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Pesquisa : D08.811.277.656.300.760.247 [Categoria DeCS]
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  1 / 2148 MEDLINE  
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[PMID]:29216587
[Au] Autor:Voitechovic E; Korepanov A; Kirsanov D; Legin A
[Ad] Endereço:St. Petersburg State University, St. Petersburg, Russia; Institute of Microelectronics of Barcelona, Barcelona, Spain. Electronic address: voitechovic.edita@gmail.com.
[Ti] Título:Quantification of immobilized protein in pharmaceutical production by bio-assisted potentiometric multisensor system.
[So] Source:J Pharm Biomed Anal;150:67-71, 2018 Feb 20.
[Is] ISSN:1873-264X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1-6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.
[Mh] Termos MeSH primário: Ensaio de Imunoadsorção Enzimática/métodos
Proteínas Imobilizadas/química
Potenciometria/métodos
Proteína Estafilocócica A/química
[Mh] Termos MeSH secundário: Endopeptidase K/química
Proteínas Imobilizadas/análise
Proteínas Recombinantes/análise
Proteínas Recombinantes/química
Reprodutibilidade dos Testes
Proteína Estafilocócica A/análise
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Immobilized Proteins); 0 (Recombinant Proteins); 0 (Staphylococcal Protein A); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180130
[Lr] Data última revisão:
180130
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171208
[St] Status:MEDLINE


  2 / 2148 MEDLINE  
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[PMID]:28470335
[Au] Autor:Han GG; Song AA; Kim EB; Yoon SH; Bok JD; Cho CS; Kil DY; Kang SK; Choi YJ
[Ad] Endereço:Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
[Ti] Título:Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling.
[So] Source:Appl Microbiol Biotechnol;101(13):5353-5363, 2017 Jul.
[Is] ISSN:1432-0614
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.
[Mh] Termos MeSH primário: Antibiose
Embaralhamento de DNA
Mutagênese
Pediococcus acidilactici/genética
Pediococcus acidilactici/fisiologia
Probióticos
Salmonella/fisiologia
[Mh] Termos MeSH secundário: Animais
Antibacterianos/farmacologia
Anti-Infecciosos
Bacteriocinas/biossíntese
Bacteriocinas/farmacologia
Ceco/microbiologia
Galinhas/microbiologia
Meios de Cultura/química
Endopeptidase K/metabolismo
Genoma Bacteriano
Pediococcus acidilactici/efeitos dos fármacos
Pediococcus acidilactici/efeitos da radiação
Doenças das Aves Domésticas/microbiologia
Doenças das Aves Domésticas/terapia
Probióticos/química
Salmonella/efeitos dos fármacos
Salmonelose Animal/microbiologia
Salmonelose Animal/terapia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Anti-Bacterial Agents); 0 (Anti-Infective Agents); 0 (Bacteriocins); 0 (Culture Media); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180104
[Lr] Data última revisão:
180104
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1007/s00253-017-8293-6


  3 / 2148 MEDLINE  
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[PMID]:29072706
[Au] Autor:Schopper S; Kahraman A; Leuenberger P; Feng Y; Piazza I; Müller O; Boersema PJ; Picotti P
[Ad] Endereço:Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.
[Ti] Título:Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry.
[So] Source:Nat Protoc;12(11):2391-2410, 2017 Nov.
[Is] ISSN:1750-2799
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.
[Mh] Termos MeSH primário: Proteólise
Proteoma/análise
Proteômica/métodos
Espectrometria de Massas em Tandem/métodos
[Mh] Termos MeSH secundário: Biomarcadores/análise
Misturas Complexas/química
Desenho de Drogas
Endopeptidase K/química
Ficina/química
Células HeLa
Seres Humanos
Pronase/química
Estrutura Secundária de Proteína
Estrutura Terciária de Proteína
Proteoma/química
Proteômica/instrumentação
Controle de Qualidade
Saccharomyces cerevisiae/química
Saccharomyces cerevisiae/metabolismo
Termolisina/química
Tripsina/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Biomarkers); 0 (Complex Mixtures); 0 (Proteome); EC 3.4.21.4 (Trypsin); EC 3.4.21.64 (Endopeptidase K); EC 3.4.22.3 (Ficain); EC 3.4.24.- (Pronase); EC 3.4.24.27 (Thermolysin)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171102
[Lr] Data última revisão:
171102
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171027
[St] Status:MEDLINE
[do] DOI:10.1038/nprot.2017.100


  4 / 2148 MEDLINE  
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[PMID]:28910420
[Au] Autor:Groveman BR; Raymond GJ; Campbell KJ; Race B; Raymond LD; Hughson AG; Orrú CD; Kraus A; Phillips K; Caughey B
[Ad] Endereço:Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
[Ti] Título:Role of the central lysine cluster and scrapie templating in the transmissibility of synthetic prion protein aggregates.
[So] Source:PLoS Pathog;13(9):e1006623, 2017 Sep.
[Is] ISSN:1553-7374
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90-160. Substitution of 4 lysines within residues 101-110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into "hamsterized" Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.
[Mh] Termos MeSH primário: Encéfalo/metabolismo
Lisina/metabolismo
Proteínas Priônicas/metabolismo
Scrapie/metabolismo
[Mh] Termos MeSH secundário: Amiloide/química
Animais
Encéfalo/patologia
Endopeptidase K/metabolismo
Camundongos Transgênicos
Proteínas PrPSc/metabolismo
Agregados Proteicos/fisiologia
Proteínas Recombinantes/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Amyloid); 0 (PrPSc Proteins); 0 (Prion Proteins); 0 (Protein Aggregates); 0 (Recombinant Proteins); EC 3.4.21.64 (Endopeptidase K); K3Z4F929H6 (Lysine)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171026
[Lr] Data última revisão:
171026
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170915
[St] Status:MEDLINE
[do] DOI:10.1371/journal.ppat.1006623


  5 / 2148 MEDLINE  
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[PMID]:28814578
[Au] Autor:Thackray AM; Cardova A; Wolf H; Pradl L; Vorberg I; Jackson WS; Bujdoso R
[Ad] Endereço:Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, U.K.
[Ti] Título:Genetic human prion disease modelled in PrP transgenic .
[So] Source:Biochem J;474(19):3253-3267, 2017 Sep 20.
[Is] ISSN:1470-8728
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene and accumulation of PrP , an abnormal isomer of the normal host protein PrP , in the brain of affected individuals. PrP is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Adult transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic and show that inherited human prion disease can be modelled in this invertebrate host.
[Mh] Termos MeSH primário: Drosophila melanogaster/genética
Doenças Priônicas/genética
Proteínas Priônicas/genética
[Mh] Termos MeSH secundário: Animais
Animais Geneticamente Modificados
Western Blotting
Cricetinae
Drosophila melanogaster/citologia
Drosophila melanogaster/efeitos dos fármacos
Endopeptidase K/metabolismo
Seres Humanos
Locomoção/efeitos dos fármacos
Camundongos
Microscopia Confocal
Mutação/genética
Neurotoxinas/toxicidade
Transgenes
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Neurotoxins); 0 (Prion Proteins); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170929
[Lr] Data última revisão:
170929
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170818
[St] Status:MEDLINE
[do] DOI:10.1042/BCJ20170462


  6 / 2148 MEDLINE  
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[PMID]:28765278
[Au] Autor:Dias AF; Rodrigues TA; Pedrosa AG; Barros-Barbosa A; Francisco T; Azevedo JE
[Ad] Endereço:From the Instituto de Investigação e Inovação em Saúde (i3S) and.
[Ti] Título:The peroxisomal matrix protein translocon is a large cavity-forming protein assembly into which PEX5 protein enters to release its cargo.
[So] Source:J Biol Chem;292(37):15287-15300, 2017 Sep 15.
[Is] ISSN:1083-351X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5-cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1-197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1-125)) still interacted correctly with the DTM; however, this species was largely accessible to exogenously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1-125)-DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138-639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5-DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo.
[Mh] Termos MeSH primário: Membranas Intracelulares/metabolismo
Proteínas de Membrana/metabolismo
Modelos Biológicos
Peroxissomos/metabolismo
Receptores Citoplasmáticos e Nucleares/metabolismo
Proteínas Repressoras/metabolismo
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Substituição de Aminoácidos
Transporte Biológico
Endopeptidase K/metabolismo
Deleção de Genes
Seres Humanos
Concentração de Íons de Hidrogênio
Proteínas de Membrana/química
Proteínas de Membrana/genética
Mutagênese Sítio-Dirigida
Mutação
Mutação de Sentido Incorreto
Fragmentos de Peptídeos/química
Fragmentos de Peptídeos/genética
Fragmentos de Peptídeos/metabolismo
Receptor 1 de Sinal de Orientação para Peroxissomos
Domínios e Motivos de Interação entre Proteínas
Multimerização Proteica
Receptores Citoplasmáticos e Nucleares/química
Receptores Citoplasmáticos e Nucleares/genética
Proteínas Recombinantes de Fusão/química
Proteínas Recombinantes de Fusão/metabolismo
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Proteínas Repressoras/química
Proteínas Repressoras/genética
Solubilidade
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Membrane Proteins); 0 (PEX13 protein, human); 0 (PEX14 protein, human); 0 (PEX5 protein, human); 0 (Peptide Fragments); 0 (Peroxisome-Targeting Signal 1 Receptor); 0 (Receptors, Cytoplasmic and Nuclear); 0 (Recombinant Fusion Proteins); 0 (Recombinant Proteins); 0 (Repressor Proteins); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170803
[St] Status:MEDLINE
[do] DOI:10.1074/jbc.M117.805044


  7 / 2148 MEDLINE  
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[PMID]:28669732
[Au] Autor:Fang S; Wang R; Liu H; Zhuang W; Wang Z; Zhang J; Pei L; Liu Y; Su Y
[Ad] Endereço:Novomab Biopharmaceuticals Inc, Nanjing 210042, China.
[Ti] Título:The retention of prion protein in the endoplasmic reticulum prevents N2A cells from proteasome inhibition-induced cytotoxicity.
[So] Source:Biochem Biophys Res Commun;491(2):500-507, 2017 Sep 16.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Prion disease is a fatal neurodegenerative disease that may result from the conversion of normal cellular prion protein (PrP ) to the pathogenic scrapie PrP isoform (PrP ), however, how proliferation of prion leads to neuronal apoptosis is still not clear. In this study, to explore the role of the endoplasmic reticulum (ER) in prion diseases, we engineered the KDEL ER-retention motif to the C-terminus of PrP and studied its effect on N2A cell toxicity. The KDEL retention signal led to the accumulation of PrP in the ER, and KDEL signal could effectively deplete PrP from the cell surface and trap PrP in the ER/Cis-Golgi compartment. PrP molecules were delayed in their transit along the early pathway of the secretory compartment, however, they did not aggregate, and were not resistant to Proteinase K (PK) or become detergent-insoluble. Moreover, we found that the ER was not the site where PrP became detergent-insoluble and acquired PK resistance. In addition, an MTT assay indicated cells expressing PrP /N2A were sensitive to proteasome inhibition, but not N2A cells expressing PrP . Our findings suggest that the ER is not a compartment in which wild type PrP is able to initiate aggregation, protease resistance or other scapie-like properties of PrP.
[Mh] Termos MeSH primário: Retículo Endoplasmático/metabolismo
Complexo de Golgi/metabolismo
Neurônios/metabolismo
Proteínas PrPC/metabolismo
Proteínas PrPSc/metabolismo
Complexo de Endopeptidases do Proteassoma/metabolismo
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Animais
Apoptose
Linhagem Celular Tumoral
Endopeptidase K/química
Retículo Endoplasmático/efeitos dos fármacos
Expressão Gênica
Complexo de Golgi/efeitos dos fármacos
Leupeptinas/farmacologia
Camundongos
Neurônios/citologia
Neurônios/efeitos dos fármacos
Proteínas PrPC/genética
Proteínas PrPSc/genética
Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos
Inibidores de Proteassoma/farmacologia
Engenharia de Proteínas
Transporte Proteico
Proteólise/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Leupeptins); 0 (PrPC Proteins); 0 (PrPSc Proteins); 0 (Proteasome Inhibitors); EC 3.4.21.64 (Endopeptidase K); EC 3.4.25.1 (Proteasome Endopeptidase Complex); RF1P63GW3K (benzyloxycarbonylleucyl-leucyl-leucine aldehyde)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170829
[Lr] Data última revisão:
170829
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170704
[St] Status:MEDLINE


  8 / 2148 MEDLINE  
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[PMID]:28445750
[Au] Autor:Lenton S; Grimaldo M; Roosen-Runge F; Schreiber F; Nylander T; Clegg R; Holt C; Härtlein M; García Sakai V; Seydel T; Marujo Teixeira SC
[Ad] Endereço:Environment, Physical Sciences and Applied Mathematics (EPSAM), Keele University, Staffordshire, United Kingdom; Institut Laue-Langevin, Grenoble, France; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom.
[Ti] Título:Effect of Phosphorylation on a Human-like Osteopontin Peptide.
[So] Source:Biophys J;112(8):1586-1596, 2017 Apr 25.
[Is] ISSN:1542-0086
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.
[Mh] Termos MeSH primário: Osteopontina/metabolismo
[Mh] Termos MeSH secundário: Animais
Bovinos
Elasticidade
Eletroforese em Gel de Poliacrilamida
Endopeptidase K/metabolismo
Escherichia coli
Cavalos
Seres Humanos
Simulação de Dinâmica Molecular
Difração de Nêutrons
Ressonância Magnética Nuclear Biomolecular
Osteopontina/química
Fosforilação
Proteólise
Espectroscopia de Prótons por Ressonância Magnética
Proteínas Recombinantes/química
Proteínas Recombinantes/metabolismo
Espalhamento a Baixo Ângulo
Soluções
Água/química
Difração de Raios X
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Recombinant Proteins); 0 (SPP1 protein, human); 0 (Solutions); 059QF0KO0R (Water); 106441-73-0 (Osteopontin); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170807
[Lr] Data última revisão:
170807
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170427
[St] Status:MEDLINE


  9 / 2148 MEDLINE  
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[PMID]:28411648
[Au] Autor:Küchler A; Messmer D; Schlüter AD; Walde P
[Ad] Endereço:ETH Zürich, Zürich, Switzerland.
[Ti] Título:Preparation and Applications of Dendronized Polymer-Enzyme Conjugates.
[So] Source:Methods Enzymol;590:445-474, 2017.
[Is] ISSN:1557-7988
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Dendronized polymer-enzyme conjugates are large, water-soluble macromolecular structures built from a linear, fully synthetic, dendronized polymer (denpol), and several copies of enzyme molecules covalently bound to the peripheral functional groups of the denpol. Since denpol chains comprise repeating units with regularly branched side chains (dendrons), denpols have a cylindrical shape and are much thicker than conventional linear polymers. Depending on the dendron generation and chemical structure, denpols may have a large number of functional groups on their surface, exposed to the aqueous medium in which they are dissolved. Enzymes (and also other molecules) can be attached to these functional groups, for example, via a stable bis-aryl hydrazone (BAH) bond. The dendronized polymer scaffold might also serve as a nanoarmor and stabilize the delicate enzymes. One of the denpols which can be used for the preparation of denpol-enzyme conjugates is de-PG2. It has a poly(methacrylate) backbone and consists of second-generation dendrons with four peripheral amino groups in each repeating unit. The synthesis of de-PG2 and the preparation of a de-PG2 conjugate carrying BAH-linked proteinase K (proK), as an example, are described here for applications in the field of enzyme immobilization on solid surfaces. The nanoarmored enzyme-polymer conjugate indicated high stability and retention of enzymatic activity.
[Mh] Termos MeSH primário: Dendrímeros/química
Enzimas Imobilizadas/química
Ácidos Polimetacrílicos/química
[Mh] Termos MeSH secundário: Biocatálise
Endopeptidase K/química
Ensaios Enzimáticos
Estabilidade Enzimática
Peroxidase do Rábano Silvestre/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Dendrimers); 0 (Enzymes, Immobilized); 0 (Polymethacrylic Acids); EC 1.11.1.- (Horseradish Peroxidase); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170807
[Lr] Data última revisão:
170807
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170417
[St] Status:MEDLINE


  10 / 2148 MEDLINE  
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[PMID]:28409448
[Au] Autor:Francisco T; Dias AF; Pedrosa AG; Grou CP; Rodrigues TA; Azevedo JE
[Ad] Endereço:Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
[Ti] Título:Determining the Topology of Peroxisomal Proteins Using Protease Protection Assays.
[So] Source:Methods Mol Biol;1595:27-35, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Protease protection assays are powerful tools to determine the topology of organelle proteins. Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years. Here, we provide a detailed protocol to use with mammalian peroxisomes. Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.
[Mh] Termos MeSH primário: Endopeptidases/metabolismo
Peroxissomos/metabolismo
Proteínas/metabolismo
[Mh] Termos MeSH secundário: Endopeptidase K/metabolismo
Proteólise
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Proteins); EC 3.4.- (Endopeptidases); EC 3.4.21.64 (Endopeptidase K)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170518
[Lr] Data última revisão:
170518
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170415
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6937-1_3



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