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1.
Ann Rheum Dis ; 79(9): 1194-1202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32532752

RESUMO

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.


Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Infecções por Bacteroidaceae/imunologia , Epitopos/imunologia , Porphyromonas gingivalis/imunologia , Artrite Reumatoide/microbiologia , Infecções por Bacteroidaceae/microbiologia , Citrulinação/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Peptídeos/imunologia , Desiminases de Arginina em Proteínas/imunologia
2.
Sci Rep ; 9(1): 4366, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867451

RESUMO

Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.


Assuntos
Cátions/farmacologia , Ouro/farmacologia , Nanopartículas Metálicas , Mitocôndrias/efeitos dos fármacos , Compostos de Amônio/química , Autofagia/efeitos dos fármacos , Cátions/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fenômenos Químicos , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Ouro/química , Humanos , Redes e Vias Metabólicas , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Proteoma , Proteômica/métodos , Transcriptoma
3.
J Am Soc Mass Spectrom ; 24(7): 1037-44, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23633018

RESUMO

Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Dissulfetos/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Ranidae/genética , Ranidae/fisiologia , Análise de Sequência de Proteína , Pele/química , Pele/metabolismo
4.
J Biol Chem ; 279(6): 4768-81, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14593120

RESUMO

Tetradecameric Clp protease core complexes in non-photosynthetic plastids of roots, flower petals, and in chloroplasts of leaves of Arabidopsis thaliana were purified based on native mass and isoelectric point and identified by mass spectrometry. The stoichiometry between the subunits was determined. The protease complex consisted of one to three copies of five different serine-type protease Clp proteins (ClpP1,3-6) and four non-proteolytic ClpR proteins (ClpR1-4). Three-dimensional homology modeling showed that the ClpP/R proteins fit well together in a tetradecameric complex and also indicated unique contributions for each protein. Lateral exit gates for proteolysis products are proposed. In addition, ClpS1,2, unique to land plants, tightly interacted with this core complex, with one copy of each per complex. The three-dimensional modeling show that they do fit well on the axial sites of the ClpPR cores. In contrast to plastids, plant mitochondria contained a single approximately 320-kDa homo-tetradecameric ClpP2 complex, without association of ClpR or ClpS proteins. It is surprising that the Clp core composition appears identical in all three plastid types, despite the remarkable differences in plastid proteome composition. This suggests that regulation of plastid proteolysis by the Clp machinery is not through differential regulation of ClpP/R/S gene expression, but rather through substrate recognition mechanisms and regulated interaction of chaperone-like molecules (ClpS1,2 and others) to the ClpP/R core.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Plantas/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Endopeptidase Clp , Genes de Plantas , Focalização Isoelétrica , Substâncias Macromoleculares , Mitocôndrias/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Fotossíntese , Plantas/genética , Plastídeos/enzimologia , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética
5.
Nucleic Acids Res ; 31(4): 1174-9, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12582236

RESUMO

Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.


Assuntos
Cloroplastos/genética , Nicotiana/genética , Fragmentos de Peptídeos/genética , Toxina Tetânica/genética , Animais , Northern Blotting , Western Blotting , Expressão Gênica , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Extratos Vegetais/imunologia , Folhas de Planta/química , Plantas Geneticamente Modificadas , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Toxina Tetânica/imunologia , Toxina Tetânica/metabolismo
6.
J Biol Chem ; 277(44): 41931-9, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12138166

RESUMO

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.


Assuntos
Metaloendopeptidases/isolamento & purificação , Mitocôndrias/enzimologia , Proteínas Mitocondriais/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Precursores de Proteínas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Sequência de Aminoácidos , Espectrometria de Massas , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/química
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