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1.
Biochemistry ; 54(31): 4863-76, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26174911

RESUMO

Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.


Assuntos
Bacteriocinas/química , Enterococcus faecium/química , Bacteriocinas/biossíntese , Bacteriocinas/genética , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
2.
Insect Biochem Mol Biol ; 41(11): 902-8, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21914479

RESUMO

Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9.


Assuntos
Bombyx/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Bombyx/química , Bombyx/genética , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Histonas/química , Histonas/isolamento & purificação , Espectrometria de Massas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Plant Sci ; 181(2): 125-31, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21683877

RESUMO

The esp1 mutant CM21 specifically exhibits reduced levels of cysteine-poor (CysP) prolamin bands with pIs of 6.65, 6.95, 7.10, and 7.35 in rice seed. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that the bands with pIs 6.65, 6.95, and 7.35 are encoded by different structural genes. These results suggest that the Esp1 locus encodes a regulatory factor involved in the synthesis and/or accumulation of CysP prolamin molecules. Isoelectric focusing (IEF) analysis of CysP prolamins in chromosome substitution lines showed that structural genes for bands with pI values of 6.95, 7.10, and 7.35, which are reduced in esp1 mutant lines, are located as a gene cluster in the 44.2 cM region on chromosome 5.


Assuntos
Cisteína , Oryza/genética , Proteínas de Plantas/metabolismo , Prolaminas/genética , Cromossomos de Plantas/genética , Cisteína/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Endosperma/química , Endosperma/genética , Endosperma/metabolismo , Focalização Isoelétrica , Família Multigênica , Mutação , Oryza/química , Oryza/metabolismo , Proteínas de Plantas/genética , Prolaminas/química , Prolaminas/metabolismo , RNA de Plantas/genética , Sementes/química , Sementes/genética , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
4.
Acta Histochem ; 112(6): 519-28, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19628254

RESUMO

Immunohistochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant "staining pattern". Without this latter step, each of these contradictory results would have been a priori "technically acceptable" and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.


Assuntos
Biomarcadores Tumorais/análise , Proteína de Capeamento de Actina CapZ/análise , Imuno-Histoquímica/métodos , Imuno-Histoquímica/normas , Proteínas Adaptadoras de Transdução de Sinal/análise , Reações Antígeno-Anticorpo , Neoplasias da Mama/química , Carboxiliases/análise , Carboxiliases/metabolismo , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/metabolismo , Humanos , Inclusão em Parafina , Proteínas de Ligação a RNA/análise , Reprodutibilidade dos Testes , Fixação de Tecidos , Transcetolase/análise , Transcetolase/metabolismo , Células Tumorais Cultivadas
5.
J Biosci Bioeng ; 108(6): 460-4, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19914576

RESUMO

NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacteriocinas/biossíntese , Cisteína Proteases/metabolismo , Sinais Direcionadores de Proteínas , Staphylococcus/enzimologia , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Transporte Biológico/genética , Sequência Conservada , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Homologia de Sequência de Aminoácidos , Staphylococcus/genética , Staphylococcus/metabolismo , Especificidade por Substrato
6.
Org Biomol Chem ; 7(17): 3407-12, 2009 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-19675894

RESUMO

Novel small substrates with a variety of fluorophores were designed for the covalent labeling of proteins catalyzed by microbial transglutaminase (MTG). The new design is based on the flexibility in the substrate recognition of MTG for the substitution of the N-terminal protecting group of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG). Here we report for the first time that MTG can accept diverse fluorophores (dansyl, fluorescein, and rhodamine derivatives) in place of the benzyloxycarbonyl moiety when linked via a beta-alanine or epsilon-aminocaproic acid linker. The utility of the new fluorescent substrates was demonstrated by site-specific, covalent and quantitative labeling of an MTG-reactive Lys-containing peptide tag fused to the N-terminus of a recombinant bacterial alkaline phosphatase with retention of target protein functionality.


Assuntos
Corantes Fluorescentes/síntese química , Proteínas/química , Transglutaminases/metabolismo , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias , Catálise , Reagentes de Ligações Cruzadas , Dipeptídeos , Especificidade por Substrato
7.
Nutr Neurosci ; 12(4): 155-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19622239

RESUMO

Conjugated linoleic acid (CLA) has a role of biogenic regulation through modifying prostaglandin production. However, its effects on related metabolites of arachidonate remain unclear. Therefore, the effects of CLA on brain endocannabinoid content as well as its analogs were investigated. Mice (3-week-old), provided with diets containing 3% linoleic acid or 3% CLA for 4 weeks, were sacrificed and lipids were extracted from their cerebral cortex and hypothalamus. The amounts of N-arachidonoyl-ethanolamide, 2-arachidonoyl-glycerol (2-AG), oleoyl-ethanolamide and palmitoyl-ethanolamide were determined quantitatively by LC-MS. The 2-AG level in the cerebral cortex was significantly decreased by CLA treatment, but the other compounds were unaffected in the cerebral cortex and hypothalamus. The present study indicated that dietary CLA site-selectively decreases 2-AG in the cerebral cortex.


Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Córtex Cerebral/metabolismo , Endocanabinoides , Hipotálamo/metabolismo , Ácido Linoleico/farmacologia , Ácidos Linoleicos Conjugados/farmacologia , Administração Oral , Animais , Ácidos Araquidônicos/metabolismo , Córtex Cerebral/efeitos dos fármacos , Glicerídeos/metabolismo , Hipotálamo/efeitos dos fármacos , Ácidos Linoleicos Conjugados/administração & dosagem , Camundongos
8.
J Proteome Res ; 7(4): 1518-28, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18318472

RESUMO

Proteomic and transcriptomic platforms both play important roles in cancer research, with differing strengths and limitations. Here, we describe a proteo-transcriptomic integrative strategy for discovering novel cancer biomarkers, combining the direct visualization of differentially expressed proteins with the high-throughput scale of gene expression profiling. Using breast cancer as a case example, we generated comprehensive two-dimensional electrophoresis (2DE)/mass spectrometry (MS) proteomic maps of cancer (MCF-7 and HCC-38) and control (CCD-1059Sk) cell lines, identifying 1724 expressed protein spots representing 484 different protein species. The differentially expressed cell-line proteins were then mapped to mRNA transcript databases of cancer cell lines and primary breast tumors to identify candidate biomarkers that were concordantly expressed at the gene expression level. Of the top nine selected biomarker candidates, we reidentified ANX1, a protein previously reported to be differentially expressed in breast cancers and normal tissues, and validated three other novel candidates, CRAB, 6PGL, and CAZ2, as differentially expressed proteins by immunohistochemistry on breast tissue microarrays. In total, close to half (4/9) of our protein biomarker candidates were successfully validated. Our study thus illustrates how the systematic integration of proteomic and transcriptomic data from both cell line and primary tissue samples can prove advantageous for accelerating cancer biomarker discovery.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Perfilação da Expressão Gênica/métodos , Proteoma/análise , Proteômica/métodos , Anexina A1/análise , Anexina A1/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína de Capeamento de Actina CapZ/análise , Proteína de Capeamento de Actina CapZ/genética , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/genética , Linhagem Celular Tumoral , Biologia Computacional , Eletroforese em Gel Bidimensional , Feminino , Humanos , Proteoma/genética , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Serial de Tecidos/métodos , Cadeia B de alfa-Cristalina/análise , Cadeia B de alfa-Cristalina/genética
9.
J Proteome Res ; 6(6): 2295-303, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17497908

RESUMO

The silkworm is a typical holometabolous insect going through drastic morphological changes upon metamorphosis from larvae to pupae. Comprehensive studies focusing on the changes help elucidate understanding of a biogenic mechanism. Here, we report the initial profile of the intersegmental muscle (ISM) proteins of the silkworm during larval-pupal metamorphosis. In total, 258 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifty-seven larval proteins were identified, where 3 proteins were exclusively detected in larval samples. Fifty-four other proteins were common in pupal samples. Of these, 12 proteins belonging to the contractile apparatus, metabolism, regulation, and signal transduction were altered in their contents during the metamorphosis from larvae to pupae. Three pupa-defective proteins were identified as isoforms of troponin I, followed by an immunoblotting validation. This data will be helpful in understanding the biochemistry of an insect ISM.


Assuntos
Bombyx/crescimento & desenvolvimento , Metamorfose Biológica , Proteínas Musculares/análise , Músculo Esquelético/química , Proteômica , Animais , Bombyx/metabolismo , Eletroforese em Gel Bidimensional , Larva/química , Músculo Esquelético/crescimento & desenvolvimento , Isoformas de Proteínas/análise , Pupa/química
10.
Photosynth Res ; 93(1-3): 55-67, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17211581

RESUMO

The entire genome of the unicellular cyanobacterium Synechococcus elongatus PCC 6301 (formerly Anacystis nidulans Berkeley strain 6301) was sequenced. The genome consisted of a circular chromosome 2,696,255 bp long. A total of 2,525 potential protein-coding genes, two sets of rRNA genes, 45 tRNA genes representing 42 tRNA species, and several genes for small stable RNAs were assigned to the chromosome by similarity searches and computer predictions. The translated products of 56% of the potential protein-coding genes showed sequence similarities to experimentally identified and predicted proteins of known function, and the products of 35% of the genes showed sequence similarities to the translated products of hypothetical genes. The remaining 9% of genes lacked significant similarities to genes for predicted proteins in the public DNA databases. Some 139 genes coding for photosynthesis-related components were identified. Thirty-seven genes for two-component signal transduction systems were also identified. This is the smallest number of such genes identified in cyanobacteria, except for marine cyanobacteria, suggesting that only simple signal transduction systems are found in this strain. The gene arrangement and nucleotide sequence of Synechococcus elongatus PCC 6301 were nearly identical to those of a closely related strain Synechococcus elongatus PCC 7942, except for the presence of a 188.6 kb inversion. The sequences as well as the gene information shown in this paper are available in the Web database, CYORF (http://www.cyano.genome.jp/).


Assuntos
Cromossomos Bacterianos/genética , Genes Bacterianos , Synechococcus/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Água Doce , Dados de Sequência Molecular , Fotossíntese/genética , Análise de Sequência de DNA , Fator sigma/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transposases/genética
11.
J Proteome Res ; 5(9): 2194-206, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16944931

RESUMO

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.


Assuntos
Proteínas/metabolismo , Proteômica/métodos , Tamoxifeno/metabolismo , Linhagem Celular Tumoral , Primers do DNA , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Humanos , Immunoblotting , Marcação por Isótopo , Espectrometria de Massas , Nitrobenzenos , Proteínas/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia
12.
Peptides ; 27(1): 122-30, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16043263

RESUMO

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.


Assuntos
Química Encefálica , Neuropeptídeos/isolamento & purificação , Peptídeos Opioides/líquido cefalorraquidiano , Peptídeos Opioides/isolamento & purificação , Precursores de Proteínas/líquido cefalorraquidiano , Precursores de Proteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Humanos , Metionina/química , Dados de Sequência Molecular , Neuropeptídeos/líquido cefalorraquidiano , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Peptídeos Opioides/antagonistas & inibidores , Peptídeos Opioides/metabolismo , Peptídeos Opioides/fisiologia , Dor/metabolismo , Dor/fisiopatologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/fisiologia , Precursores de Proteínas/metabolismo , Radioimunoensaio , Receptores Opioides/isolamento & purificação , Receptores Opioides/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nociceptina
13.
J Bacteriol ; 187(12): 4276-85, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15937190

RESUMO

Natural isolates of pathogenic bacteria can exhibit a broad range of phenotypic traits. To investigate the molecular mechanisms contributing to such phenotypic variability, we compared the genomes, transcriptomes, and proteomes of two natural isolates of the gram-negative bacterium Burkholderia pseudomallei, the causative agent of the human disease melioidosis. Significant intrinsic genomic, transcriptional, and proteomic variations were observed between the two strains involving genes of diverse functions. We identified 16 strain-specific regions in the B. pseudomallei K96243 reference genome, and for eight regions their differential presence could be ascribed to either DNA acquisition or loss. A remarkable 43% of the transcriptional differences between the strains could be attributed to genes that were differentially present between K96243 and Bp15682, demonstrating the importance of lateral gene transfer or gene loss events in contributing to pathogen diversity at the gene expression level. Proteins expressed in a strain-specific manner were similarly correlated at the gene expression level, but up to 38% of the global proteomic variation between strains comprised proteins expressed in both strains but associated with strain-specific protein isoforms. Collectively, >65 hypothetical genes were transcriptionally or proteomically expressed, supporting their bona fide biological presence. Our results provide, for the first time, an integrated framework for classifying the repertoire of natural variations existing at distinct molecular levels for an important human pathogen.


Assuntos
Burkholderia pseudomallei/genética , Perfilação da Expressão Gênica , Variação Genética , Transcrição Gênica , Proteínas de Bactérias/genética , Genoma Bacteriano , Fenótipo , Isoformas de Proteínas/genética , Proteoma , Especificidade da Espécie
14.
DNA Res ; 10(1): 49-57, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12693554

RESUMO

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.


Assuntos
Perfilação da Expressão Gênica , Baço/metabolismo , Adulto , Clonagem Molecular , DNA Complementar , Humanos , Análise de Sequência de DNA
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