RESUMO
Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.
Assuntos
Ácido Aspártico Endopeptidases/química , Membrana Celular/enzimologia , Complexo de Golgi/enzimologia , Isoenzimas/química , Metaloendopeptidases/química , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/análise , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Células CHO , Linhagem Celular , Clonagem Molecular , Cricetinae , Endotelina-1 , Enzimas Conversoras de Endotelina , Endotelinas/metabolismo , Imunofluorescência , Humanos , Isoenzimas/análise , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Metaloendopeptidases/análise , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Precursores de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Análise de Sequência de DNAAssuntos
Genes Fúngicos , RNA de Transferência de Leucina/genética , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Treonina/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA FúngicoRESUMO
The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.
Assuntos
Produtos do Gene rev/genética , Genes rev , HIV-2/genética , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Produtos do Gene rev/isolamento & purificação , Dados de Sequência Molecular , Fosforilação , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Transfecção , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.
Assuntos
Produtos do Gene rev/metabolismo , HIV-1/genética , HIV-2/genética , RNA Mensageiro/genética , Transativadores/metabolismo , Proteínas do Envelope Viral/genética , Sequência de Bases , Sítios de Ligação , Deleção Cromossômica , Clonagem Molecular , Escherichia coli/genética , Produtos do Gene rev/genética , Produtos do Gene rev/isolamento & purificação , Genes Virais , Vetores Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Vírus da Imunodeficiência Símia/genética , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication. Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned. Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells. TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat. In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation. The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion.
Assuntos
Proteínas de Ligação a DNA/genética , Produtos do Gene tat/metabolismo , Complexo de Endopeptidases do Proteassoma , Transativadores/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Expressão Gênica , Biblioteca Gênica , HIV/genética , Humanos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
A region in the human immunodeficiency virus (HIV) env message, with the potential to form a complex secondary structure (designated RRE), interacts with the rev protein (Rev). This interaction is believed to mediate export of HIV structural messenger RNAs from the nucleus to the cytoplasm. In this report the regions essential for Rev interaction with the RRE are further characterized and the functional significance of Rev-RRE interaction in vivo is examined. A single hairpin loop structure within the RRE was found to be a primary determinant for Rev binding in vitro and Rev response in vivo. Maintenance of secondary structure, rather than primary nucleotide sequence alone, appeared to be necessary for Rev-RNA interaction, which distinguishes it from the mechanism for cis-acting elements in DNA. Limited changes within the 200 nucleotides, which preserved the proper RRE conformational structure, were well tolerated for Rev binding and function. Thus, variation among the RRE elements present in the diverse HIV isolates would have little, if any, effect on Rev responsiveness.
Assuntos
Produtos do Gene rev/metabolismo , HIV/genética , RNA Mensageiro/genética , Transativadores/metabolismo , Composição de Bases , Sequência de Bases , Deleção Cromossômica , Produtos do Gene rev/genética , Genes rev , HIV/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Plasmídeos , Conformação Proteica , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
In the yeast Saccharomyces cerevisiae, the majority of the tRNA genes are found associated with transposable elements one of which is the Ty element. We noticed that the transcriptional activity of several of these genes was rather different depending on the type of the accompanying element(s) [Nelböck et al. (1985) Biol. Chem. Hoppe-Seyler 366, 1041-1051]. In order to study the influence of a Ty element on tRNA gene expression, we used the method of micro-injection into Xenopus oocytes taking advantage of a Ty+/Ty- allelic pair of a tRNALys1 gene recently isolated in our laboratory. A direct comparison showed that the expression of the plus allele was c. sixfold higher than that of the minus allele. In order to determine sequences of the Ty responsible for this effect, we constructed a number of variants in which distinct segments of the Ty were deleted or rearranged. The activating ability was localized to a 590-bp segment of the Ty containing the 'promoter delta', independent of its orientation or location towards the tRNA gene thus resembling the effects described for transcriptional enhancers. This is the first time that a long-range effect has been observed on the expression of a gene transcribed by RNA polymerase III.
Assuntos
Elementos Facilitadores Genéticos , Genes Reguladores , RNA Fúngico/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Animais , Elementos de DNA Transponíveis , Microinjeções , Oócitos , Plasmídeos , Transcrição Gênica , XenopusRESUMO
We have extended the catalogue of yeast tRNA genes that are found associated with repetitive (transposable) elements. We determined the nucleotide sequences of loci containing the genes for a tRNAGln and a tRNASer2 (pY66), a tRNAGlu3 (pY80), and a tRNALys1 (pY109). Our analyses revealed that complex patterns exist in which different types of elements (Ty, delta, sigma, and tau) are involved. We could further demonstrate that in several there are alleles of which one contains a particular element and the other lacks it; such differences are also found when comparing hybridization patterns of DNA from a diploid and a haploid yeast strain. In order to investigate a possible functional role of the elements in conjunction with the tRNA genes, we compared the transcriptional activities of several tRNA genes by microinjection into Xenopus oocyte nuclei. The observed differences in expression may be attributed to the presence or absence of different elements in the vicinity of the tRNA genes.
