showed the highest SLC26A11 transcript levels in placenta, kidney, and brain. The SLC26A11 gene mapped to human chromosome 17q25, very close to the hereditary hearing loss diseases loci DFNA20, DFNA26, and USH1G. RT-PCR analysis of SLC26 sulfate transporters in human HEVEC revealed coexpression of SLC26A11 with SLC26A2/DTDST and lack of SLC26A1/SAT1, SLC26A3/DRA, and SLC26A8/TAT1. Together, our results indicate that SLC26A11 is a novel Na+-independent sulfate transporter that may cooperate with SLC26A2 to mediate DIDS-sensitive sulfate uptake into HEVEC.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte/genética , Endotélio Vascular/metabolismo , Proteínas de Membrana Transportadoras , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica , Predisposição Genética para Doença/genética , Perda Auditiva/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Sódio/farmacologia , Spodoptera , Transportadores de SulfatoRESUMO
We have recently described an evolutionarily conserved protein motif, designated the THAP domain, which defines a previously uncharacterized family of cellular factors (THAP proteins). The THAP domain exhibits similarities to the site-specific DNA-binding domain of Drosophila P element transposase, including a putative metal-coordinating C2CH signature (CX(2-4)CX(35-53)CX(2)H). In this article, we report a comprehensive list of approximately 100 distinct THAP proteins in model animal organisms, including human nuclear proapoptotic factors THAP1 and DAP4/THAP0, transcriptional repressor THAP7, zebrafish orthologue of cell cycle regulator E2F6, and Caenorhabditis elegans chromatin-associated protein HIM-17 and cell-cycle regulators LIN-36 and LIN-15B. In addition, we demonstrate the biochemical function of the THAP domain as a zinc-dependent sequence-specific DNA-binding domain belonging to the zinc-finger superfamily. In vitro binding-site selection allowed us to identify an 11-nucleotide consensus DNA-binding sequence specifically recognized by the THAP domain of human THAP1. Mutations of single nucleotide positions in this sequence abrogated THAP-domain binding. Experiments with the zinc chelator 1,10-o-phenanthroline revealed that the THAP domain is a zinc-dependent DNA-binding domain. Site-directed mutagenesis of single cysteine or histidine residues supported a role for the C2CH motif in zinc coordination and DNA-binding activity. The four other conserved residues (P, W, F, and P), which define the THAP consensus sequence, were also found to be required for DNA binding. Together with previous genetic data obtained in C. elegans, our results suggest that cellular THAP proteins may function as zinc-dependent sequence-specific DNA-binding factors with roles in proliferation, apoptosis, cell cycle, chromosome segregation, chromatin modification, and transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Motivos de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Caenorhabditis elegans , Quelantes/farmacologia , Cisteína/química , DNA/química , Proteínas de Ligação a DNA/química , Bases de Dados Genéticas , Histidina/química , Humanos , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/química , Fenantrolinas/farmacologia , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transcrição Gênica , Peixe-Zebra , Zinco/química , Zinco/farmacologia , Dedos de ZincoRESUMO
Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fase S/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Chlorocebus aethiops , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/genética , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Estrutura Terciária de Proteína , Fase de Repouso do Ciclo Celular/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Fase S/fisiologiaRESUMO
Endothelial cells play a central role in chronic inflammation: for example, they express adhesion molecules and present chemokines leading to enhanced leukocyte recruitment into tissues. Numerous markers of endothelial cells have been reported but there has been a lack of comparative data on their specificity. The present study compared the specificity of seven endothelial cell markers in the rheumatoid synovium and the colon of patients with Crohn's disease. These markers were: the sulphated epitope MECA-79, the Duffy antigen receptor for chemokines (DARC), von Willebrand factor, CD31 (PECAM-1), CD34, CD105 (endoglin) and CD146. MECA-79, DARC and von Willebrand factor showed a specific endothelial cell distribution. MECA-79, which recognizes sulphated ligands for leukocyte adhesion receptor L-selectin (CD62L), was selective for a subset of venules in highly inflamed tissue and was present in rheumatoid but not control osteoarthritic synovia. DARC was also specific for venules but had a more widespread distribution than MECA-79, and was present in rheumatoid and control synovia. The other markers all labelled endothelial cells in venules, arterioles and capillaries. However, they also localized to other cell types. For example, CD34 stained fibroblasts, CD146 was expressed by the pericytes and smooth muscle cells of vessel walls and CD31 and CD105 labelled a broad range of cell types.
Assuntos
Antígenos CD/análise , Antígenos de Superfície/análise , Artrite/patologia , Doença de Crohn/patologia , Sistema do Grupo Sanguíneo Duffy/análise , Células Endoteliais/química , Receptores de Superfície Celular/análise , Fator de von Willebrand/análise , Antígenos CD34/análise , Artrite Reumatoide/patologia , Biomarcadores/análise , Antígeno CD146 , Colo/química , Colo/patologia , Endoglina , Células Endoteliais/patologia , Humanos , Imuno-Histoquímica/métodos , Proteínas de Membrana , Moléculas de Adesão de Célula Nervosa/análise , Osteoartrite do Joelho/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Membrana Sinovial/química , Membrana Sinovial/patologia , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.
Assuntos
DNA Ribossômico/metabolismo , Oócitos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição , RNA Polimerase I/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Animais , Western Blotting , Células CHO , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cricetinae , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Histonas/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Genéticos , Fosfoproteínas/farmacologia , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Xenopus laevis , NucleolinaRESUMO
It is generally believed that, during Xenopus laevis oogenesis, polymerase I transcription is high in the early vitellogenic oocytes (stages III and IV) and very low in later stages. We used a combination of RNA labeling, nuclease S1 protection assays, Northern blot, and half-life measurement of preribosomal RNA to reinvestigate the pattern of polymerase I activity during oogenesis. Unexpectedly, when we compared the amount of 40S pre-rRNA produced in stages IV and VI by direct labeling or with a probe that hybridizes with the 5' external transcribed spacer, we found a high level of 40S pre-rRNA in stage VI oocytes. This precursor ribosomal RNA transcribed in stage VI oocytes is processed to give the matured 18S and 28S species. These results suggest that the activity of RNA polymerase I in stage VI oocytes is similar or very close to that found in stage IV, which is probably required to maintain the huge number of ribosomes during oogenesis.
Assuntos
DNA Ribossômico/genética , Oogênese/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/genética , Animais , Feminino , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese/fisiologia , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Transcrição Gênica , Xenopus laevis/metabolismoRESUMO
Despite the identification of numerous factors involved in ribosomal RNA synthesis and maturation, the molecular mechanisms of ribosome biogenesis, and in particular the relationship between the different steps, are still largely unknown. We have investigated the consequences of an increased amount of a major nucleolar non-ribosomal protein, nucleolin, in Xenopus laevisstage VI oocytes on the production of ribosomal subunits. We show that a threefold increase in nucleolin leads to the complete absence of pre-rRNA maturation in addition to significant repression of RNA polymerase I transcription. Observation of "Christmas trees" by electron microscopy and analysis of the sedimentation properties of 40S pre-ribosomal particles suggest that an increased amount of nucleolin leads to incorrect packaging of the 40S particle. Interestingly, nucleolin affects the maturation of the 40S particle only when it is present at the time of transcription. These results indicate that nucleolin participates in the co-transcriptional packaging of the pre-rRNA, and that the quality of this packaging will determine whether the 40S precursor undergoes maturation or is degraded. The interaction of nucleolin with nascent pre-rRNA could help the co-transcriptional assembly on pre-rRNA of factors necessary for the subsequent maturation of the pre-ribosomal particle containing the 40S pre-rRNA.
Assuntos
Fosfoproteínas/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Transcrição Gênica , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fosfoproteínas/farmacologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Transporte de RNA/efeitos dos fármacos , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/farmacologia , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Transcrição Gênica/efeitos dos fármacos , Xenopus laevis , NucleolinaRESUMO
A unique characteristic of endothelial cells from high endothelial venules (HEVEC) in lymphoid organs and chronically inflamed tissues is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that HEVEC express two functional classes of sulfate transporters: sodium/sulfate cotransporters and sulfate/anion exchangers. Here, we report the molecular cloning from human HEVEC of a 2.9-kb cDNA encoding SLC26A7, a novel member of the SLC26 (solute carrier 26) sulfate/anion exchanger family. SLC26A7 exhibits 30% identity with three known sulfate transporters from the SLC26 family: SLC26A2 (also known as DTDST), SLC26A1 (also known as SAT1), and SLC26A3 (also known as DRA). Northern blot analysis revealed specific expression of SLC26A7 mRNA in kidney. Alternative splicing and polyadenylation of SLC26A7 pre-mRNA in kidney suggest the existence of two protein isoforms, SLC26A7.1 and SLC26A7.2, differing in their carboxy termini.
Assuntos
Antiporters/genética , Proteínas de Transporte/genética , Endotélio Vascular/química , Rim/química , Proteínas de Membrana Transportadoras , Vênulas/química , Processamento Alternativo , Sequência de Aminoácidos , Animais , Antiporters/análise , Proteínas de Transporte/análise , Antiportadores de Cloreto-Bicarbonato , Clonagem Molecular , DNA Complementar , Humanos , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Homologia de Sequência , Transportadores de Sulfato , Distribuição TecidualRESUMO
Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues "speak" to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule-specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type-specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Tonsila Palatina/irrigação sanguínea , Tonsila Palatina/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno/genética , Regulação para Baixo/fisiologia , Sistema do Grupo Sanguíneo Duffy/genética , Fucosiltransferases/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vênulas/citologiaRESUMO
Endothelial cells are active participants in chronic inflammatory diseases. These cells undergo phenotypic changes that can be characterised as activated, angiogenic, apoptotic and leaky. In the present review, these phenotypes are described in the context of human rheumatoid arthritis as the disease example. Endothelial cells become activated in rheumatoid arthritis pathophysiology, expressing adhesion molecules and presenting chemokines, leading to leukocyte migration from the blood into the tissue. Endothelial cell permeability increases, leading to oedema formation and swelling of the joints. These cells proliferate as part of the angiogenic response and there is also a net increase in the turnover of endothelial cells since the number of apoptotic endothelial cells increases. The endothelium expresses various cytokines, cytokine receptors and proteases that are involved in angiogenesis, proliferation and tissue degradation. Associated with these mechanisms is a change in the spectrum of genes expressed, some of which are relatively endothelial specific and others are widely expressed by other cells in the synovium. Better knowledge of molecular and functional changes occurring in endothelial cells during chronic inflammation may lead to the development of endothelium-targeted therapies for rheumatoid arthritis and other chronic inflammatory diseases.
Assuntos
Apoptose/genética , Artrite Reumatoide/genética , Permeabilidade da Membrana Celular/genética , Células Endoteliais/química , Células Endoteliais/metabolismo , Neovascularização Patológica/genética , Membrana Sinovial/patologia , Animais , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Fenótipo , Membrana Sinovial/química , Membrana Sinovial/metabolismoRESUMO
Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.