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1.
J Med Genet ; 45(8): 525-34, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18456717

RESUMO

BACKGROUND: The m.3243A>G mutation in the mitochondrial tRNA(Leu(UUR)) gene is an example of a mutation causing a very heterogeneous phenotype. It is the most frequent cause (80%) of the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), but it can also lead in addition or separately to type 2 diabetes, deafness, renal tubulopathy and/or cardiomyopathy. METHODS: To identify pathogenic processes induced by this mutation, we compared global gene expression levels of muscle biopsies from affected and unaffected mutation carriers with controls. RESULTS AND CONCLUSIONS: Gene expression changes were relatively subtle. In the asymptomatic group 200 transcripts were upregulated and 12 were downregulated, whereas in the symptomatic group 15 transcripts were upregulated and 52 were downregulated. In the asymptomatic group, oxidative phosphorylation (OXPHOS) complex I and IV genes were induced. Protein turnover and apoptosis were elevated, most likely due to the formation of dysfunctional and reactive oxygen species (ROS) damaged proteins. These processes returned to normal in symptomatic patients. Components of the complement system were upregulated in both groups, but the strongest in the symptomatic group, which might indicate muscle regeneration--most likely, protein damage and OXPHOS dysfunction stimulate repair (protein regeneration) and metabolic adaptation (OXPHOS). In asymptomatic individuals these processes suffice to prevent the occurrence of symptoms. However, in affected individuals the repair process terminates, presumably because of excessive damage, and switches to muscle regeneration, as indicated by a stronger complement activation. This switch leaves increasingly damaged tissue in place and muscle pathology becomes manifest. Therefore, the expression of complement components might be a marker for the severity and progression of MELAS clinical course.


Assuntos
Síndrome MELAS/genética , Mutação Puntual , RNA de Transferência de Leucina/genética , Adolescente , Adulto , Idoso , Apoptose , Criança , Pré-Escolar , Ativação do Complemento , Feminino , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Síndrome MELAS/fisiopatologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Fosforilação Oxidativa , Proteínas/metabolismo , RNA de Transferência de Leucina/metabolismo
2.
Genomics ; 88(4): 480-8, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16781840

RESUMO

Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.


Assuntos
Cardiomegalia/genética , Regulação da Expressão Gênica , Animais , Aorta/cirurgia , Cardiomegalia/etiologia , Modelos Animais de Doenças , Metabolismo Energético/genética , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/genética , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Pressão Ventricular
3.
Arterioscler Thromb Vasc Biol ; 26(6): 1226-35, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16574897

RESUMO

During the past 6 years, gene expression profiling of atherosclerosis has been used to identify genes and pathways relevant in vascular (patho)physiology. This review discusses some critical issues in the methodology, analysis, and interpretation of the data of gene expression studies that have made use of vascular specimens from animal models and humans. Analysis of gene expression studies has evolved toward the genome-wide expression profiling of large series of individual samples of well-characterized donors. Despite the advances in statistical and bioinformatical analysis of expression data sets, studies have not yet fully exploited the potential of gene expression data sets to obtain novel insights into the molecular mechanisms underlying atherosclerosis. To assess the potential of published expression data, we compared the data of a CC chemokine gene cluster between 18 murine and human gene expression profiling articles. Our analysis revealed that an adequate comparison is mainly hindered by the incompleteness of available data sets. The challenge for future vascular genomic profiling studies will be to further improve the experimental design, statistical, and bioinformatical analysis and to make data sets freely accessible.


Assuntos
Aterosclerose/genética , Expressão Gênica , Genoma , Animais , Biologia Computacional , Interpretação Estatística de Dados , Perfilação da Expressão Gênica , Humanos , Camundongos/genética
4.
Mol Biol Cell ; 11(1): 117-29, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10637295

RESUMO

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Sinais Direcionadores de Proteínas , Ativação Transcricional , Células 3T3 , Sequência de Aminoácidos , Animais , Fusão Gênica Artificial , Sítios de Ligação , Células CACO-2 , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Ácidos Graxos Insaturados/farmacologia , Expressão Gênica , Glicoproteínas , Células HL-60 , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Proteínas com Domínio LIM , Células LLC-PK1 , Metaloproteínas/química , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Coelhos , Suínos , Células Vero , Zixina
5.
Oncogene ; 18(36): 5076-87, 1999 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-10490844

RESUMO

The HMGIC gene has been implicated in the control of cell proliferation and development. We show here that HMGIC has multiple mRNA isoforms that arise by transcription initiation from alternative tandem promoters. These transcripts are not only differentially expressed between cell lines, but they can also differ within an individual cell line, in response to particular stimuli. Whereas quiescent 3T3-L1 preadipocytes express low levels of HMGIC mRNA, stimulation by serum results in a dramatic upregulation with the characteristics of a delayed-early response gene. Characterization of involved signal transduction pathways showed that both FGF-1 and PDGF-BB are strong inducers of HMGIC expression mediated via both the PI-3 kinase and MAP kinase pathways. In order to characterize the regulatory elements, sequences upstream of the translation initiation site of HMGIC were assayed for promoter activity. The HMGIC 5' flanking sequences had constitutive promoter activity in all cell lines tested, suggesting that HMGIC is regulated by negative regulatory elements that were not present in the 5'-flanking regions analysed here.


Assuntos
Ciclo Celular/genética , Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica , Células 3T3 , Adipócitos/citologia , Animais , Sequência de Bases , Sangue , Diferenciação Celular/genética , Primers do DNA , Camundongos , Regiões Promotoras Genéticas , Transdução de Sinais , Transcrição Gênica
6.
Biochim Biophys Acta ; 1399(1): 83-7, 1998 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-9714752

RESUMO

Mutations in the genes for high mobility group protein I-C (HMGI-C) and insulin-like growth factor 1 (IGF1) are known to be responsible for dwarf phenotypes in the mouse. Because the locus for autosomal dwarfism (adw) in the chicken maps to a region which is syntenic to a region in the human and mouse in which the HMGI-C and IGF1 genes are located, HMGI-C and IGF1 are likely candidate genes for adw in the chicken. In this study their possible role in the establishment of this phenotype has been investigated. We have cloned and sequenced the complete coding region of the chicken HMGI-C cDNA. Comparison with its human counterpart revealed a nucleotide sequence conservation of 84%. Only nine amino acids are present principally in the N-terminal segment before the first DNA-binding domain. Northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild-type chicken embryos. Also no mutations in either the HMGI-C or the IGF1 RNA nucleotide sequence were detected in adw chicken embryos.


Assuntos
Galinhas/genética , DNA Complementar/química , Proteínas de Grupo de Alta Mobilidade/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , Expressão Gênica , Proteína HMGA2 , Fator de Crescimento Insulin-Like I/genética , Dados de Sequência Molecular , RNA/isolamento & purificação , RNA Mensageiro/análise , Alinhamento de Sequência
7.
Biochem J ; 326 ( Pt 2): 507-14, 1997 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9291125

RESUMO

The bait region of the general protease inhibitor alpha 2-macroglobulin (alpha 2M) was mutated by introducing a recognition sequence of furin. This did not interfere with folding, S-ester formation or tetramerization of the mutant recombinant alpha 2M (r alpha 2M). Mutant r alpha 2M inhibited furin in vitro, by a similar mechanism to that used by plasma alpha 2M to inhibit high-molecular-mass proteases. The mutant alpha 2M was intracellularly active in COS-1 cells in inhibiting the endogenous processing of the soluble substrates for furin (von Willebrand factor, transforming growth factor beta1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant alpha 2M strongly indicated that alpha 2M attains its native conformation, and thus that the unusual internal S-ester is formed, before alpha 2M passes through the cleavage compartment(s). Our results show for the first time that modulation of the bait region of alpha 2M allows the creation of an inhibitor against membrane-bound proteases. It can be expected that the use of alpha 2M-bait mutants will become important as a technique for the study of various proteolytic processes and for the identification of the proteases involved.


Assuntos
Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Subtilisinas/genética , Subtilisinas/metabolismo , alfa-Macroglobulinas/genética , alfa-Macroglobulinas/farmacologia , Animais , Células COS , Furina , Humanos , Hidrólise , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Leucemia Eritroblástica Aguda , Mutagênese Insercional , Precursores de Proteínas/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/farmacologia , Solubilidade , Subtilisinas/antagonistas & inibidores , Células Tumorais Cultivadas , alfa-Macroglobulinas/química
8.
J Biol Chem ; 272(4): 2500-8, 1997 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-8999965

RESUMO

The proximal promoter region of the neuroendocrine-specific human prohormone convertase 1 (PC1) gene contains two distinct cAMP response elements (CRE-1 and CRE-2). Both elements are essential in directing the cAMP-mediated hormonal regulation of PC1 gene transcription. In this study, we have demonstrated that CRE-1 binds several trans-acting factors. In electrophoretic mobility shift assay experiments with nuclear extracts prepared from neuroendocrine AtT-20 and beta-TC3 cells and non-neuroendocrine COS-1 cells, three specific protein-DNA complexes (I-III) were detected. Complexes II and III were shown to contain CREB-1 and ATF-1, respectively. The most slowly migrating complex I was only detected with the neuroendocrine cell lines and appeared to comprise a c-Jun-containing heterodimer. In addition, CRE-2 was shown to bind a protein that was only detected in nuclear extracts derived from the neuroendocrine cell lines. Antibody supershift experiments indicated that both the c-Jun-interacting protein in CRE-1 complex I and the CRE-2-interacting protein are distinct from known members of the basic domain, leucine zipper family of transcription factors. UV cross-linking experiments demonstrated that these potential novel proteins are approximately 100 and 60 kDa in size, respectively. Site-specific mutagenesis experiments demonstrated that the formation of both CRE-1 and CRE-2 complexes is correlated with the transcriptional activity of the proximal PC1 promoter as has been shown in transient transfections with wild-type and mutant promoter constructs. In addition, it was shown that both CREB-1 and ATF-1 transactivate the human PC1 promoter in transient transfection experiments.


Assuntos
Ácido Aspártico Endopeptidases/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Fator 1 Ativador da Transcrição , Fator 2 Ativador da Transcrição , Sítios de Ligação , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Humanos , Pró-Proteína Convertases , Conformação Proteica , Células Tumorais Cultivadas , Raios Ultravioleta
9.
Biochem J ; 328 ( Pt 1): 69-74, 1997 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-9359835

RESUMO

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 2 (PC2) gene and established characteristics of the PC2 promoter region. The proximal promoter region is very G+C-rich and does not contain a canonical TATA box or a CAAT box. Transient expression assays with a set of human PC2 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, we show that the transcription factor EGR-1 interacts with two distinct elements within the proximal human PC2 promoter region. Transfection experiments also demonstrate that EGR-1 is able to enhance PC2 promoter activity.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Imediatamente Precoces , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Subtilisinas/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Células COS , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Insulinoma , Dados de Sequência Molecular , Sistemas Neurossecretores/metabolismo , Adeno-Hipófise , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Pró-Proteína Convertase 2 , Ligação Proteica/genética , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
10.
J Pathol ; 179(2): 145-50, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8758205

RESUMO

Chromosomal abnormalities involving 3q27 have recently been associated with diffuse large B-cell lymphomas and, less frequently, with follicular lymphomas. Molecular studies have led to the identification of the BCL-6/LAZ-3 gene, located at 3q27 and coding for a putative zinc-finger protein that might act as a transcriptional regulator during cell differentiation and development. Rearrangement of BCL-6 results in truncation of the gene in its 5' portion, leaving the protein intact; a resultant deregulation of its expression has been hypothesized. In order to test this hypothesis, the expression of BCL-6 protein was investigated in human reactive lymphoid tissue and compared with a group of non-Hodgkin's lymphomas (NHLs) with or without 3q27 anomalies and/or BCL-6 gene rearrangement. BCL-6 protein is consistently expressed in reactive lymphoid tissues, where it is restricted to the follicle centre. The protein is also widely expressed in NHL: all follicular lymphomas tested showed a pattern of expression similar to the reactive B follicle, independently of the presence of BCL-6 gene rearrangement and/or 3q27 anomalies. In the diffuse large B-cell lymphomas, there was more variation in BCL-6 expression, but a correlation with 3q27 anomalies and/or BCL-6 rearrangement was not found. Deregulation of the BCL-6 gene did not result in an aberrant tissue expression as detected by immunohistochemistry.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Tecido Linfoide/metabolismo , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Especificidade de Anticorpos , Western Blotting , Aberrações Cromossômicas , Cromossomos Humanos Par 3 , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Linfoma de Células B/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Fatores de Transcrição/genética
11.
Genomics ; 32(2): 191-9, 1996 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8833145

RESUMO

Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described. This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts. The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons. Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis. Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries. The NSP exons were found to be dispersed over a genomic region of about 275 kb. The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms. Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity. Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes.


Assuntos
Éxons , Íntrons , Família Multigênica , Proteínas do Tecido Nervoso/genética , Sequência de Bases , Cromossomos Artificiais de Levedura , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas
12.
FASEB J ; 10(4): 453-60, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8647344

RESUMO

Promoters have been defined as modulatory DNA structures containing a complex array of cis-acting regulatory elements required for accurate and efficient initiation of transcription and for controlling expression of a gene. It is becoming increasingly evident that they also constitute prime target elements through which diversity and flexibility in the complex patterns of gene expression in multicellular organisms are created. The use of multiple promoters and transcription start sites is apparently a frequently used mechanism, whereas at the same time there is considerable variation and complexity in the patterns of alternative promoter usage. This review discusses the use of alternative promoters as a versatile mechanism to create diversity and flexibility in the regulation of gene expression. Alternative promoter usage can influence gene expression in very diverse ways. The level of transcription initiation can vary between alternative promoters, the turnover or translation efficiency of mRNA isoforms with different leader exons can differ, alternative promoters can have different tissue specificity and react differently to some signals, and finally, alternative promoter usage can lead to the generation of protein isoforms differing at the amino terminus.


Assuntos
Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Animais , Homeostase , Humanos , Transcrição Gênica
13.
Mol Biol Rep ; 23(2): 87-95, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8983022

RESUMO

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.


Assuntos
Células CHO/fisiologia , Proteínas Recombinantes/metabolismo , Subtilisinas/metabolismo , Animais , Cricetinae , Toxina Diftérica/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Furina , Humanos , Processamento de Proteína Pós-Traducional , Transfecção , Fator de Crescimento Transformador beta/biossíntese , Fator de von Willebrand/biossíntese
14.
J Biol Chem ; 270(25): 15391-7, 1995 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-7797529

RESUMO

Prohormone convertases are involved in the tissue-specific endoproteolytic processing of prohormones and neuropeptide precursors within the secretory pathway. In the present study, we have isolated genomic clones comprising the 5'-terminal region of the human prohormone convertase 1 (PC1) gene and identified and characterized the PC1 promoter region. We found multiple transcription start sites located within a 15-base pair region, 205 base pairs upstream of the translation start codon. The promoter region is not G+C-rich and does not contain a canonical TATA box nor a CAAT box. Transient expression assays with a set of human PC1 gene fragments containing progressive 5' deletions demonstrate that the proximal promoter region is capable of directing high levels of neuroendocrine-specific expression of reporter gene constructs. In addition, the proximal promoter region confers both basal and hormone-regulated promoter activity. Site-specific mutagenesis experiments demonstrate that two closely spaced cAMP response elements within the proximal promoter region direct cAMP-mediated hormonal regulation of transcription of the PC1 gene.


Assuntos
Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/genética , AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Sistemas Neurossecretores/enzimologia , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Bromocriptina/farmacologia , Linhagem Celular , Clonagem Molecular , Primers do DNA , Biblioteca Genômica , Humanos , Luciferases/biossíntese , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Proteína Convertases , Receptores de Dopamina D2/biossíntese , Receptores de Dopamina D2/metabolismo , Proteínas Recombinantes/biossíntese , Deleção de Sequência , TATA Box , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas
15.
DNA Cell Biol ; 14(3): 223-34, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7880443

RESUMO

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.


Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Genes de Insetos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Subtilisinas/genética , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Drosophila melanogaster/enzimologia , Embrião não Mamífero/enzimologia , Éxons , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Íntrons , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Subtilisinas/metabolismo
16.
J Biol Chem ; 270(6): 2695-702, 1995 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-7852339

RESUMO

The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.


Assuntos
Retículo Endoplasmático/metabolismo , Precursores de Proteínas/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Bovinos , Linhagem Celular , Primers do DNA , Furina , Glicosilação , Humanos , Hidrólise , Dados de Sequência Molecular , Mutação , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Subtilisinas/biossíntese , Subtilisinas/genética , Suínos
18.
J Biol Chem ; 269(12): 9298-303, 1994 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-8132667

RESUMO

The prototype mammalian proprotein processing enzyme furin is shown to be encoded by three distinct FUR mRNA isoforms which differ only in their 5'-untranslated regions. By primer extension analysis, the transcription start sites of the three mRNA isoforms were defined. The genomic regions located immediately upstream of the three alternative transcriptional start sites were shown to possess promoter activity in transfection experiments using the luciferase encoding gene as reporter. In a liver cell line, the P1 promoter appeared to be the strongest; in a lung cell line, the P1A promoter. Human FUR promoter P1 but not P1A or P1B was transactivated by transcription factor C/EBP beta. Other members of this family of bZIP transcription factors, C/EBP alpha and C/EBP delta, were not able to transactivate the P1 promoter. Promoter P1A and P1B have characteristics of promoters of housekeeping genes. They lack TATA or CAAT boxes upstream of the transcription start site but are very GC-rich and contain several SP1 sites. Promoter P1, on the other hand, has a TATA box in the proximal promoter region. In electromobility shift assays and DNase I footprinting analysis, transcription factor SP1 was found to bind to the proximal region of the P1 promoter. Altogether, our results indicate that expression of the human FUR gene is directed by alternative promoters, housekeeping (GC-rich) as well as regulated (TATA-containing) promoters, suggesting that their differential use may be a mechanism to modulate levels of the furin enzyme.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , Subtilisinas/genética , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Primers do DNA/química , Proteínas de Ligação a DNA/metabolismo , Furina , Genes , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Fator de Transcrição Sp1/metabolismo
19.
Biochimie ; 76(3-4): 210-6, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7819325

RESUMO

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.


Assuntos
Processamento de Proteína Pós-Traducional , Subtilisinas/química , Subtilisinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Furina , Humanos , Mamíferos , Estrutura Secundária de Proteína , Subtilisinas/genética
20.
J Biol Chem ; 268(29): 21826-34, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8408037

RESUMO

The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. Finally, we made mutants in the substrate binding region of human furin and studied their ability to process von Willebrand factor (pro-vWF) substrates, including wild-type pro-vWF as well as pro-vWF mutants in which the P1 (vWFR-1G), P2 (vWFK-2A), or P4 (vWFR-4A) basic residue with respect to the pro region cleavage site had been mutated. It is demonstrated that particular negatively charged residues in or near the substrate binding region of furin are critical for cleavage activity and specificity of the enzyme for multiple basic residues in the substrate. Furthermore, substrate binding region mutants of furin were obtained, which cleaved either the pro-vWFK-2A or pro-vWFR-4A mutant of pro-vWF more efficiently than wild-type pro-vWF.


Assuntos
Processamento de Proteína Pós-Traducional , Subtilisinas/metabolismo , Aminoácidos/química , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Primers do DNA , Furina , Humanos , Hidrólise , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Precipitina , Precursores de Proteínas/metabolismo , Deleção de Sequência , Subtilisinas/biossíntese , Subtilisinas/genética , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
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