RESUMO
The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.
Assuntos
Apoptose/efeitos dos fármacos , Príons/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Células de Purkinje/efeitos dos fármacos , Fatores Etários , Análise de Variância , Animais , Contagem de Células , Cerebelo/citologia , Frutose-Bifosfato Aldolase/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Príons/toxicidadeRESUMO
Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.
Assuntos
Apoptose/fisiologia , Príons/fisiologia , Células de Purkinje/fisiologia , Proteína X Associada a bcl-2/fisiologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Feminino , Imunofluorescência , Proteínas Ligadas por GPI , Genótipo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Príons/genética , Príons/metabolismo , Células de Purkinje/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with several X linked mental retardation syndromes, the best known being alpha thalassaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fingers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8, and the beginning of exon 9) while 35% of the mutations have been found in the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific genotype-phenotype correlation has been found, suggesting that gene alteration leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific attention to the functional consequences of mutations to the zinc finger domain. We used monoclonal antibodies directed against the XNP/ATR-X protein and performed immunocytochemical and western blot analyses, which showed altered or absent XNP/ATR-X expression in cells of affected patients. In addition, we used in vitro experiments to show that the zinc finger domain can mediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide insights into the understanding of the functional significance of XNP/ATR-X mutations.
Assuntos
Núcleo Celular/metabolismo , Deficiência Intelectual/genética , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Talassemia/genética , Transporte Ativo do Núcleo Celular , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Western Blotting , Células Cultivadas , DNA/genética , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/imunologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Epitopos/imunologia , Imunofluorescência , Expressão Gênica , Humanos , Masculino , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Ligação Proteica , Síndrome , Proteína Nuclear Ligada ao X , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
The ternary complex factors (TCFs), Elk-1, Sap-1a and Net, are key integrators of the transcriptional response to different signalling pathways. Classically, three MAP kinase pathways, involving ERK, JNK, and p38, transduce various extracellular stimuli to the nucleus. Net is a repressor that is converted into an activator by Ras/ERK signalling. Net is also exported from the nucleus in response to stress stimuli transduced through the JNK pathway, leading to relief from repression. Here we show that ERK and p38 bind to the D box and that binding is required for phosphorylation of the adjacent C-terminally located C-domain. The D box as well as the phosphorylation sites in the C-domain (the DC element) are required for transcription activation by Ras. On the other hand, JNK binds to the J box in the middle of the protein, and binding is required for phosphorylation of the adjacent EXport motif. Both the binding and phosphorylation sites (the JEX element) are important for Net export. In conclusion, specific targeting of Net by MAP kinase pathways involves two different docking sites and phosphorylation of two different domains. These two elements, DC and JEX, mediate two distinct functional responses.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Oncogênicas , Fatores de Transcrição/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , MAP Quinase Quinase 4 , MAP Quinase Quinase 6 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas ras/genética , Proteínas ras/metabolismo , Proteínas ras/fisiologiaRESUMO
The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.
Assuntos
DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Isoenzimas/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Apêndice/metabolismo , Neoplasias da Mama/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT , Células COS/metabolismo , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Feto , Fibroblastos/metabolismo , Biblioteca Gênica , Células HeLa/metabolismo , Humanos , Isoenzimas/genética , Células Jurkat/metabolismo , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/isolamento & purificação , Timo/químicaRESUMO
The E6 protein of cancer-associated human papillomavirus type 16 (HPV16) binds to cellular p53 and promotes its degradation through the ubiquitin pathway. In an attempt to identify the regions of E6 that could be targetted for functional inhibition, we generated monoclonal antibodies to the HPV16 E6 oncoprotein (16E6) and analysed their effect on E6-mediated p53 in vitro degradation. The isolated antibodies recognize the 16E6 oncoprotein expressed in the CaSki carcinoma cell line and strongly inhibit the proteolysis of p53 in vitro by binding specifically to a region of 10 residues located at the N-terminal end of 16E6. The variable regions of these antibodies were cloned and expressed in E. coli as single chain Fvs (scFvs). Purified scFvs were present in monomeric form and totally abolished 16E6-mediated p53 degradation by preventing the formation of E6/p53 protein complexes. Our results demonstrate that monovalent binding of scFvs to the N-terminal end of 16E6 abrogates the biological mechanisms leading to the degradation of p53, and they suggest that this region of 16E6 may be a useful in vivo target for blocking the oncogenic activity of HPV16 E6 protein.
Assuntos
Anticorpos Monoclonais/farmacologia , Fragmentos de Imunoglobulinas/farmacologia , Proteínas Oncogênicas Virais/antagonistas & inibidores , Papillomaviridae/fisiologia , Proteínas Repressoras , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/patologia , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Fragmentos de Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas Virais/farmacologia , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/patologiaRESUMO
Matrix metalloproteinase 2 (MMP2) activity is associated with the aggressiveness of human cancers. Therefore, the mechanisms regulating its activation are of great interest for a better understanding of malignant invasive processes. MT1-MMP, a membrane-bound MMP, is involved in the conversion of the latent form of MMP2 to the active one. In the present study, we have raised 3 monoclonal antibodies (Mabs) directed against 3 different epitopes of human MT1-MMP, which we used to investigate the expression and cellular localization of MT1-MMP protein in human carcinomas. MT1-MMP protein was present in all invasive carcinomas tested, and it was almost exclusively located to the stromal cells and not to cancer cells as previously reported, suggesting that MMP2 activation might be a peri-fibroblastic event.
Assuntos
Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Fibroblastos/enzimologia , Metaloendopeptidases/análise , Invasividade Neoplásica , Proteínas de Neoplasias/análise , Animais , Anticorpos Monoclonais/imunologia , Neoplasias da Mama/patologia , Células COS , Carcinoma/patologia , Carcinoma Ductal de Mama/enzimologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/enzimologia , Carcinoma Lobular/patologia , Ativação Enzimática , Indução Enzimática , Epitopos/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/imunologia , Proteínas Recombinantes/imunologia , Células Estromais/enzimologia , TransfecçãoRESUMO
Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular/metabolismo , Drosophila , Eucromatina , Interfase , Metáfase , Camundongos , Nuclease do Micrococo/metabolismo , XenopusRESUMO
Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.
Assuntos
Núcleo Celular/química , Receptores do Ácido Retinoico/imunologia , Animais , Anticorpos/química , Células COS , Embrião de Mamíferos/química , Humanos , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Receptores do Ácido Retinoico/análise , Transfecção , Células Tumorais CultivadasRESUMO
In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. Here we show that the homeobox genes ladybird early and ladybird late are expressed in only one muscle progenitor and its progeny: the segmental border muscle founder cell and two precursors of adult muscles. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. Unlike so far described progenitors but similar to the neuroblasts, the ladybird-positive progenitor undergoes morphologically asymmetric division. We demonstrate that the ectopic ladybird expression is sufficient to change the identity of a subset of progenitor/founder cells and to generate an altered pattern of muscle precursors. When ectopically expressed, ladybird transforms the identity of neighbouring, Krüppel-positive progenitors leading to the formation of giant segmental border muscles and supernumerary precursors of lateral adult muscles. In embryos lacking ladybird gene function, specification of two ladybird-expressing myoblast lineages is affected. The segmental border muscles do not form or have abnormal shapes and insertion sites while the number of lateral precursors of adult muscles is dramatically reduced. Altogether our results provide new insights into the genetic control of diversification of muscle precursors and indicate a further similarity between the myogenic and neurogenic pathways.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Homeodomínio/fisiologia , Proteínas de Insetos/fisiologia , Músculos/embriologia , Fatores de Transcrição/fisiologia , Animais , Padronização Corporal/genética , Diferenciação Celular , Drosophila/genética , Embrião não Mamífero/embriologia , Células Epidérmicas , Proteínas de Homeodomínio/genética , Proteínas de Insetos/genética , Mesoderma/citologia , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
NSD1, a novel 2588 amino acid mouse nuclear protein that interacts directly with the ligand-binding domain (LBD) of several nuclear receptors (NRs), has been identified and characterized. NSD1 contains a SET domain and multiple PHD fingers. In addition to these conserved domains found in both positive and negative Drosophila chromosomal regulators, NSD1 contains two distinct NR interaction domains, NID-L and NID+L, that exhibit binding properties of NIDs found in NR corepressors and coactivators, respectively. NID-L, but not NID+L, interacts with the unliganded LBDs of retinoic acid receptors (RAR) and thyroid hormone receptors (TR), and this interaction is severely impaired by mutations in the LBD alpha-helix 1 that prevent binding of corepressors and transcriptional silencing by apo-NRs. NID+L, but not NID-L, interacts with the liganded LBDs of RAR, TR, retinoid X receptor (RXR), and estrogen receptor (ER), and this interaction is abrogated by mutations in the LBD alpha-helix 12 that prevent binding of coactivators of the ligand-induced transcriptional activation function AF-2. A novel variant (FxxLL) of the NR box motif (LxxLL) is present in NID+L and is required for the binding of NSD1 to holo-LBDs. Interestingly, NSD1 contains separate repression and activation domains. Thus, NSD1 may define a novel class of bifunctional transcriptional intermediary factors playing distinct roles in both the presence and absence of ligand.
Assuntos
Proteínas de Transporte/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Tretinoína/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS/citologia , Proteínas de Transporte/química , Receptor alfa de Estrogênio , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores de Estrogênio/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Receptor alfa de Ácido Retinoico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , LevedurasRESUMO
Stromelysin-3 (STR-3) is a recently characterized matrix metalloproteinase (MMP) with a unique pattern of expression and substrate specificity. Unlike other MMPs, STR-3 is consistently and dramatically overexpressed by multiple epithelial malignancies, including carcinomas of the breast, lung, colon, head and neck, and skin. Recent studies suggest that STR-3 promotes the local establishment of epithelial malignancies, contributing to tumor cell survival and implantation in host tissues; however, STR-3's mechanism of action remains undefined. STR-3 is a stromal cell product, prompting speculation that infiltrating stromal cells secrete STR-3 in response to tumor-derived factors. To explore this possibility, we developed a tumor/"stroma" coculture assay in which non-small cell lung cancer (NSCLC) cell lines were grown on confluent monolayers of normal pulmonary fibroblasts. In these tumor/stroma cocultures, NSCLCs stimulate normal pulmonary fibroblasts to secrete STR-3 and release extracellular basic fibroblast growth factor. Thereafter, STR-3 is processed at a unique internal sequence via a basic fibroblast growth factor- and MMP-dependent mechanism to a previously unidentified 35-kDa protein that lacks enzymatic activity. 35-kDa STR-3 is the most abundant STR-3 protein in tumor/stroma cocultures and is only detected when normal pulmonary fibroblasts are cultured with malignant bronchial epithelial cells. Therefore, the tumor-specific processing of STR-3 to the 35-kDa protein is likely to be an important regulatory mechanism.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/enzimologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/enzimologia , Metaloendopeptidases/biossíntese , Células Estromais/enzimologia , Sequência de Aminoácidos , Carcinoma Pulmonar de Células não Pequenas/patologia , Catálise , Comunicação Celular , Técnicas de Cocultura , Indução Enzimática , Humanos , Neoplasias Pulmonares/patologia , Metaloproteinase 11 da Matriz , Metaloendopeptidases/química , Dados de Sequência Molecular , Células Tumorais Cultivadas , alfa-Macroglobulinas/metabolismoRESUMO
Efficient pulsed room-temperature laser emission at 946 nm is obtained from a Nd:YAG rod pumped by a Ti-doped sapphire laser in the free-running mode. Three bonded YAG rods of 3-mm diameter with different Nd concentrations and active lengths were tested. A maximum output energy of 83.5 mJ at 3 Hz was obtained with a slope efficiency of 32.3% in an end-pumping configuration.
RESUMO
Spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disorder caused by an unstable and expanded CAG trinucleotide repeat that leads to the expansion of a polyglutamine tract in a protein of unknown function, ataxin-3. We have generated and characterized a panel of monoclonal and polyclonal antibodies raised against ataxin-3 and used them to analyze its expression and localization. In Hela cells, multiple isoforms are expressed besides the major 55-kDa form. While the majority of ataxin-3 is cytosolic, both immunocytofluorescence and subcellular fractionation studies indicate the presence of ataxin-3, in particular, of some of the minor isoforms, in the nuclear and mitochodrial compartments. We also show that ataxin-3 can be phosphorylated. In the brain, only one ataxin-3 isoform containing the polyglutamine stretch was detected, and normal and mutated proteins were found equally expressed in all patient brain regions analyzed. In most neurons, ataxin-3 had a cytoplasmic, dendritic, and axonal localization. Some neurons presented an additional nuclear localization. Ataxin-3 is widely expressed throughout the brain, with a variable intensity specific for subpopulations of neurons. Its expression is, however, not restricted to regions that show intranuclear inclusions and neurodegeneration in SCA3/MJD.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos/genética , Ataxina-3 , Células HeLa/metabolismo , Humanos , Imuno-Histoquímica , Isomerismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares , Proteínas Repressoras , Frações Subcelulares/metabolismo , Distribuição Tecidual/fisiologiaRESUMO
The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous alpha globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/fisiologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Galinhas , Sequência Consenso , Dimerização , Regulação para Baixo , Globinas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão , Relação Estrutura-Atividade , Transcrição Gênica , Dedos de ZincoRESUMO
Huntington's disease is an inherited disorder caused by expansion of a CAG trinucleotide repeat in the IT15 gene, which leads to expansion of a polyglutamine tract within the protein called huntingtin. Despite the characterization of the IT15 gene and the mutation involved in the disease, the normal function of huntingtin and the effects of the mutation on its function and on its neuronal location remain unknown. To study whether mutated huntingtin has the same neuronal distribution and intracellular location as normal huntingtin, we analyzed immunohistochemically both forms of this protein in the brain of 5 controls and 5 patients with Huntington's disease. We show that the distribution of mutated huntingtin is, like that of the normal form, heterogeneous throughout the brain, but is not limited to vulnerable neurons in Huntington's disease, supporting the hypothesis that the presence of the mutated huntingtin in a neuron is not in itself sufficient to lead to neuronal death. Moreover, whereas normal huntingtin is detected in some neuronal perikarya, nerve fibers, and nerve endings, the mutated form is observed in some neuronal perikarya and proximal nerve processes but is not detectable in nerve endings. Our results suggest that the expression or processing of the mutated huntingtin in perikarya and nerve endings differs quantitatively or qualitatively from the expression of the normal form in the same neuronal compartments.
Assuntos
Química Encefálica , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Repetições de Trinucleotídeos , Adulto , Idoso , Especificidade de Anticorpos , Morte Celular/genética , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/imunologia , Proteínas Nucleares/imunologiaRESUMO
Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.
Assuntos
Ataxia de Friedreich/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Ligação ao Ferro , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Células COS , Imunofluorescência , Células HeLa , Humanos , Proteínas de Membrana/imunologia , Camundongos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , FrataxinaRESUMO
The MLN64 gene, which is localized in q12-q21 of the human chromosome 17, encodes a novel protein containing 2 distinct domains. At the N-terminal, MLN64 exhibits a potential trans-membrane region, while at the C-terminal, it shares homology with the F26F4.4 protein of Coenorhabditis elegans and the steroidogenic acute regulatory (StAR) protein, a mitochondrial protein which is involved in steroid-hormone synthesis. By comparing the C-terminal part of these proteins, we defined a novel protein domain, which we termed SHD for "StAR Homology Domain". Of the 93 primary invasive breast carcinomas that were examined, 14 were found to over-express MLN64. These 14 tumors also expressed high c-erbB-2 transcript levels, which were not detected in the MLN64-negative tumors. MLN64 mRNA and protein were specifically detected in malignant cells of breast carcinomas. MLN64 protein was localized within bundle-like structures distributed throughout the cell cytoplasm and condensed in a perinuclear patch, suggesting an association with a specific cell compartment. When the N-terminal part of MLN64 was deleted, MLN64 was uniformly distributed in the cell cytoplasm, indicating that N-terminal part is involved in the specific cytoplasmic localization of MLN64. The homology between the C-terminal part of MLN64 and the functional StAR domain (SHD) suggests that MLN64 and StAR, although distributed in different cellular compartments, may both play a role in steroidogenesis. In this case, the high levels of MLN64 observed in some breast carcinomas could contribute to the progression of these tumors through increased intratumoral steroidogenesis.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Proteínas de Transporte , Fibroadenoma/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Células Cultivadas , Feminino , Fibroadenoma/metabolismo , Fibroadenoma/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Receptor ErbB-2/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.
Assuntos
Precursores Enzimáticos/metabolismo , Gelatinases/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Pele/citologia , Cicatrização/fisiologia , Animais , Northern Blotting , Células COS/fisiologia , Clonagem Molecular , Colagenases/metabolismo , DNA Complementar , Ativação Enzimática , Feminino , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Biblioteca Gênica , Tecido de Granulação/enzimologia , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Dados de Sequência Molecular , Inibidores de Proteases/metabolismo , Proteínas/metabolismo , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Pele/enzimologia , Organismos Livres de Patógenos Específicos , Células Estromais/enzimologia , Inibidor Tecidual de Metaloproteinase-2 , Inibidor Tecidual de Metaloproteinase-3RESUMO
TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.