RESUMEN
NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.
Asunto(s)
Proteínas I-kappa B , Ligasas/metabolismo , Péptido Sintasas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitinas/fisiología , Ciclosoma-Complejo Promotor de la Anafase , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Ligasas/genética , Proteína NEDD8 , Inhibidor NF-kappaB alfa , Proteínas Ligasas SKP Cullina F-box , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismoRESUMEN
A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.
Asunto(s)
Péptido Sintasas/metabolismo , Schizosaccharomyces/enzimología , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Proteínas Ligasas SKP Cullina F-box , Homología de Secuencia de Aminoácido , Ubiquitinas/químicaRESUMEN
Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/genética , Regulación de la Expresión Génica , Humanos , Proteína NEDD8 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitinas/genéticaAsunto(s)
Complejos de Ubiquitina-Proteína Ligasa , Ubiquitina-Proteína Ligasas , Ubiquitinas , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas de Ciclo Celular/metabolismo , Elonguina , Humanos , Ligasas/fisiología , Datos de Secuencia Molecular , Proteína NEDD8 , Proteínas/fisiología , Proteína SUMO-1 , Factores de Transcripción/fisiología , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas/fisiologíaRESUMEN
NEDD8 is a ubiquitin (Ub)-like protein. Here we report a novel ubiquitinylation-related pathway for modification by NEDD8. NEDD8 was activated by an E1 (Ub-activating enzyme)-like complex, consisting of APP-BP1 and hUba3 with high respective homologies to the amino- and carboxy-terminal regions of E1 and then linked to hUbc12 (a human homolog of yeast Ub-conjugating enzyme Ubc12p). The major target protein modified by NEDD8 was found to be Hs-cullin-4A (Cul-4A), a member of the family of human cullin/Cdc53 proteins functioning as an essential component of a multifunctional Ub-protein ligase E3 complex that has a critical role in Ub-mediated proteolysis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Técnicas In Vitro , Ligasas/genética , Ligasas/metabolismo , Datos de Secuencia Molecular , Proteína NEDD8 , Conejos , Homología de Secuencia de Aminoácido , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína Ligasas , Ubiquitinas/genéticaRESUMEN
Many mites were found on bookshelves near the windows in 2 rooms of the Department of Pediatrics on the 4th floor in the Tokai University Hospital. Fortunately, nobody reported being bitten by, or experiencing itching due to, the mites. The arthropods were tentatively identified as adults of the northern fowl mite, Ornithonyssus sylviarum (Canestrini et Frazago, 1877), commonly called "Torisashi-dani" in Japanese. It appears the mites came from an empty wagtail nest which was outside the windows. Since migrating birds like the wagtail may carry O. sylviarum and other pests and pathogens, and their possible transfer to domestic birds, more attention should be paid to the danger posed by bird mites.
Asunto(s)
Hospitales Universitarios , Ácaros , Habitaciones de Pacientes , Animales , Humanos , Ácaros/anatomía & histología , Ácaros/clasificaciónRESUMEN
A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.
Asunto(s)
Anafase , Ligasas/metabolismo , Mitosis , Proteínas Nucleares , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Anafase/genética , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ciclina B , Ciclinas/genética , Ciclinas/metabolismo , ADN Complementario/química , ADN Complementario/aislamiento & purificación , ADN de Hongos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G2 , Ligasas/genética , Sustancias Macromoleculares , Mitosis/genética , Datos de Secuencia Molecular , Mutagénesis , Proteínas Quinasas/metabolismo , Mapeo Restrictivo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Temperatura , Ubiquitinas/metabolismoRESUMEN
The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Apoptosis/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Ubiquitinas/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos de Neoplasias , Extractos Celulares , Cisteína Endopeptidasas/efectos de los fármacos , ADN Complementario , Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Genes bcl-2 , Humanos , Hidrólisis , Complejos Multienzimáticos/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal , Transfección , Células Tumorales CultivadasRESUMEN
cDNA encoding a novel human ubiquitin-conjugating enzyme has been cloned from an epidermoid carcinoma KB cDNA library. This clone encodes a protein of 152 amino acids with a calculated M(r) of 17,137. The amino acid sequence showed 80% identity with the Drosophila's bendless gene product (ubiquitin-conjugating enzyme E2). The corresponding transcripts are highly expressed in heart, skeletal muscle, and testis. The product expressed in Escherichia coli exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin-activating enzyme E1-dependent manner. These results suggest that the obtained cDNA encodes a novel human E2 which may be involved in protein degradation mainly in the muscles and testis.
Asunto(s)
Proteínas de Drosophila , Drosophila/enzimología , Expresión Génica , Ligasas/biosíntesis , Ligasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Drosophila/genética , Escherichia coli , Femenino , Humanos , Células KB , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Miocardio/enzimología , Especificidad de Órganos , Mapeo Restrictivo , Testículo/enzimología , Transcripción Genética , Enzimas Ubiquitina-ConjugadorasRESUMEN
Eukaryotic proteasomes from an evolutionarily conserved multi-gene family and are thought to have originated from a common ancestral gene and diverged into alpha-type and beta-type subgroups. To understand the molecular basis of the proteasome genes, we isolated and characterized two human proteasome genes econding the alpha-type HC3 and beta-type HC5 subunit. The functional genes for HC3 and HC5 are similar in being approximately 15 kb in length, but differ in having exon numbers of 9 and 6, respectively. Analyses of about 2.5 to 3.0 kb of the 5'-flanking regions of these two genes revealed the absence of TATA and CAAT promoter elements. However, two or three GC boxes were found. By analysis of the transcriptional regulatory activities in the 5'-flanking regions of the two genes, these GC boxes were found to function coordinately as promoters of the two genes. Interestingly, the HC3 gene possesses an additional silencer element in the 5'-upstream region near the first exon. This element is also able to repress the promoter activities of other genes, such as the HC5 and the type 1 glucose transporter genes, irrespective of whether it has a sense or antisense orientation, indicating that it acts as a general transcriptional silencer. The HC5 gene does not have this silence element, and its promoter activity is five to ten times that of HC3. These results show that the human proteasomal HC3 and HC5 genes differ not only in their genomic structures, such as their numbers of exons and their exon-intron organizations, but also in the mechanisms regulating their transcription, suggesting that they diverged at an early stage of evolution.
Asunto(s)
Cisteína Endopeptidasas/genética , Regulación Enzimológica de la Expresión Génica , Complejos Multienzimáticos/genética , Familia de Multigenes/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Cisteína Endopeptidasas/biosíntesis , Drosophila/genética , Genoma Humano , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
In order to learn the biological effect of photochemical oxidants on living bodies, we exposed newborn and adult rats, of both sexes, to ozone at a concentration of 0.25 ppm, which can be encountered in an urban environment, and then measured the osmotic resistance of their erythrocytes. The results of experiments using newborn rats indicated a positive increase in the osmotic resistance of erythrocytes in whole blood following ozone exposure for 4 weeks. An increase in the osmotic resistance of erythrocytes in the top part obtained by centrifugation was observed following ozone exposure for 12 weeks. This tendency was especially evident among male rats. On the other hand, no increase in the osmotic resistance of erythrocytes was recognized in the adult animals which had been exposed to the same concentration of ozone for 18 months.
Asunto(s)
Membrana Eritrocítica/efectos de los fármacos , Ozono/farmacología , Envejecimiento , Animales , Animales Recién Nacidos , Femenino , Hemólisis/efectos de los fármacos , Masculino , Ósmosis , Ratas , Ratas Sprague-Dawley , Factores SexualesRESUMEN
The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.
Asunto(s)
Precursores Enzimáticos/genética , Mitocondrias/enzimología , Procesamiento Proteico-Postraduccional , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Clonación Molecular , ADN/genética , Sondas de ADN , Biblioteca de Genes , Humanos , Riñón/enzimología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Conformación Proteica , Señales de Clasificación de Proteína/genética , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat factor 6 as a probe. The sequence was composed of 466 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions on the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame were found to consist of 108 and 76 amino acid residues with molecular weights of 12,596 and 8,969, respectively. The presequence of 32 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN/genética , Precursores Enzimáticos/genética , ATPasas de Translocación de Protón Mitocondriales , Factores de Acoplamiento de la Fosforilación Oxidativa , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN/aislamiento & purificación , Sondas de ADN , Biblioteca de Genes , Humanos , Riñón/enzimología , Mitocondrias , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , Conformación Proteica , ATPasas de Translocación de Protón/genética , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components. Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes. The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes. The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low. These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.
Asunto(s)
Cisteína Endopeptidasas/genética , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Clonación Molecular , Cisteína Endopeptidasas/química , Drosophila , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejo de la Endopetidasa Proteasomal , Ratas , Saccharomyces cerevisiae , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN/genética , Precursores Enzimáticos/genética , Mitocondrias Hepáticas/enzimología , ATPasas de Translocación de Protón Mitocondriales , Factores de Acoplamiento de la Fosforilación Oxidativa , ATPasas de Translocación de Protón/genética , Adenosina Trifosfatasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular/métodos , Biblioteca de Genes , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Conformación Proteica , Señales de Clasificación de Proteína/genética , ATPasas de Translocación de Protón/aislamiento & purificación , Ratas , Mapeo RestrictivoRESUMEN
The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.
Asunto(s)
ADN/análisis , Biblioteca de Genes , Mitocondrias Hepáticas/enzimología , Señales de Clasificación de Proteína/genética , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/análisis , ATPasas de Translocación de Protón/análisis , RatasAsunto(s)
Calefacción/instrumentación , Inmunoglobulina E/análisis , Niño , Femenino , Humanos , MasculinoRESUMEN
This study was conducted through regular pneumonoconiosis examination according to the law on 1,096 employees of medium and small-sized ceramic enterprises in Tokai district in 1981-82. Interview examination with BMRC questionnaire, X-ray examination and measurement of urinary hydroxyproline to creatinine ratio (HOP ratio) were carried out in order to elucidate the relationship between silicosis and urinary HOP ratio and to demonstrate the effect of smoking on pneumofibrosis. Grade of silicosis was classified into five types (0 to 4) based on the Japanese Classification of Radiographs of Pneumoconioses. In evaluating the behavior of urinary HOP ratio, when smoking factor is added in the early grade of pneumofibrosis (type 1 and type 2), collagen decomposition rate is rapidly repressed and fibroplastic conditions develop to the final grade as type 3 and 4, although smoking itself does not seem to induce pneumofibrosis. To exclude the effects of smoking, nonsmoking group was used for measurement of HOP ratio by grade. The HOP ratio in type 0 was lowest and HOP ratio increased in the order of type 1 and type 2. The turning point was found in type 2 and their HOP ratio decreased one after another. The turning point shifted from type 2 to type 3 in the case of non-smokers without any index symptoms by BMRC questionnaire and also shifted to type 1, in the case of non-smokers with them. Shifting of turning point suggests that index symptoms also promote fibroplastic activities.