RESUMO
Interferon-stimulated gene 15 (ISG15) encodes a protein that is most upregulated by type I interferon stimulation and, upon activation, is conjugated to various target proteins in a process known as ISGylation. ISGylation has been shown to have roles in various biological phenomena such as viral infection and cancer. To gain further insight into the function of ISGylation, it would be useful to be able to identify ISGylated proteins. Here, we describe a method for the identification of proteins modified with ISG15. This method involves the generation of stable ISG15-transfectant cells, followed by affinity purification, and then identification of the ISGylated proteins by mass spectrometry.
Assuntos
Citocinas/metabolismo , Ubiquitinas/metabolismo , Animais , Citocinas/química , Citocinas/genética , Citocinas/isolamento & purificação , Humanos , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Proteínas Recombinantes de Fusão , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/isolamento & purificaçãoRESUMO
Senescent cells exhibit dramatic changes in protein post-translational modifications. Here, we describe a method, stable isotope labeling with amino acids in cell culture (SILAC) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS), to identify changes in the ubiquitinome in cells that have undergone oncogene-induced senescence.
Assuntos
Senescência Celular , Oncogenes/genética , Proteoma , Proteômica , Ubiquitinas/metabolismo , Linhagem Celular , Senescência Celular/genética , Cromatografia de Afinidade , Cromatografia Líquida/métodos , Fibroblastos/metabolismo , Genes ras , Humanos , Marcação por Isótopo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Ubiquitinas/isolamento & purificaçãoRESUMO
Cancer/testis (CT) antigens are considered promising target molecules for immunotherapy. To efficiently identify potential CT antigens, a testis cDNA library was immunoscreened with sera from hepatocellular carcinoma (HCC) patients. We isolated 3 different antigens, AKAP3, CTp11, and UBQLN3. Although AKAP3 and CTp11 have been previously reported as CT antigens, this is the first time that these 2 antigens have been isolated from HCC patients by SEREX. Conventional RT-PCR analysis showed that AKAP3 was frequently present in HCC cell lines (5/7) and HCC tissues (5/10), and the gene was broadly expressed in several cancer types, including breast cancer cell lines (3/6), breast cancer tissues (6/9), colon cancer cell lines (3/10), colon cancer tissues (5/6), ovary cancer cell lines (6/8), ovary cancer tissues (11/16), lung cancer cell lines (4/7) and lung cancer tissues (6/13). By phage plaque analysis, anti-AKAP3 antibody was detected in sera from 15 of 27 HCC patients and 8 of 27 healthy donors. These data suggest that AKAP3 may be useful for diagnosis and immunotherapy in HCC patients.
Assuntos
Proteínas de Ancoragem à Quinase A/imunologia , Antígenos de Neoplasias/sangue , Carcinoma Hepatocelular/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Ubiquitinas/imunologia , Proteínas de Ancoragem à Quinase A/sangue , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/isolamento & purificação , Idoso , Antígenos de Neoplasias/imunologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Masculino , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Neoplasias/metabolismo , RNA Mensageiro/genética , Ubiquitinas/sangue , Ubiquitinas/genética , Ubiquitinas/isolamento & purificaçãoRESUMO
The protein modifier ubiquitin is a signal for proteasome-mediated degradation in eukaryotes. Proteasome-bearing prokaryotes have been thought to degrade proteins via a ubiquitin-independent pathway. We have identified a prokaryotic ubiquitin-like protein, Pup (Rv2111c), which was specifically conjugated to proteasome substrates in the pathogen Mycobacterium tuberculosis. Pupylation occurred on lysines and required proteasome accessory factor A (PafA). In a pafA mutant, pupylated proteins were absent and substrates accumulated, thereby connecting pupylation with degradation. Although analogous to ubiquitylation, pupylation appears to proceed by a different chemistry. Thus, like eukaryotes, bacteria may use a small-protein modifier to control protein stability.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismo , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Glicina/metabolismo , Lisina/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/isolamento & purificaçãoRESUMO
The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.
Assuntos
Polímeros/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sequência de Aminoácidos , Extratos Celulares , Núcleo Celular/metabolismo , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Proteína SUMO-1/química , Proteína SUMO-1/isolamento & purificação , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/química , Ubiquitinas/isolamento & purificação , Ubiquitinas/metabolismoRESUMO
Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14kDa) and ubiquitinated H2A (23kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A(1-87) and 12-kDa H2A(1-114), suggesting that both were produced by limited proteolysis of intact H2A(1-129). The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A(1-87) in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A(1-114) was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) but not with carbobenzoxy-l-isoleucyl-gamma-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.
Assuntos
Anticorpos/química , Cromatina/química , Histonas/química , Histonas/isolamento & purificação , Monócitos/química , Ubiquitinas/química , Ubiquitinas/isolamento & purificação , Anticorpos/imunologia , Antineoplásicos/farmacologia , Carcinógenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , Histonas/biossíntese , Histonas/imunologia , Humanos , Leupeptinas , Monócitos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Ubiquitinas/biossíntese , Ubiquitinas/imunologiaRESUMO
Ubiquitin-like proteins modify target proteins, altering their activities or causing them to be slated for degradation. These modifications are used to efficiently regulate key events in the cell. To explore the set of proteins modified by a small ubiquitin-like protein, we have developed a proteomic approach. Affinity purification of an epitope-tagged Nedd8 allowed the identification of the majority of proteins known to be involved with the neddylation pathway. This purification not only isolated the known targets of neddylation but also the constellation of enzymes and complexes known to regulate neddylation and deneddylation, including the COP9 signalosome, Nub1, and enzymes in the neddylation cascade. This purification scheme can be applied to other small ubiquitin-like proteins, especially those with limited protein targets such as the SUMOs (1, 2, and 3), Isg15, or FAT10.
Assuntos
Peptídeos/análise , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Complexo do Signalossomo COP9 , Células Cultivadas , Cromatografia de Afinidade , Proteínas Culina/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Proteína NEDD8 , Oligopeptídeos , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Especificidade por Substrato , Ubiquitinas/análise , Ubiquitinas/isolamento & purificaçãoRESUMO
Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.
Assuntos
Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Cisteína/metabolismo , Humanos , Hidrólise , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Rotação , Alinhamento de Sequência , Especificidade por Substrato , Ubiquitina/análogos & derivados , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitinas/química , Ubiquitinas/isolamento & purificaçãoRESUMO
A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.
Assuntos
Agaricales/química , Ribonucleases/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/farmacologia , Animais , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Temperatura Alta , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Peptídeos/farmacologia , Ribonucleases/imunologia , Ribonucleases/isolamento & purificação , Ubiquitinas/isolamento & purificaçãoRESUMO
To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.
Assuntos
Núcleo Celular/química , Histonas/isolamento & purificação , Histonas/metabolismo , Leucemia/patologia , Ubiquitinas/isolamento & purificação , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Extratos Celulares , Linhagem Celular , Cromatina/metabolismo , Histonas/química , Histonas/imunologia , Humanos , Soros Imunes/imunologia , Immunoblotting , Masculino , Dados de Sequência Molecular , Frações Subcelulares , Ubiquitinas/química , Ubiquitinas/imunologiaRESUMO
The chemokine receptor CXCR4 is a primary coreceptor for the HIV-1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T-lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, antiubiquitin antibodies reacted with the 62-kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101-kDa CXCR4 isoform in immunoblots by three- to sevenfold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV-1 envelope-expressing cells. Similar increases in the intensity of the 101-kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.
Assuntos
Acetilcisteína/análogos & derivados , Receptores CXCR4/genética , Receptores CXCR4/isolamento & purificação , Acetilcisteína/farmacologia , Heterogeneidade Genética , Humanos , Células Jurkat , Linfócitos/química , Macrófagos/química , Peso Molecular , Monócitos/química , Testes de Precipitina , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Transporte Proteico , Receptores CXCR4/metabolismo , Ubiquitinas/isolamento & purificaçãoRESUMO
NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to cullin family members. Recently, we identified a negative regulator of the NEDD8 conjugation system, NUB1, which interacts with NEDD8 and down-regulates NEDD8 expression post-transcriptionally (Kito, K., Yeh, E. T. H., and Kamitani, T. (2001) J. Biol. Chem. 276, 20603-20609). Here, we show that NUB1 possesses a ubiquitin-like domain at the N-terminal region and binds to S5a of the 19 S proteasome activator (PA700). A GST pull-down assay revealed that the overexpression of NUB1 leads to a greater precipitation of NEDD8 conjugates with GST-S5a, suggesting that NUB1 might have an adaptor function between S5a and NEDD8. Furthermore, proteasome inhibitors completely block NUB1-mediated down-regulation of NEDD8 expression. These results suggest that NUB1 recruits NEDD8 and its conjugates to the proteasome for degradation, providing a direct functional link between the NEDD8 conjugation system and the proteasomal degradation pathway.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Células COS , Glutationa Transferase/metabolismo , Humanos , Hidrólise , Proteína NEDD8 , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Fatores de Transcrição/isolamento & purificação , Células Tumorais Cultivadas , Ubiquitinas/isolamento & purificaçãoRESUMO
A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (>30 kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin-UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin-UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein-ubiquitin complexes.
Assuntos
Leucemia Eritroblástica Aguda/enzimologia , Ligases/isolamento & purificação , Ubiquitinas/isolamento & purificação , Sequência de Aminoácidos , Cromatografia de Afinidade , Ésteres/isolamento & purificação , Humanos , Células K562 , Dados de Sequência Molecular , Análise de Sequência de Proteína , Compostos de Sulfidrila/isolamento & purificação , Enzimas de Conjugação de UbiquitinaRESUMO
It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of (125)I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme.
Assuntos
Óxido Nítrico Sintase/metabolismo , Ubiquitinas/metabolismo , Animais , Encéfalo/enzimologia , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Citosol/enzimologia , Humanos , Rim , Complexos Multienzimáticos/metabolismo , Óxido Nítrico Sintase/isolamento & purificação , Óxido Nítrico Sintase Tipo I , Complexo de Endopeptidases do Proteassoma , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , Transfecção , Ubiquitinas/isolamento & purificaçãoRESUMO
We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Genes Supressores de Tumor , Ligases , Proteínas/genética , Proteínas/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismo , Doença de von Hippel-Lindau/genética , Substituição de Aminoácidos , Anticorpos , Anticorpos Monoclonais , Sítios de Ligação , Proteínas de Transporte/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Humanos , Mutagênese Sítio-Dirigida , Proteína NEDD8 , Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Ubiquitinas/isolamento & purificação , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Alcoholic extracts of bakers' yeast (Saccharomyces cerevisiae) have been used for over 60 years in over-the-counter medications for the treatment of hemorrhoids, burns, and wounds. Although previous studies suggested that small peptides were responsible for the medical observations, the peptides were never resolved into separate fractions and identified. In the present report, a protein fraction was prepared by RPC18 chromatography of the extract which enhances wound closure in both diabetic and non-diabetic littermates. The peptides are active in nanomolar amounts and are 600 times more active than the initial extract. SDS-PAGE and N-terminal amino acid sequencing identified 4 polypeptides in the extract. Three of the proteins were small molecular weight stress-associated proteins: copper, zinc superoxide-dismutase, ubiquitin, and glucose lipid regulated protein (HSP 12). The fourth protein, acyl-CoA binding protein II, has not been previously associated with stress proteins.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Saccharomyces cerevisiae , Sequência de Aminoácidos , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/farmacologia , Proteínas de Transporte/uso terapêutico , Inibidor da Ligação a Diazepam , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/uso terapêutico , Proteínas Fúngicas/farmacologia , Proteínas Fúngicas/uso terapêutico , Proteínas de Choque Térmico/isolamento & purificação , Proteínas de Choque Térmico/farmacologia , Proteínas de Choque Térmico/uso terapêutico , Humanos , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae , Superóxido Dismutase/isolamento & purificação , Superóxido Dismutase/farmacologia , Superóxido Dismutase/uso terapêutico , Ubiquitinas/isolamento & purificação , Ubiquitinas/farmacologia , Ubiquitinas/uso terapêutico , Ferimentos e Lesões/tratamento farmacológicoRESUMO
Challenging tomato or tobacco varieties with ethylene-inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death. Using the yeast two-hybrid system, we isolated a novel protein, tomato small ubiquitin-related modifier protein (T-SUMO), which specifically interacts with EIX. T-SUMO, a cytoplasmic protein, is a member of the ubiquitin-like protein family. It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor-induced cell death. Transgenic plants that express T-SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX-induced ethylene biosynthesis. These results indicate that T-SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses.
Assuntos
Morte Celular , Etilenos/biossíntese , Solanum lycopersicum/genética , Ubiquitinas/isolamento & purificação , Ubiquitinas/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Mapeamento Cromossômico , Humanos , Solanum lycopersicum/química , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Ubiquitinas/química , Xilano Endo-1,3-beta-XilosidaseRESUMO
The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Ligases , NF-kappa B/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina , Ubiquitinas/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Células HeLa , Humanos , Mutação , Inibidor de NF-kappaB alfa , Fosforilação , Sistemas do Segundo Mensageiro , Especificidade da Espécie , Ubiquitinas/genética , Ubiquitinas/fisiologiaRESUMO
Using a tobacco ubiquitin cDNA clone as a probe, a genomic clone in EMBL3 coding for a tobacco polyubiquitin protein was isolated. Southern blot hybridization of the genomic clone with the cDNA clone identified a BamHI/EcoRI fragment of 2.5 kb to contain the coding region of polyubiquitin, and thus the fragment was subcloned into a plasmid vector. Nucleotide sequence determination of the clone identified an open reading frame for the four head-to-tail repeats of ubiquitin monomer of 76 amino acids interrupted by an intron sequence of 55 nucleotides. The four ubiquitin units were completely conserved except for the extra glutamine at the carboxy terminus of the last ubiquitin monomer. At the 5'-region upstream of the open reading frame, a sequence of 630 nucleotides was determined. In this region, well-known regulatory sequences such as the CCAAT box, TATA box and heat-shock elements could not be located; instead, a region very rich in C and T and repeats of CA was noticed. In the 3'-downstream region of the open reading frame, a sequence of 474 nucleotides was determined which contained putative polyadenylation signals and a GU-rich region.
Assuntos
Biopolímeros/genética , Genes de Plantas , Nicotiana/genética , Plantas Tóxicas , Ubiquitinas/genética , Sequência de Aminoácidos , Sequência de Bases , Biopolímeros/química , Biopolímeros/isolamento & purificação , Clonagem Molecular , Éxons , Íntrons , Dados de Sequência Molecular , Poliubiquitina , Análise de Sequência de DNA , Ubiquitinas/química , Ubiquitinas/isolamento & purificaçãoRESUMO
We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.