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1.
Insect Biochem Mol Biol ; 43(4): 344-51, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23376333

RESUMO

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Bombyx/microbiologia , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos
2.
J Proteome Res ; 11(9): 4553-66, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22823882

RESUMO

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H2¹8O, IGOT) and finally LC-MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.


Assuntos
Bases de Dados de Proteínas , Glicoproteínas/análise , Proteoma/análise , Animais , Cromatografia Líquida , Glicômica , Glicoproteínas/sangue , Glicoproteínas/química , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Proteoma/química , Interface Usuário-Computador
3.
Exp Cell Res ; 317(20): 2853-63, 2011 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21996351

RESUMO

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.


Assuntos
Diacilglicerol Quinase/metabolismo , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Nucleossomos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteína 1 de Modelagem do Nucleossomo/genética , Nucleossomos/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transporte Proteico , Proteômica/métodos , Transdução de Sinais , Células Tumorais Cultivadas , alfa Carioferinas/metabolismo
4.
J Proteomics ; 74(10): 1985-93, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21621025

RESUMO

We performed here MS-based cell surface proteome profiling of HCT-116 cells by two distinct methods based on biotin labeling and glycoprotein capturing. In total, 742 biotinylated and 219 glycosylated proteins were identified by the biotin labeling and glycoprotein capturing, of which 224 and 138 proteins known to be located on plasma membrane were included, respectively, according to ingenuity pathway analysis. Although 104 plasma membrane proteins were identified by both methods, the rest of 154 were identified only by one. Almost all the identified plasma membrane proteins possessed consensus N-glycosylation sites, and proteins having various numbers of glycosylation sites were identified by both methods. Thus, the discrepancies of the identified proteins obtained from those two methods might not be only due to the number of glycosylation sites, but also to the expression and/or glycosylation level of the cell surface proteins. We also identified 312 N-glycosylated proteins from xenograft samples by glycoprotein capturing of which 135 were known as plasma membrane proteins. Although a number of highly-expressed plasma membrane proteins were common between culture and xenograft cells, some proteins showed culture- or xenograft-specific expression, suggesting that those proteins might contribute to grow in different environment.


Assuntos
Biotinilação/métodos , Proteínas de Membrana/análise , Proteômica/métodos , Animais , Cromatografia Líquida , Glicoproteínas/metabolismo , Glicosilação , Células HCT116 , Humanos , Camundongos , Espectrometria de Massas em Tandem , Transplante Heterólogo
5.
J Proteomics ; 74(3): 319-26, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21118733

RESUMO

We performed here MS-based phosphoproteomics using both metal oxide affinity chromatography (pSTY proteomics) and anti-phosphotyrosine antibody (pY proteomics). The former method identified mainly phospho-serine and -threonine of nuclear or cytoplasmic proteins, whereas the latter did phosphotyrosine including more plasma membrane proteins and kinases. The overlap between these two methods was limited (24 tyrosine phosphorylation sites out of 325) and, by combining the two, coverage of the signaling molecules was enhanced as exemplified by Erk signaling. We also performed whole cell proteomics using an off-gel fractionator, and found 68.9% of the proteins identified by phosphoproteomics. Thus, the expression levels of phosphoproteins were roughly estimated. In addition to many uncharacterized phosphorylation sites, the dataset includes 136 sites that were experimentally verified elsewhere to be phosphorylated by a total of 83 kinases and kinase groups out of the 256 registered in the Phospho.ELM database. With the integration of various proteomic analyses and information from database, the responsible kinases of the identified phosphorylation sites and possibly their activity status were predicted by phosphorylation status and expression levels of their substrates, and thus our method may be able to monitor the activity status of phosphorylation signaling.


Assuntos
Proteômica/métodos , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas/métodos , Metais/química , Óxidos/química , Fosforilação , Fosfotirosina/química , Proteoma , Transdução de Sinais , Tripsina/química , Tirosina/química
6.
Proc Natl Acad Sci U S A ; 106(40): 17211-6, 2009 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-19805073

RESUMO

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.


Assuntos
Biomarcadores/análise , Cones de Crescimento/metabolismo , Proteoma/análise , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Cateninas/análise , Cateninas/genética , Células Cultivadas , Cromatografia Líquida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Imuno-Histoquímica/métodos , Espectrometria de Massas , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Proteoma/genética , Interferência de RNA , Ratos , Ratos Transgênicos , Fatores de Tempo
7.
J Proteomics ; 73(2): 357-60, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19733703

RESUMO

We developed a software program (titled Precursor Ion Calibration software for LTQ or, in short, PICsL) that increases the reliability of precursor ion assignations from LC-MS analysis using ultra zoom scanning of LTQ linear ion trap MS and automatically corrects the assignations. Although existing software calculates the theoretical isotopic distribution according to m/z with a computational algorithm, our method simply searches for ions close to the theoretical mass value using both MS/MS raw data and Mascot search result files, followed by a second database search that identifies the proteins using the regenerated peak list files. Our software program mimics the manual inspection of the spectral data of precursor ions and is expected to be applicable not only for low resolution MS, such as LTQ, but also for a wide variety of MS instruments.


Assuntos
Íons/análise , Espectrometria de Massas/métodos , Software , Algoritmos , Cromatografia Líquida , Isótopos/análise
8.
J Biol Chem ; 284(40): 27360-9, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19667067

RESUMO

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.


Assuntos
Sistema do Grupo Sanguíneo Lutheran/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade , Regulação Enzimológica da Expressão Gênica , Humanos , Sistema do Grupo Sanguíneo Lutheran/análise , Sistema do Grupo Sanguíneo Lutheran/isolamento & purificação , Espectrometria de Massas , Metaloproteinase 14 da Matriz/isolamento & purificação , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/isolamento & purificação , Especificidade por Substrato
9.
Cancer Sci ; 100(7): 1284-90, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19432894

RESUMO

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1-MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1-MMP was affinity purified together with associating proteins using an anti-FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1-MMP when biotin-labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano-flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1-MMP, although most had not previously been identified. Six of these membrane proteins, including one previously shown to interact with MT1-MMP, were co-expressed with MT1-MMP in HT1080 cells. Five of the latter were found to associate with MT1-MMP in an immunoprecipitation assay. Immunostaining of cells expressing each of these test proteins revealed that one colocalized with MT1-MMP at the ruffling membrane and the other at the perinuclear vesicles. In contrast, another protein which did not coprecipitate with MT1-MMP showed no colocalization. Recombinant MT1-MMP cleaved two of the tested proteins at least in vitro. Thus, we provide a valuable resource to identify substrates and regulators of MT1-MMP in tumor cells.


Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Proteínas de Neoplasias/análise , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Melanoma/metabolismo , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Transfecção
10.
Proteomics ; 9(10): 2861-74, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19415658

RESUMO

Here, we report for the first time a comparative phosphoproteomic analysis of distinct tumor cell lines in the presence or absence of the microtubule-interfering agent nocodazole. In total, 1525 phosphorylation sites assigned to 726 phosphoproteins were identified using LC-MS-based technology following phosphopeptide enrichment. Analysis of the amino acid composition surrounding the identified in vivo phosphorylation sites revealed that they could be classified into two motif groups: pSer-Pro and pSer-Asp/Glu. Phosphoproteomic change resulting from nocodazole treatment varied among cell lines in terms of the numbers of total phosphopeptides identified, motif groups, and functional annotation groups; however, the cell lines were equally sensitive to nocodazole. The identified phosphoproteome subset contained major signaling proteins and proteins known to be involved in mitosis, but did not always exhibit the same changes in the tumor cells from nocodazole treatment. In spite of the complex changes observed in the phosphorylation of many of the proteins, possible common features induced by nocodazole were found, including phosphorylation of nucleophosmin (NPM) S254 and coatomer protein complex, subunit alpha (COPA) S173, suggesting that the events are not cell-type specific but events generally occurring in mitosis or induced by a microtubule-interfering agent. Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M-phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M-phase arrest.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/análise , Nocodazol/farmacologia , Fosfoproteínas/análise , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromatografia Líquida , Células HCT116 , Células HeLa , Humanos , Espectrometria de Massas , Mitose , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Proteômica , Transdução de Sinais , Fatores de Tempo
11.
Mol Biol Cell ; 20(11): 2639-49, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19369418

RESUMO

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Qa-SNARE/metabolismo , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Glicosilação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Lisossomos/metabolismo , Espectrometria de Massas , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/genética , Ligação Proteica , Transporte Proteico , Proteínas Q-SNARE/genética , Proteínas Q-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Interferência de RNA , Transfecção
12.
Mol Cell Proteomics ; 8(7): 1552-65, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19369196

RESUMO

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.


Assuntos
Evolução Molecular , Peptidilprolil Isomerase/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Camundongos , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Estrutura Terciária de Proteína , Proteômica/métodos , Interferência de RNA , Precursores de RNA/genética , RNA Ribossômico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Espectrometria de Massas em Tandem
13.
Biochem J ; 413(3): 505-16, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18419580

RESUMO

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.


Assuntos
RNA Polimerase II/metabolismo , RecQ Helicases/metabolismo , Transcrição Gênica , Linhagem Celular , Imunoprecipitação da Cromatina , Células HeLa , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/química , RecQ Helicases/química , RecQ Helicases/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ultracentrifugação
14.
Mol Cell Proteomics ; 6(12): 2100-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17761667

RESUMO

Protein glycosylation is one of the most common post-translational modifications in eukaryotes and affects various aspects of protein structure and function. To facilitate studies of protein glycosylation, we paired glycosylation site-specific stable isotope tagging of lectin affinity-captured N-linked glycopeptides with mass spectrometry and determined 1,465 N-glycosylated sites on 829 proteins expressed in Caenorhabditis elegans. The analysis shows the diversity of protein glycosylation in eukaryotes in terms of glycosylation sites and oligosaccharide structures attached to polypeptide chains and suggests the substrate specificity of oligosaccharyltransferase, a single multienzyme complex in C. elegans that incorporates an oligosaccharide moiety en bloc to newly synthesized polypeptides. In addition, topological analysis of 257 N-glycosylated proteins containing a putative single transmembrane segment that were identified based on the relative positions of glycosylation sites and transmembrane segments suggests that an atypical non-cotranslational mechanism translocates large N-terminal segments from the cytosol to the endoplasmic reticulum lumen in the absence of signal sequence function.


Assuntos
Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteômica , Animais , Glicosilação , Transporte Proteico
15.
J Plant Res ; 120(4): 575-9, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17558543

RESUMO

The female gamete, the egg cell, is a specially differentiated haploid cell that develops into an embryo following fertilization. In the present study, we analyzed egg cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology and identified the major proteins expressed in rice egg cells. The proteins identified included glyceraldehyde-3-phosphate dehydrogenase, ascorbate peroxidase and heat shock protein 90. The abundant existence of chaperons and antioxidant enzymes in plant egg cells indicates that the major protein components of plant egg cells are partly analogous to those of mammalian eggs and oocytes.


Assuntos
Oryza/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Oryza/citologia , Oryza/fisiologia , Espectrometria de Massas em Tandem
16.
J Proteome Res ; 4(5): 1826-31, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16212438

RESUMO

We describe the software, STEM (STrategic Extractor for Mascot's results), which efficiently processes large-scale mass spectrometry-based proteomics data. V (View)-mode evaluates the Mascot peptide identification dataset, removes unreliable candidates and redundant assignments, and integrates the results with key information in the experiment. C (Comparison)-mode compares peptide coverage among multiple datasets and displays proteins commonly/specifically found therein, and processes data for quantitative studies that utilize conventional isotope tags or tags having a smaller mass difference. STEM significantly improves throughput of proteomics study.


Assuntos
Biologia Computacional/métodos , Proteoma , Proteômica/métodos , Algoritmos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Cromatografia Líquida , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Modelos Estatísticos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/química , Software
17.
Proteomics ; 5(5): 1346-61, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15742316

RESUMO

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.


Assuntos
Proteoma/análise , Células-Tronco/fisiologia , Animais , Linhagem Celular , Embrião de Mamíferos , Regulação da Expressão Gênica , Humanos , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Análise Serial de Proteínas , Processamento de Proteína Pós-Traducional , Células-Tronco/citologia , Fatores de Transcrição/análise
18.
J Biol Chem ; 280(13): 13187-94, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15677482

RESUMO

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.


Assuntos
Proteínas 14-3-3/fisiologia , Canais de Sódio/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Catálise , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Canais Epiteliais de Sódio , Genes Dominantes , Glutationa Transferase/metabolismo , Humanos , Proteínas Imediatamente Precoces , Ubiquitina-Proteína Ligases Nedd4 , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Células PC12 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Serina/química , Coloração pela Prata , Sódio/metabolismo , Fatores de Tempo , Ubiquitina/metabolismo , Xenopus , Proteínas de Xenopus , Xenopus laevis
19.
Plant Cell Physiol ; 45(10): 1406-12, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15564524

RESUMO

In most flowering plants, the female gametophyte develops in an ovule deeply embedded in the ovary. Through double fertilization, the egg cell fuses with the sperm cell, resulting in a zygote, which develops into the embryo. In the present study, we analyzed egg cell lysates by polyacrylamide gel electrophoresis and subsequent mass spectrometry-based proteomics technology, and identified major protein components expressed in the egg cell. The identified proteins included three cytosolic enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase beta-subunit and an adenine nucleotide transporter, and annexin p35. In addition, expression levels of these proteins in the egg cell were compared with those in the early embryo, the central cell and the suspension cell. Annexin p35 was highly expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the adenine nucleotide transporter were expressed at higher levels in egg cells than in central and cultured cells. These results indicate that annexin p35 in the egg cell and zygote is involved in the exocytosis of cell wall materials, which is induced by a fertilization-triggered increase in cytosolic Ca2+ levels, and that the egg cell is rich in an enzyme subset for the energy metabolism.


Assuntos
Oócitos/química , Oócitos/metabolismo , Proteínas de Plantas/análise , Sementes/química , Sementes/metabolismo , Zea mays/metabolismo , Translocador 1 do Nucleotídeo Adenina/metabolismo , Anexinas/metabolismo , Sinalização do Cálcio/fisiologia , Parede Celular/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Citosol/metabolismo , Metabolismo Energético/genética , Exocitose/fisiologia , Gliceraldeído-3-Fosfato Desidrogenase (NADP+)(Fosforiladora)/análise , Gliceraldeído-3-Fosfato Desidrogenase (NADP+)(Fosforiladora)/metabolismo , Ácidos Glicéricos/metabolismo , Oócitos/citologia , Fosfotransferases/análise , Fosfotransferases/genética , Proteínas de Plantas/metabolismo , Proteômica/métodos , Sementes/citologia , Triose-Fosfato Isomerase/metabolismo , Zea mays/citologia , Zea mays/embriologia
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