RESUMO
BACKGROUND: The global spread of COVID-19 created an explosion in rapid tests with results in < 1 hour, but their relative performance characteristics are not fully understood yet. Our aim was to determine the most sensitive and specific rapid test for the diagnosis of SARS-CoV-2. METHODS: Design: Rapid review and diagnostic test accuracy network meta-analysis (DTA-NMA). ELIGIBILITY CRITERIA: Randomized controlled trials (RCTs) and observational studies assessing rapid antigen and/or rapid molecular test(s) to detect SARS-CoV-2 in participants of any age, suspected or not with SARS-CoV-2 infection. INFORMATION SOURCES: Embase, MEDLINE, and Cochrane Central Register of Controlled Trials, up to September 12, 2021. OUTCOME MEASURES: Sensitivity and specificity of rapid antigen and molecular tests suitable for detecting SARS-CoV-2. Data extraction and risk of bias assessment: Screening of literature search results was conducted by one reviewer; data abstraction was completed by one reviewer and independently verified by a second reviewer. Risk of bias was not assessed in the included studies. DATA SYNTHESIS: Random-effects meta-analysis and DTA-NMA. RESULTS: We included 93 studies (reported in 88 articles) relating to 36 rapid antigen tests in 104,961 participants and 23 rapid molecular tests in 10,449 participants. Overall, rapid antigen tests had a sensitivity of 0.75 (95% confidence interval 0.70-0.79) and specificity of 0.99 (0.98-0.99). Rapid antigen test sensitivity was higher when nasal or combined samples (e.g., combinations of nose, throat, mouth, or saliva samples) were used, but lower when nasopharyngeal samples were used, and in those classified as asymptomatic at the time of testing. Rapid molecular tests may result in fewer false negatives than rapid antigen tests (sensitivity: 0.93, 0.88-0.96; specificity: 0.98, 0.97-0.99). The tests with the highest sensitivity and specificity estimates were the Xpert Xpress rapid molecular test by Cepheid (sensitivity: 0.99, 0.83-1.00; specificity: 0.97, 0.69-1.00) among the 23 commercial rapid molecular tests and the COVID-VIRO test by AAZ-LMB (sensitivity: 0.93, 0.48-0.99; specificity: 0.98, 0.44-1.00) among the 36 rapid antigen tests we examined. CONCLUSIONS: Rapid molecular tests were associated with both high sensitivity and specificity, while rapid antigen tests were mainly associated with high specificity, according to the minimum performance requirements by WHO and Health Canada. Our rapid review was limited to English, peer-reviewed published results of commercial tests, and study risk of bias was not assessed. A full systematic review is required. REVIEW REGISTRATION: PROSPERO CRD42021289712.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Metanálise em Rede , Viés , Testes Diagnósticos de Rotina , Sensibilidade e Especificidade , Teste para COVID-19RESUMO
BACKGROUND: Authentic researcher-youth partnerships in patient-oriented research (POR) where the research responds to the needs expressed by youth themselves are essential to make research meaningful. While patient-oriented research (POR) is increasingly practiced, few training programs exist in Canada and none, to our knowledge, are tailored for youth with neurodevelopmental disabilities (NDD). Our primary objective was to explore the training needs of youth (ages 18-25) with NDD to enhance their knowledge, confidence, and skills as research partners. Our secondary objective was to identify the benefits and challenges of engaging youth with NDD in a POR approach. METHODS: Our team of four youth and one parent with lived experience [Youth Engagement in Research (YER) partners] and six researchers engaged in POR to investigate the primary objective via two phases: (1) individual interviews with youth living with NDD and (2) a two-day virtual symposium with focus groups with youth and researchers. Collaborative qualitative content analysis was employed to synthesize the data. Our secondary objective was assessed by asking our YER partners to complete the Public and Patient Engagement Evaluation Tool (PPEET) survey and participate in reflective discussions. RESULTS: Phase 1 participants (n = 7) identified various barriers and facilitators to their engagement in research and offered suggestions to meet their needs through minimizing barriers and integrating facilitators, which would subsequently enhance their knowledge, confidence, and skills as research partners. Informed by phase 1, phase 2 participants (n = 17) prioritized the following POR training needs: researcher-youth communication, research roles and responsibilities, and finding partnership opportunities. For delivery methods, participants stated the importance of youth representation, using Universal Design for Learning, and co-learning between youth and researchers. Based on the PPEET data and subsequent discussions, YER partners agreed that they were able to express views freely, feel that their views were heard, and that their participation made a meaningful difference. Challenges included scheduling difficulties, ensuring multiple methods for engagement, and working under short timelines. CONCLUSION: This study identified important training needs for youth with NDD and for researchers to engage in meaningful POR, which can subsequently inform the co-production of accessible training opportunities with and for youth.
Partnerships between researchers and youth, known as patient-oriented research (POR), are needed to make sure research is meaningful to youth. Our main goal was to explore the training needs of youth (ages 18-25) with neurodevelopmental disabilities (NDD) to enhance their knowledge, confidence, and skills as research partners. To find out, our team of four youth and one parent with lived experience (YER partners) and six researchers completed this project in two parts: 1) interviews with youth and 2) a two-day virtual workshop with youth and researchers. Data from the two parts were reviewed to answer our question. We learned from Part 1 that the needs of participants can be met by providing support and reducing barriers in POR. From Part 2, the top three important topics in partnerships were: researcher-youth communication, research roles and responsibilities, and finding partnership opportunities. Participants in the workshop emphasized having different youth represented, using a framework that allows learning for everyone, and co-learning between youth and researchers in the creation of learning materials. Our second goal was to understand the benefits and challenges of our partnership. To assess, YER partners completed a survey and reflected about their experiences. YER partners agreed on being able to express views, feel that their views were heard, and that their participation made a meaningful difference. Challenges included scheduling difficulties, providing multiple ways to partner, and working under short timelines. Overall, the study described important POR needs for youth and researchers, which can inform future training opportunities.
RESUMO
CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/fisiologia , Diferenciação Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas de Capeamento de Actina , Fatores de Despolimerização de Actina , Actinas/análise , Sequência de Aminoácidos , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Caseína Quinase II/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Destrina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Osteossarcoma , Fosforilação , Mapeamento de Interação de Proteínas , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Serina/metabolismoRESUMO
Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.
Assuntos
Arachis/química , Peroxidase/química , Polissacarídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Configuração de Carboidratos , Cromatografia Líquida de Alta Pressão , Meios de CulturaAssuntos
Proteínas de Ligação ao Cálcio/química , Sequência de Aminoácidos , Animais , Calmodulina/química , Calmodulina/metabolismo , Bovinos , Humanos , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/química , Parvalbuminas/química , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.
Assuntos
Proteína B Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida , Dados de Sequência Molecular , Nanotecnologia , Alinhamento de Sequência , Ovinos , Especificidade da EspécieRESUMO
BACKGROUND: Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE: These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.
Assuntos
Proteína Quinase CDC2/metabolismo , Cinesinas/metabolismo , Microtúbulos/enzimologia , Óvulo/enzimologia , Fuso Acromático/enzimologia , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animais , Soluções Tampão , Extratos Celulares , Ciclina B/metabolismo , Cinesinas/deficiência , Fosforilação , Ligação Proteica , Proteínas de Xenopus/deficiênciaRESUMO
Exposing BLES (bovine lipid extract surfactant), a clinical surfactant, to reactive oxygen species (ROS) alters surfactant protein B (SP-B), as indicated by Coomassie Blue staining, silver staining, and Western analysis. Hypochlorous acid (HOCl) treatment leads to elevated maximum surface tension (gammamax) and a deterioration in minimum gamma (gammamin) during surface area cycling. Fenton reaction resulted in immediate increases in gammamin and gammamax. Intrinsic fluorescence measurements indicated Fenton, but not HOCl, induced conversion of Trp9 of SP-B to hydroxyTrp (OHTrp), N-formylkynurenine (NFKyn), and kynurenine (Kyn). Electrospray ionization mass spectrometry (ESI-MS) revealed molecular weight alterations consistent with oxidation of Met (HOCl, Fenton) and Trp (Fenton) residues. Oxidative alterations to Met29 and Met65 (HOCl, Fenton) and to Trp9 (OHTrp with HOCL and NFKyn plus Kyn with Fenton) were confirmed by matrix-assisted laser desorption mass spectrometry (MALDI-MS) studies on SP-B tryptic fragments. Some Met oxidation was observed with control SP-B. When taken together with captive bubble tensiometer measurements, these studies suggest that Met oxidation of SP-B by HOCl or Fenton interferes with phospholipid respreading during compression-expansion of surfactant films, while Fenton oxidation, which produces more extensive Met oxidation and disruption of the indole ring of Trp9, further abrogated the ability of such films to attain low surface tensions during compression. These studies provide insight into the manner by which ROS generated during acute lung injury and the acute respiratory distress syndrome act to inhibit not only endogenous surfactant but also therapeutic surfactants administered to counteract these conditions.
Assuntos
Metionina/metabolismo , Proteína B Associada a Surfactante Pulmonar/antagonistas & inibidores , Proteína B Associada a Surfactante Pulmonar/metabolismo , Triptofano/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Peróxido de Hidrogênio/química , Ácido Hipocloroso/química , Ferro/química , Metionina/química , Dados de Sequência Molecular , Oxirredução , Fosfolipídeos/química , Proteína B Associada a Surfactante Pulmonar/química , Espécies Reativas de Oxigênio/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano/químicaRESUMO
Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.
Assuntos
Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/isolamento & purificação , Sequência de Bases , Quitina/química , Quitina/metabolismo , Colina O-Acetiltransferase/química , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Histidina/química , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform a comprehensive analysis of the post-translational modification of OPN purified from rat bone. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203, S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150 was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.
Assuntos
Osso e Ossos/química , Osso e Ossos/metabolismo , Processamento de Proteína Pós-Traducional , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Assialoglicoproteínas/metabolismo , Configuração de Carboidratos , Bovinos , Cromatografia Líquida/métodos , Glicosilação , Proteínas do Leite/química , Proteínas do Leite/metabolismo , Dados de Sequência Molecular , Osteopontina , Polissacarídeos/metabolismo , Ratos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sulfatos/metabolismoRESUMO
A two-dimensional gel electrophoresis (2-DE)-based proteomic approach was used to study a transgenic mouse model of acerbated dilated cardiomyopathy in which the small monomeric GTPase, Rac1, was constitutively expressed exclusively in the myocardium. A subfractionation procedure allowed for the focused analysis of both cytoplasmic and myofilament protein-enriched extracts of ventricular tissue from Rac1 transgenic and age-matched nontransgenic (NTG) mice. The majority of these mice displayed severe hypertrophy (heart-to-body weight ratios >2-fold greater in the Rac1 mice) and died from overt heart failure between days 14 and 17. Comparative 2-DE analysis (pH 3-10, 12% SDS-PAGE) derived from Rac1 (n = 4) and NTG (n = 4) groups revealed differences in mean protein spot intensities. Twelve proteins from the cytoplasmic protein-enriched extract met our criteria for robustness and spot resolution and were identified. These proteins represent a broad distribution of cellular functions with only some previously implicated in myocardial hypertrophy. The myofilament subproteome displayed no change in posttranslational modification, but further analysis by one-dimensional Western blot showed increased quantities of myofilament proteins in the Rac1 mouse ventricles. Additionally, three proteins with different functionality that were altered in the cytoplasmic protein-enriched subproteome, tubulin beta-chain, manganese superoxide dismutase, and malate dehydrogenase, were analyzed at days 7, 9, and 11 to assess their role in the development of the dilated cardiomyopathic phenotype. The quantity of all three proteins peaked at day 9, suggesting an early response in cardiac hypertrophic failure.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Miocárdio/metabolismo , Neuropeptídeos/metabolismo , Proteoma/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Cardiomiopatia Dilatada/mortalidade , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Proteínas Recombinantes/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
Here, we demonstrate the application of the proteomic approach to the study of a transgenic mouse model of heart failure and provide an example of a disease-associated protein alteration that can be observed using this approach. Specifically, we applied the proteomic approach to the analysis of a mouse model of dilated cardiomyopathy in which the small GTPase, Rac1, was constitutively expressed specifically in the myocardium. We utilized the methods of two-dimensional gel electrophoresis (2-DE) for protein separation, silver-staining for protein visualization and mass spectrometry (MALDI-TOF and MS/MS) for protein spot identification. Computer-generated composite images were created which represent a normalized average of four 2-DE gel images derived from analysis of either Rac1 transgenic (n = 4) or non-transgenic (n = 4) mice. Analysis of composite images derived from NTG and Rac1 experimental groups revealed numerous statistically significant differences in mean protein spot intensities. Here, we report a statistically significant increase, of approximately 1.6-fold, in the mean protein spot intensity for creatine kinase M-chain in the composite image of Rac1 transgenic mice compared to control. This protein alteration may be consistent with an end-stage heart failure phenotype in which maximal myocardial reserve is employed to sustain survival.
Assuntos
Cardiomiopatia Dilatada/enzimologia , Creatina Quinase/análise , Camundongos Transgênicos , Cadeias Leves de Miosina/análise , Proteômica , Proteínas rac1 de Ligação ao GTP/análise , Animais , Cardiomiopatia Dilatada/metabolismo , Creatina Quinase/metabolismo , Eletroforese em Gel Bidimensional , Expressão Gênica , Insuficiência Cardíaca/metabolismo , Processamento de Imagem Assistida por Computador , Espectrometria de Massas , Camundongos , Miocárdio/enzimologia , Miocárdio/metabolismo , Cadeias Leves de Miosina/metabolismo , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We report the use of electrospray ionization (ESI) mass spectrometry (MS) in conjunction with online rapid mixing to monitor the kinetics of acid-induced ferrihemoglobin denaturation. Under equilibrium conditions, the hemoglobin mass spectrum is dominated by the intact heterotetramer. Dimeric and monomeric species are also observed at lower intensities. In addition, ionic signals corresponding to hexameric (tetramer-dimer) and octameric (tetramer x 2) hemoglobin species are observed. These complexes may represent weak solution-phase assemblies. The acid-induced denaturation process was monitored for reaction time ranging from 9 ms to approximately 3 s. The data obtained were subjected to a global analysis procedure which simultaneously fit all kinetic (ESI-MS intensity vs time) profiles to multiexponential expressions. Results of the global analysis are consistent with the coexistence of two subpopulations of tetrameric hemoglobin which differ in their disassembly rates and ESI charge states. The higher-charge state tetramer ions preferentially dissociate via a rapid pathway (tau(1) = 51 ms), resulting in the transient formation of a heme-saturated dimer, holo-alpha-globin, and a heme-deficient dimer. The latter is shown by MS/MS to be comprised of a heme-bound alpha-subunit complexed with an apo-beta-chain. The slow-decaying tetramer population, apparent at a slightly lower average charge state, breaks down into its monomeric constituents with no observable intermediate species (tau(2) = 390 ms). Surprisingly, unfolded apo-alpha-globin is formed more rapidly than unfolded apo-beta-globin. The appearance of the latter occurs with a relaxation time tau(3) of 1.2 s. It is postulated that accumulation of unfolded apo-beta-globin is delayed by transient population of an undetected unfolding intermediate.
Assuntos
Metemoglobina/química , Dobramento de Proteína , Subunidades Proteicas/química , Ácido Acético/química , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Bovinos , Dimerização , Globinas/química , Globinas/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Cinética , Metemoglobina/metabolismo , Desnaturação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Molecular determinants underlying the production of siderophores in the human and animal pathogen Staphylococcus aureus and the contribution of siderophore production to the virulence of this bacterium have, until now, remained undefined. Here, we show that S. aureus strains RN6390 and Newman produce siderophore when the cells are starved for iron. We further identified and characterized a nine-gene, iron-regulated operon, designated sbn and situated between sirABC and galE on the S. aureus chromosome, that is involved in the production of a siderophore. Mutation of the sbnE gene, in both RN6390 and Newman, eliminates the ability of these strains to produce a siderophore under iron-limited growth conditions, while introduction of multicopy sbnE into sbnE mutants complemented the inability of the mutants to produce the siderophore. sbnE mutants, in both the RN6390 and Newman backgrounds, displayed a drastic growth deficiency, compared to the wild type, in iron-restricted growth medium, whereas no such deficiency was observed during growth in iron-replete medium. Complemented mutants showed a restored ability to grow under iron restriction. We further showed that an sbnE mutant was compromised in a murine kidney abscess model of S. aureus infection, illustrating the importance of siderophore production to the pathogenicity of S. aureus. sbn genes were present in all S. aureus strains tested (and all S. aureus genome sequences) but were undetectable in any of the 13 coagulase-negative staphylococci tested, including Staphylococcus epidermidis.
Assuntos
Proteínas de Bactérias/genética , Óperon , Sideróforos/biossíntese , Staphylococcus aureus/patogenicidade , Abscesso/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Meios de Cultura , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Ferro/metabolismo , Nefropatias/microbiologia , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/crescimento & desenvolvimento , VirulênciaRESUMO
Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.
Assuntos
Colina O-Acetiltransferase/química , Colina O-Acetiltransferase/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico/genética , Linhagem Celular , Colina O-Acetiltransferase/genética , Ativação Enzimática , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Mutagênese Sítio-Dirigida , Síndromes Miastênicas Congênitas/enzimologia , Síndromes Miastênicas Congênitas/genética , Neurônios/enzimologia , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Espectrometria de Massas por Ionização por Electrospray , Treonina/químicaRESUMO
A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.
Assuntos
Espectrometria de Massas/instrumentação , Peptídeos/química , Análise de Sequência de Proteína/métodos , Software , Sequência de Aminoácidos , Dados de Sequência Molecular , Análise de Sequência de Proteína/instrumentaçãoRESUMO
Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.
Assuntos
Hidrolases de Éster Carboxílico/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Biotina/biossíntese , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Elétrons , Ligação de Hidrogênio , Cinética , Espectrometria de Massas , Modelos Moleculares , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory beta-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2alpha with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2alpha becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2alpha do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2alpha that had been phosphoradiolabelled by incubation with [(32)P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248-268 sequence, phosphorylated previously by c-Fgr at Tyr(255) in vitro. The identification of Tyr(255) as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248-268 tryptic fragments from CK2alpha and by on-target phosphatase treatment. A CK2alpha mutant in which Tyr(255) was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.