Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
Mais filtros










Intervalo de ano de publicação
2.
J Appl Microbiol ; 2018 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-30472814

RESUMO

AIMS: To examine the antioxidant activity of Bacterioruberin (Bctr)-rich extracts isolated from a hyperpigmented-genetically modified Haloferax volcanii strain (HVLON3) and to investigate the effect on cold-sensitive ram sperm cells. METHODS AND RESULTS: The strain HVLON3 produces higher Bctr amounts than most haloarchaea (220 ± 13 mg/g DW). HVLON3-Bctr extract has higher antioxidant activity than ß-carotene (3-fold) as evaluated using 2,2 diphenyl-1-picrylhydrazyl combined with Electron Paramagnetic Resonance analysis (EC50 4.5 x 10-5 mol l-1 vs. 13.9 x 10-5 mol l-1 , respectively). Different concentrations of HVLON3-Bctr extracts were assayed on ram sperm after freezing/thawing and physiologically relevant parameters were examined. Extracts containing 7 and 20 µmol l-1 Bctr significantly improved cell viability (p<0.0001), total and progressive motility (p<0.0001) and sperm velocities (p=0.0172 for curvilinear velocity VCL, p=0.0268 for average path velocity VAP and p=0.0181 for straight-line velocity VSL) and did not affect other parameters evaluated. CONCLUSIONS: HVLON3 is an excellent source of natural microbial C50 carotenoids with applicability in Biotechnology, Biomedical and Veterinary fields. HVLON3 Bctr-extract improves the quality of cryopreserved ram sperm cells and could be applied to increase insemination yields. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insight on the bioactive properties of a bioproduct derived from haloarchaea (carotenoids) which are so far underexploited. This article is protected by copyright. All rights reserved.

3.
Proteomics ; 18(14): e1800116, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29888524

RESUMO

The dynamic changes that take place along the phases of microbial growth (lag, exponential, stationary, and death) have been widely studied in bacteria at the molecular and cellular levels, but little is known for archaea. In this study, a high-throughput approach was used to analyze and compare the proteomes of two haloarchaea during exponential and stationary growth: the neutrophilic Haloferax volcanii and the alkaliphilic Natrialba magadii. Almost 2000 proteins were identified in each species (≈50% of the predicted proteome). Among them, 532 and 432 were found to be differential between growth phases in H. volcanii and N. magadii, respectively. Changes upon entrance into stationary phase included an overall increase in proteins involved in the transport of small molecules and ions, stress response, and fatty acid catabolism. Proteins related to genetic processes and cell division showed a notorious decrease in amount. The data reported in this study not only contributes to our understanding of the exponential-stationary growth phase transition in extremophilic archaea but also provides the first comprehensive analysis of the proteome composition of N. magadii. The MS proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier JPST000395.

4.
Int J Biol Macromol ; 113: 1134-1141, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29524492

RESUMO

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.


Assuntos
Espaço Extracelular/enzimologia , Halobacteriaceae/citologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sais/farmacologia , Relação Dose-Resposta a Droga , Halobacteriaceae/enzimologia , Cinética , Solventes/química , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Interface (Botucatu, Online) ; 22(64): 97-108, jan.-mar. 2018. ilus
Artigo em Português | LILACS | ID: biblio-893465

RESUMO

O objetivo foi descrever e analisar modelos e estratégias internacionais de envolvimento de usuários de sistemas de saúde nos processos de avaliação para incorporação de tecnologias de saúde registrados na literatura científica. Realizou-se levantamento da literatura em sete bases científicas, seleção de artigos, identificação e descrição dos modelos adotados em diferentes países. De 392 artigos identificados, oito foram selecionados segundo critérios de elegibilidade. As estratégias e modelos identificados foram classificados segundo o domínio do envolvimento; tipo de público e nível de envolvimento. A descrição dos modelos permitiu identificar uma ampla diversidade de experiências para envolvimento do público em processos de ATS. Os resultados apontam para a troca de conhecimentos e informações como forma de reduzir o distanciamento entre os usuários e os processos de avaliação mediante estratégias diversificadas de incentivo à participação.(AU)


This paper aims to describe and analyze international models and strategies for the involvement of patients of healthcare systems in HTA processes. A literature review was conducted on papers from seven databases, followed by selection of papers, identification and description of the models adopted in different countries. Based on the 392 articles identified, eight were selected for analysis based on eligibility criteria. The models and strategies were classified according to the domains of the involvement; type of audiences and level of involvement. The description of the models allowed identifying a wide range of experiences for citizens' involvement in the HTA processes. The results point to knowledge and information exchange as ways to reduce the distance between patients and HTA processes through diversified strategies to foster participation.(AU)


El objetivo fue describir y analizar modelos y estrategias internacionales de envolvimiento de usuarios de sistemas de salud en los procesos de evaluación para incorporación de tecnologías de salud registrados en la literatura científica. Se realizó un levantamiento de la literatura en siete bases científicas, selección de artículos, identificación y descripción de los modelos adoptados en diferentes países. De 392 artículos identificados, se seleccionaron ocho según criterios de elegibilidad. Las estrategias y modelos identificados se clasificaron según el dominio del envolvimiento, tipo de público y nivel de envolvimiento. La descripción de los modelos permitió identificar una amplia diversidad de experiencias para envolvimiento del público en procesos de ETS. Los resultados señalan el intercambio de conocimientos e informaciones como forma de reducir el distanciamiento entre los usuarios y los procesos de evaluación mediante estrategias diversificadas de incentivo a la participación.(AU)


Assuntos
Humanos , Tecnologia Biomédica/métodos , Sistemas de Saúde , Participação do Paciente
6.
J Proteome Res ; 17(3): 1158-1171, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29411617

RESUMO

The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis.


Assuntos
Proteínas Arqueais/metabolismo , Carotenoides/biossíntese , Regulação da Expressão Gênica em Archaea , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Haloferax volcanii/enzimologia , Protease La/metabolismo , Proteoma/metabolismo , Proteínas Arqueais/genética , Cromatografia Líquida , Ontologia Genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Haloferax volcanii/genética , Marcação por Isótopo/métodos , Anotação de Sequência Molecular , Mutação , Protease La/genética , Biossíntese de Proteínas , Proteólise , Proteoma/genética , Especificidade por Substrato , Espectrometria de Massas em Tandem
7.
J Proteome Res ; 17(3): 961-977, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29301397

RESUMO

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.


Assuntos
Proteínas Arqueais/genética , Deleção de Genes , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/genética , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteínas Arqueais/classificação , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Adesão Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endopeptidases/deficiência , Endopeptidases/genética , Ontologia Genética , Glicosilação , Haloferax volcanii/química , Haloferax volcanii/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Anotação de Sequência Molecular , Proteoma/classificação , Proteoma/isolamento & purificação , Proteoma/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
8.
Extremophiles ; 19(6): 1133-43, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26369649

RESUMO

The biodiversity and biotechnological potential of microbes from central Argentinean halophilic environments have been poorly explored. Salitral Negro and Colorada Grande salterns are neutral hypersaline basins exploded for NaCl extraction. As part of an ecological analysis of these environments, two bacterial and seven archaeal representatives were isolated, identified and examined for their biotechnological potential. The presence of hydrolases (proteases, amylases, lipases, cellulases and nucleases) and bioactive molecules (surfactants and antimicrobial compounds) was screened. While all the isolates exhibited at least one of the tested activities or biocompounds, the species belonging to Haloarcula genus were the most active, also producing antimicrobial compounds against their counterparts. In general, the biosurfactants were more effective against olive oil and aromatic compounds than detergents (SDS or Triton X-100). Our results demonstrate the broad spectrum of activities with biotechnological potential exhibited by the microorganisms inhabiting the Argentinean salterns and reinforce the importance of screening pristine extreme environments to discover interesting/novel bioactive molecules.


Assuntos
Haloarcula/metabolismo , Microbiologia Industrial/métodos , Tensoativos/metabolismo , Anti-Infecciosos/metabolismo , Proteínas Arqueais/metabolismo , Haloarcula/enzimologia , Haloarcula/genética , Haloarcula/isolamento & purificação , Hidrolases/metabolismo , Tolerância ao Sal
9.
Data Brief ; 4: 50-3, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26217762

RESUMO

This data article provides information in support of the research article "Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii" [1]. The proteome composition of a wt and a LonB protease mutant strain (suboptimal expression) in the archaeon Haloferax volcanii was assessed by a quantitative shotgun proteomic approach. Membrane and cytosol fractions of H. volcanii strains were examined at two different growth stages (exponential and stationary phase). Data is supplied in the present article. This study represents the first proteome examination of a Lon-deficient cell of the Archaea Domain.

10.
J Proteomics ; 121: 1-14, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25829260

RESUMO

UNLABELLED: The membrane-associated LonB protease is essential for viability in Haloferax volcanii, however, the cellular processes affected by this protease in archaea are unknown. In this study, the impact of a lon conditional mutation (down-regulation) on H. volcanii physiology was examined by comparing proteomes of parental and mutant cells using shotgun proteomics. A total of 1778 proteins were identified (44% of H. volcanii predicted proteome) and 142 changed significantly in amount (≥2 fold). Of these, 66 were augmented in response to Lon deficiency suggesting they could be Lon substrates. The "Lon subproteome" included soluble and predicted membrane proteins expected to participate in diverse cellular processes. The dramatic stabilization of phytoene synthase (57 fold) in concert with overpigmentation of lon mutant cells suggests that Lon controls carotenogenesis in H. volcanii. Several hypothetical proteins, which may reveal novel functions and/or be involved in adaptation to extreme environments, were notably increased (300 fold). This study, which represents the first proteome examination of a Lon deficient archaeal cell, shows that Lon has a strong impact on H. volcanii physiology evidencing the cellular processes controlled by this protease in Archaea. Additionally, this work provides a platform for the discovery of novel targets of Lon proteases. BIOLOGICAL SIGNIFICANCE: The proteome of a Lon-deficient archaeal cell was examined for the first time showing that Lon has a strong impact on H. volcanii physiology and evidencing the proteins and cellular processes controlled by this protease in Archaea. This work will facilitate future investigations aiming to address Lon function in archaea and provides a platform for the discovery of endogenous targets of the archaeal-type Lon as well as novel targets/processes regulated by Lon proteases. This knowledge will advance the understanding on archaeal physiology and the biological function of membrane proteases in microorganisms.


Assuntos
Proteínas Arqueais/metabolismo , Haloferax volcanii/enzimologia , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Proteoma/metabolismo , Aminoácidos/química , Carotenoides/química , Membrana Celular/metabolismo , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Metabolismo dos Lipídeos , Mutação , Proteômica , Espectrometria de Massas por Ionização por Electrospray
11.
Front Microbiol ; 6: 39, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25774151

RESUMO

The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

12.
Rev. enferm. UFPE on line ; 9(1): 237-242, jan. 2015.
Artigo em Português | BDENF - Enfermagem | ID: biblio-998939

RESUMO

Objetivo: descrever o perfil reprodutivo das mulheres participantes da Campanha Outubro Rosa. Método: estudo descritivo, tipo informativo, realizado de outubro a dezembro de 2013, no município de Rio das Ostras/RJ, por discentes do Curso de Enfermagem da Universidade Federal Fluminense - Campus Rio das Ostras. Resultados: destacam-se entre as usuárias: a multiparidade em 64,3%; a nuliparidade em 10,5%; taxa de abortamentos, excluindo nulíparas, de 19%; e incidência de parto cesárea de 47%. Conclusão: evidencia-se a necessidade de que o município de Rio das Ostras (RJ) amplie o acesso e a adesão das usuárias aos serviços de saúde. A vivência foi extremamente rica na formação profissional dos acadêmicos de enfermagem participantes, por proporcionar o contato e a interação com as usuárias, fundamental para a caracterização e ampliação do conhecimento sobre a saúde reprodutiva da população feminina.(AU)


Assuntos
Humanos , Neoplasias da Mama , Saúde Reprodutiva , Saúde da Mulher , Enfermagem
13.
Electrophoresis ; 35(24): 3518-22, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25224925

RESUMO

Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Haloferax volcanii/química , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas Arqueais/análise , Concentração de Íons de Hidrogênio , Proteínas de Membrana/análise
14.
J Biol Chem ; 289(16): 11304-17, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24596091

RESUMO

Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family.


Assuntos
Endopeptidases/metabolismo , Haloferax volcanii/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Endopeptidases/genética , Técnicas de Silenciamento de Genes , Glicosilação , Haloferax volcanii/genética , Glicoproteínas de Membrana/genética , Oligossacarídeos/genética
15.
Environ Microbiol ; 16(6): 1779-92, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24428705

RESUMO

Although homologs of the ATP-dependent Lon protease exist in all domains of life, the relevance of this protease in archaeal physiology remains a mystery. In this study, we have constructed and phenotypically characterized deletion and conditional lon mutants in the model haloarchaeon Haloferax volcanii to elucidate the role of the unusual membrane-bound LonB protease in archaea. Hvlon could be deleted from the chromosome only when a copy of the wild type gene was provided in trans suggesting that Lon is essential for survival in this archaeon. Successful complementation of the lethal phenotype of ΔHvlon was attained by expression of the heterologous protease gene Nmlon from the haloalkaliphilic archaeon Natrialba magadii, meaning that the biological function of Lon is conserved in these organisms. Suboptimal cellular levels of Lon protein affected growth rate, cell shape, cell pigmentation, lipid composition and sensitivity to various antibiotics. The contents of bacterioruberins and some polar lipids were increased in the lon mutants suggesting that Lon is linked to maintenance of membrane lipid balance which likely affects cell viability in this archaeon. The phenotypes associated to a membrane-bound LonB protease mutant were examined for the first time providing insight on the relevance of this protease in archaeal physiology.


Assuntos
Proteínas Arqueais/genética , Haloferax volcanii/enzimologia , Lipídeos de Membrana/metabolismo , Peptídeo Hidrolases/genética , Antibacterianos/farmacologia , Proteínas Arqueais/metabolismo , Bacitracina/farmacologia , Sequência de Bases , Expressão Gênica , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/efeitos dos fármacos , Lovastatina/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Novobiocina/farmacologia , Peptídeo Hidrolases/metabolismo , Pigmentação , Ligação Proteica , Puromicina/farmacologia
16.
Protein Pept Lett ; 20(10): 1098-107, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23590280

RESUMO

Kallikrein-related peptidases (KLKs) are trypsin-like and chymotrypsin-like serine proteases which are expressed in several tissues. Their activity is tightly controlled by inhibitors including members of the serine protease Kazal-type (SPINK) family. These enzymes are promising targets for the treatment of skin desquamation, inflammation and cancer. Spink3 or caltrin I is expressed in mouse pancreas and males accessory glands and the resulting mature protein has been associated with different activities such as an inhibitor of trypsin and acrosin activity, calcium transport inhibitor in sperm and inhibitor of cell proliferation during embryogenesis. In this study, we produced a soluble recombinant Spink3 from mouse seminal vesicle (rmSpink3) that inhibited the activity of human KLKs. Using FRET substrates, rmSpink3 exhibited a potent inhibitory activity against human KLK2, KLK3, KLK5 (Ki ranging from 260 to 1500 nM), and to a lesser extent against KLK6, KLK1 and KLK7 (Ki around 3000 nM). As shown by mass spectrometry analysis of rmSpink3 incubated with trypsin, the inhibitor was not truncated by the target enzyme. Based on the in silico analysis of the expression of Spink3/SPINK1 and KLKs it is speculated that some KLKs may be natural targets of Spink3/SPINK1, however experimental confirmation using both proteins from mouse or human origin is needed. This work shows that rmSpink3 is a potent inhibitor of various human KLK members suggesting the potential of this molecule in the diagnosis/prevention of several human diseases.


Assuntos
Glicoproteínas/farmacologia , Calicreínas/antagonistas & inibidores , Proteínas Secretadas pela Próstata/farmacologia , Inibidores de Serino Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Humanos , Calicreínas/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Secretadas pela Próstata/química , Proteínas Secretadas pela Próstata/genética , Proteínas Secretadas pela Próstata/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/isolamento & purificação , Inibidor da Tripsina Pancreática de Kazal
17.
J Bacteriol ; 194(14): 3700-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22582277

RESUMO

Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as a model protease to examine the secretion and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing a modification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase.


Assuntos
Archaea/metabolismo , Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Serina Endopeptidases/metabolismo , Archaea/genética , Proteínas Arqueais/genética , Transporte Proteico , Serina Endopeptidases/genética
19.
Biochimie ; 94(3): 798-805, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22177966

RESUMO

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3-1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.


Assuntos
Halobacteriaceae/enzimologia , Subtilisinas/química , Subtilisinas/metabolismo , Catálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Estrutura Terciária de Proteína , Temperatura Ambiente
20.
Barbarói ; (35): 91-108, ago.-dez. 2011.
Artigo em Português | LILACS | ID: lil-619498

RESUMO

Este artigo é um relato de experiência de uma estudante de psicologia em seus encontros com seu primeiro paciente. Deste percurso emerge a construção feita por esta estagiária, que, a partir da escuta clínica e do enlace com as histórias de Alice no País das Maravilhas e Alice através do Espelho, narra paralelamente seu crescimento enquanto terapeuta e o de seu paciente que passa da infância à adolescência durante o tempo de atendimento. Nesta aventura a estudante faz suas primeiras construções teóricas ancoradas na Psicanálise e sustentadas com sua supervisora e seu grupo de supervisão. Uma curiosa aventura narrada ao sabor dos ventos literários de Lewis Carroll.


This article is an experience of a psychology student in her meetings with her first patient. Of this route emerges the construction made by the internship, who hears from the clinical and linkage with the stories of Alice in Wonderland and Alice Through the Looking Glass, tells parallel their growth as a therapist and his patient passing from childhood to adolescence during the treatment. In this adventure the student makes his first theoretical constructs anchored in Psychoanalysis and sustained with her supervisor and supervision group. A curious adventure narrated with the winds of Lewis Carroll’s literary.


Assuntos
Estágio Clínico , Assistência ao Paciente
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA