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1.
J Biol Chem ; 299(4): 104577, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36871759

RESUMO

MOV10 is an RNA helicase required for organismal development and is highly expressed in postnatal brain. MOV10 is an AGO2-associated protein that is also necessary for AGO2-mediated silencing. AGO2 is the primary effector of the miRNA pathway. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs, but no other posttranslational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex. To examine its role in cells, our RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 enhanced Cross-Linking Immunoprecipitation targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole-cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation. S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region that likely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence whereby MOV10 phosphorylation facilitates AGO2 association with the 3'UTR of translating mRNAs that leads to their degradation.


Assuntos
MicroRNAs , RNA Helicases , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , MicroRNAs/genética , Encéfalo/metabolismo , DNA Helicases/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo
2.
J Clin Microbiol ; 59(11): e0045821, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34432487

RESUMO

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.


Assuntos
Babesia , Babesiose , Animais , Anticorpos Antiprotozoários , Babesia/genética , Babesiose/diagnóstico , Cricetinae , Eritrócitos , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G , Estados Unidos
3.
Biochem Biophys Res Commun ; 508(2): 421-429, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30503337

RESUMO

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.


Assuntos
Chlamydia muridarum/patogenicidade , Chlamydia trachomatis/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Infecções por Chlamydia/etiologia , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia muridarum/metabolismo , Chlamydia trachomatis/metabolismo , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Miosina Tipo II/metabolismo , Sistemas de Secreção Tipo III/metabolismo
4.
Biochemistry ; 57(5): 781-790, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29345920

RESUMO

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Mycobacterium tuberculosis/metabolismo , Acetilação , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Clonagem Molecular , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Histona Desacetilases/genética , Histonas/genética , Cinética , Lisina/química , Modelos Moleculares , Mycobacterium tuberculosis/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem
5.
Infect Immun ; 86(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29084894

RESUMO

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.


Assuntos
Infecções por Chlamydia/complicações , Infecções por Chlamydia/patologia , Chlamydia/patogenicidade , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/etiologia , Fibrose/patologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Infecções por Chlamydia/microbiologia , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose/microbiologia , Camundongos , MicroRNAs/metabolismo , Miofibroblastos/microbiologia , Miofibroblastos/patologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fator de Crescimento Transformador beta/metabolismo
6.
PLoS Negl Trop Dis ; 17(1): e0011028, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36696390

RESUMO

Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication.


Assuntos
Alphavirus , Febre de Chikungunya , Vírus Chikungunya , Culicidae , Humanos , Animais , Culicidae/metabolismo , Mosquitos Vetores , Vírus Chikungunya/metabolismo , Alphavirus/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
7.
PLoS Biol ; 7(4): e95, 2009 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-19402752

RESUMO

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.


Assuntos
Aminoglicosídeos/farmacologia , Defensinas/biossíntese , HIV-1/imunologia , Sequência de Aminoácidos , Colo do Útero/metabolismo , Códon sem Sentido , Defensinas/genética , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Células Precursoras de Granulócitos , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Células HL-60 , Humanos , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Transfecção , Vagina/metabolismo
8.
Viruses ; 14(2)2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35215793

RESUMO

Influenza A virus (IAV), like other viruses, depends on the host cellular machinery for replication and production of progeny. The relationship between a virus and a host is complex, shaped by many spatial and temporal interactions between viral and host proteome, ultimately dictating disease outcome. Therefore, it is imperative to identify host-virus interactions as crucial determinants of disease pathogenies. Heterogeneous ribonucleoprotein A1 (hnRNPA1) is an RNA binding protein involved in the life cycle of many DNA and RNA viruses; however, its role in IAV remains undiscovered. Here we report that human hnRNPA1 physically interacts with the nucleoprotein (NP) of IAV in mammalian cells at different time points of the viral replication cycle. Temporal distribution studies identify hnRNPA1 and NP co-localize in the same cellular milieu in both nucleus and mitochondria in NP-transfected and IAV-infected mammalian cells. Interestingly, hnRNPA1 influenced NP gene expression and affected viral replication. Most importantly, hnRNPA1 knockdown caused a significant increase in NP expression and enhanced viral replication (93.82%) in IAV infected A549 cells. Conversely, hnRNPA1 overexpression reduced NP expression at the mRNA and protein levels and impeded virus replication by (60.70%), suggesting antagonistic function. Taken together, results from this study demonstrate that cellular hnRNPA1 plays a protective role in the host hitherto unknown and may hold potential as an antiviral target to develop host-based therapeutics against IAV.


Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Células A549 , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/virologia , Proteínas do Nucleocapsídeo/genética , Ligação Proteica , Replicação Viral
9.
Biochem Biophys Res Commun ; 409(3): 526-31, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21600877

RESUMO

Streptococcus pneumoniae type 2 pili are recently identified fimbrial structures extending from the bacterial surface and formed by polymers of the structural protein PitB. Intramolecular isopeptide bonds are a characteristic of the related pilus backbone protein Spy0128 of group A streptococci. Based on the identification of conserved residues in PitB, we predicted two intramolecular isopeptide bonds in PitB. Using a combination of tandem mass spectrometry and Edman sequencing, we show that these bonds were formed between Lys(63)-Asn(214) and Lys(243)-Asn(372) in PitB. Mutant proteins lacking the intramolecular isopeptide bonds retained the proteolytic stability observed with the wild type protein. However, absence of these bonds substantially decreased the melting temperature of the PitB-derivatives, indicating a stabilizing function of these bonds in PitB of the pneumococcal type 2 pilus.


Assuntos
Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Peptídeos/química , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Temperatura Alta , Hidrólise , Dados de Sequência Molecular , Mutação , Peptídeos/genética , Estabilidade Proteica , Espectrometria de Massas em Tandem
10.
Am J Trop Med Hyg ; 103(6): 2294-2304, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32901602

RESUMO

The success of the Guinea Worm (GW) Eradication Program over the past three decades has been tempered by the persistence of GW disease in a few African nations and the potential for a future resurgence in cases. Domestic dogs are now a major concern as a disease reservoir as large numbers of cases of canine GW disease are now reported each year, mainly along the Chari River in Chad. As a first step toward the development of a serologic assay for dogs, archived human plasma samples from dracunculiasis-positive donors from Togo were used to select adult female GW antigens for peptide sequencing and cloning. Eight protein sequences of interest were expressed as recombinant glutathione-S-transferase (GST) fusion proteins, and the most promising proteins were coupled to carboxylated microspheres for use in multiplex assays. A thioredoxin-like protein (TRXL1) and a domain of unknown function (DUF148) were assessed for total IgG and IgG4 reactivities using a panel of specimens from GW cases, uninfected donors, and individuals infected with various nematode worms, including Onchocerca volvulus. Both the DUF148-GST and the TRXL1-GST assays cross-reacted with O. volvulus sera, but the latter assay was always the more specific. The IgG4 and total IgG TRXL1-GST assays both had sensitivities > 87% and specificities > 90%. Maximum specificity (> 96%) was obtained with the total IgG assay when reactivity to both antigens was used to define a positive case. Given the good performance of the human assay, we are now working to modify the assay for dog assessments.


Assuntos
Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Dracunculíase/diagnóstico , Dracunculíase/veterinária , Imunoglobulina G/imunologia , Animais , Antígenos de Helmintos/genética , Western Blotting , Reações Cruzadas , Erradicação de Doenças , Reservatórios de Doenças/parasitologia , Doenças do Cão/diagnóstico , Doenças do Cão/imunologia , Cães , Dracunculíase/imunologia , Dracunculíase/prevenção & controle , Dracunculus , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/genética , Humanos , Onchocerca volvulus , Oncocercose/diagnóstico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , Tiorredoxinas/genética
11.
Front Microbiol ; 11: 581867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33101257

RESUMO

Influenza A virus (IAV) poses a major threat to global public health and is known to employ various strategies to usurp the host machinery for survival. Due to its fast-evolving nature, IAVs tend to escape the effect of available drugs and vaccines thus, prompting the development of novel antiviral strategies. High-throughput mass spectrometric screen of host-IAV interacting partners revealed host Filamin A (FLNA), an actin-binding protein involved in regulating multiple signaling pathways, as an interaction partner of IAV nucleoprotein (NP). In this study, we found that the IAV NP interrupts host FLNA-TRAF2 interaction by interacting with FLNA thus, resulting in increased levels of free, displaced TRAF2 molecules available for TRAF2-ASK1 mediated JNK pathway activation, a pathway critical to maintaining efficient viral replication. In addition, siRNA-mediated FLNA silencing was found to promote IAV replication (87% increase) while FLNA-overexpression impaired IAV replication (65% decrease). IAV NP was observed to be a crucial viral factor required to attain FLNA mRNA and protein attenuation post-IAV infection for efficient viral replication. Our results reveal FLNA to be a host factor with antiviral potential hitherto unknown to be involved in the IAV replication cycle thus, opening new possibilities of FLNA-NP interaction as a candidate anti-influenza drug development target.

12.
PLoS Negl Trop Dis ; 12(12): e0006984, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30550539

RESUMO

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms.


Assuntos
Encéfalo/virologia , Nucleoproteínas/química , Vírus da Raiva/isolamento & purificação , Raiva/virologia , Espectrometria de Massas em Tandem/métodos , Proteínas Virais/química , Proteínas Virais/metabolismo , Humanos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Proteínas Virais/genética
13.
Biochem J ; 382(Pt 3): 923-32, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15339253

RESUMO

We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) alpha and epsilon from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCalpha or epsilon activation. Intact cell phorbol ester treatment caused a decline in the in vitro (32)P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca(2+) and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated (32)P in particulate fractions isolated from 4beta-PMA-treated cells in a Ca(2+)-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCepsilon-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962-24966] into NCMs before 4beta-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4beta-PMA-treated NCMs, the PKCepsilon isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCepsilon function in NCMs. Furthermore, PKCepsilon probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/fisiologia , Imunoprecipitação , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Mitocôndrias Cardíacas/enzimologia , Fosforilação , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C-épsilon , Ratos , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia
14.
J Microbiol Methods ; 117: 14-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26183765

RESUMO

The Bruker Biotyper MALDI-TOF MS (Biotyper) system, with a modified 30 minute formic acid extraction method, was evaluated by its ability to identify 216 clinical Staphylococcus isolates from the CDC reference collection comprising 23 species previously identified by conventional biochemical tests. 16S rDNA sequence analysis was used to resolve discrepancies. Of these, 209 (96.8%) isolates were correctly identified: 177 (84.7%) isolates had scores ≥2.0, while 32 (15.3%) had scores between 1.70 and 1.99. The Biotyper identification was inconsistent with the biochemical identification for seven (3.2%) isolates, but the Biotyper identifications were confirmed by 16S rDNA analysis. The distribution of low scores was strongly species-dependent, e.g. only 5% of Staphylococcus epidermidis and 4.8% of Staphylococcus aureus isolates scored below 2.0, while 100% of Staphylococcus cohnii, 75% of Staphylococcus sciuri, and 60% of Staphylococcus caprae produced low but accurate Biotyper scores. Our results demonstrate that the Biotyper can reliably identify Staphylococcus species with greater accuracy than conventional biochemicals. Broadening of the reference database by inclusion of additional examples of under-represented species could further optimize Biotyper results.


Assuntos
Tipagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus/química , Staphylococcus/classificação
15.
PLoS One ; 10(12): e0145198, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26681200

RESUMO

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.


Assuntos
Infecções por Chlamydia/metabolismo , Transição Epitelial-Mesenquimal , Animais , Caderinas/genética , Caderinas/metabolismo , Caspases/metabolismo , Infecções por Chlamydia/patologia , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fatores de Transcrição da Família Snail , Trombospondina 1/genética , Trombospondina 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco
16.
Biosecur Bioterror ; 12(1): 49-62, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24552362

RESUMO

Abrin is a heterodimeric toxin present in the seeds of the Abrus precatorius plant. The easily obtainable seeds can yield a highly toxic product that can be used in various types of biocrimes and terrorism-related activities, including "white-powder" letters. Although the vast majority of these threats are hoaxes, the lack of rapid and reliable detection assays for abrin, such as lateral flow assays (LFAs), can be an impediment to accurate and rapid hazard assessment. One of the complicating factors associated with LFAs is the use of antibodies of poor affinity and specificity that cross-react with near neighbors or that bind to plant lectins, which are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the critical need to promote public safety and public health, we conducted a comprehensive laboratory evaluation of a commercial LFA for the rapid detection of abrin. This study was conducted using comprehensive inclusivity and exclusivity panels of abrin and near-neighbor plant materials, along with panels of lectins, related proteins, white powders, and environmental background material, to determine the sensitivity, specificity, limit of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples for the presumptive presence of abrin.


Assuntos
Abrina/isolamento & purificação , Pós/química , Kit de Reagentes para Diagnóstico/normas , Terrorismo Químico , Pós/intoxicação , Fitas Reagentes , Sensibilidade e Especificidade , Estados Unidos
17.
Virology ; 444(1-2): 55-63, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23791018

RESUMO

In July, 2012 three isolations were made from mosquitoes collected in Brazoria, Orange and Montgomery counties, Texas, USA. Data from immunofluorescence testing suggested that these isolates are members of the genus Orthobunyavirus. Expanded analyses confirmed that these isolates comprise three independent isolations of the same virus; a novel orthobunyavirus. The genetic organization of the M and L segments of this virus is similar to that of other orthobunyaviruses. However, the S segment (~1.7 kb) is nearly twice the length of known orthobunyavirus S segments, encoding a significantly larger nucleocapsid, N (~50 kDa) and putative non-structural NSs (~20 kDa) proteins in a novel strategy by which the NSs ORF precedes the N ORF. The N protein appears to consist of two functional domains; an amino portion that possesses motifs similar to other orthobunyavirus N proteins and a carboxyl portion that possesses a glutamine-rich domain with no known homologue among Bunyaviridae.


Assuntos
Proteínas do Nucleocapsídeo/genética , Orthobunyavirus/classificação , Orthobunyavirus/isolamento & purificação , Animais , Análise por Conglomerados , Culicidae/virologia , Ordem dos Genes , Peso Molecular , Proteínas do Nucleocapsídeo/química , Orthobunyavirus/química , Orthobunyavirus/genética , Filogenia , Estrutura Terciária de Proteína , RNA Viral/genética , Homologia de Sequência , Sintenia , Texas
18.
PLoS One ; 7(3): e32661, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22438882

RESUMO

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to ~300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-ß (IFN-ß) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-ß by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.


Assuntos
RNA Helicases DEAD-box/metabolismo , Genoma Viral , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sítios de Ligação/genética , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/química , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Influenza A/imunologia , Interferon beta/biossíntese , Interferon beta/genética , Cinética , Ligantes , Conformação de Ácido Nucleico , RNA Viral/química , Receptores Imunológicos , Proteínas não Estruturais Virais/genética
19.
PLoS One ; 6(1): e16285, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21283773

RESUMO

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.


Assuntos
HIV-1/fisiologia , Fragmentos de Peptídeos/farmacologia , Proteínas Tirosina Fosfatases/farmacologia , Sêmen/virologia , Fosfatase Ácida , Amiloide , Relação Dose-Resposta a Droga , Ampliador HIV , HIV-1/efeitos dos fármacos , Humanos , Antígeno Prostático Específico/metabolismo , Serina Endopeptidases/metabolismo
20.
Protein Sci ; 19(9): 1649-61, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20572021

RESUMO

The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the "cystine clusters" [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment.


Assuntos
Antivirais/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Cianobactérias/química , Dissulfetos/análise , Lectinas/química , Sequência de Aminoácidos , Antivirais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cianobactérias/metabolismo , Cistina/metabolismo , Lectinas/genética , Lectinas/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Pepsina A/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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