Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 44
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biosci Trends ; 13(3): 216-224, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31168022

RESUMO

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

2.
Anal Chem ; 91(10): 6440-6453, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31021607

RESUMO

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

3.
Fungal Genet Biol ; 124: 39-46, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30611835

RESUMO

The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suite of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains, ISSFT-021 and IF1SW-F4, isolated from HEPA filter debris and cupola surface of the ISS, respectively, is presented, along with a comparison to well-studied clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.


Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma , Astronave , Aspergillus fumigatus/isolamento & purificação , Metabolismo dos Carboidratos , Micotoxinas/metabolismo , Metabolismo Secundário , Estresse Fisiológico , Ausência de Peso
4.
J Biol Chem ; 294(12): 4368-4380, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670595

RESUMO

Angiotensin-converting enzyme (ACE) can hydrolyze many peptides and plays a central role in controlling blood pressure. Moreover, ACE overexpression in monocytes and macrophages increases resistance of mice to tumor growth. ACE is composed of two independent catalytic domains. Here, to investigate the specific role of each domain in tumor resistance, we overexpressed either WT ACE (Tg-ACE mice) or ACE lacking N- or C-domain catalytic activity (Tg-NKO and Tg-CKO mice) in the myeloid cells of mice. Tg-ACE and Tg-NKO mice exhibited strongly suppressed growth of B16-F10 melanoma because of increased ACE expression in macrophages, whereas Tg-CKO mice resisted melanoma no better than WT animals. The effect of ACE overexpression reverted to that of the WT enzyme with an ACE inhibitor but not with an angiotensin II type 1 (AT1) receptor antagonist. ACE C-domain overexpression in macrophages drove them toward a pronounced M1 phenotype upon tumor stimulation, with increased activation of NF-κB and signal transducer and activator of transcription 1 (STAT1) and decreased STAT3 and STAT6 activation. Tumor necrosis factor α (TNFα) is important for M1 activation, and TNFα blockade reverted Tg-NKO macrophages to a WT phenotype. Increased ACE C-domain expression increased the levels of reactive oxygen species (ROS) and of the transcription factor C/EBPß in macrophages, important stimuli for TNFα expression, and decreased expression of several M2 markers, including interleukin-4Rα. Natural ACE C-domain-specific substrates are not well-described, and we propose that the peptide(s) responsible for the striking ACE-mediated enhancement of myeloid function are substrates/products of the ACE C-domain.

5.
Appl Microbiol Biotechnol ; 103(3): 1363-1377, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30539259

RESUMO

The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.


Assuntos
Metabolismo dos Carboidratos/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Metabolismo Secundário/genética , Estresse Fisiológico/genética , Antraquinonas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Meio Ambiente , Genômica , Metabolômica , Proteômica , Metabolismo Secundário/fisiologia , Voo Espacial , Astronave , Xantonas/metabolismo
6.
FASEB J ; : fj201801848R, 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30452879

RESUMO

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

7.
mSystems ; 3(5)2018 Sep-Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30246146

RESUMO

The initial characterization of the Aspergillus niger isolate JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS), is reported, along with a comparison to the extensively studied strain ATCC 1015. Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement within the A. niger/welwitschiae/lacticoffeatus clade and revealed that the genome of JSC-093350089 is within the observed genetic variance of other sequenced A. niger strains. The ISS isolate exhibited an increased rate of growth and pigment distribution compared to a terrestrial strain. Analysis of the isolate's proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger on board the ISS and provide insight into the characteristics of melanized fungal species inhabiting spacecraft environments. IMPORTANCE A thorough understanding of how fungi respond and adapt to the various stimuli encountered during spaceflight presents many economic benefits and is imperative for the health of crew. As A. niger is a predominant ISS isolate frequently detected in built environments, studies of A. niger strains inhabiting closed systems may reveal information fundamental to the success of long-duration space missions. This investigation provides valuable insights into the adaptive mechanisms of fungi in extreme environments as well as countermeasures to eradicate unfavorable microbes. Further, it enhances understanding of host-microbe interactions in closed systems, which can help NASA's Human Research Program maintain a habitat healthy for crew during long-term manned space missions.

8.
Clin Cancer Res ; 24(5): 1216-1226, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29180608

RESUMO

Purpose: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp overexpression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers.Experimental Design: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival. To assess t-Darpp's metabolic effects, we used the Seahorse flux analyzer to measure glucose metabolism in trastuzumab-resistant SK-BR-3 cells (SK.HerR) that have high endogenous t-Darpp levels and SK.tDrp cells that stably overexpress exogenous t-Darpp. To investigate t-Darpp's mechanism of action, we evaluated t-Darpp:IGF-1R complexes by coimmunoprecipitation and proximity ligation assays. We used pathway-specific inhibitors to study the dependence of t-Darpp effects on IGF-1R signaling. We used siRNA knockdown to determine whether glucose reliance in SK.HerR cells was mediated by t-Darpp.Results: In breast tumors, PPP1R1B mRNA levels were inversely correlated with IGF-1R mRNA levels and directly associated with shorter overall survival. t-Darpp overexpression was sufficient to increase glucose metabolism in SK.tDrp cells and essential for the glycolytic phenotype of SK.HerR cells. Recombinant t-Darpp stimulated glucose uptake, glycolysis, and IGF-1R-Akt signaling in SK-BR-3 cells. Finally, t-Darpp stimulated IGF-1R heterodimerization with ErbB receptors and required IGF-1R signaling to confer its metabolic effects.Conclusions: t-Darpp activates IGF-1R signaling through heterodimerization with EGFR and HER2 to stimulate glycolysis and confer trastuzumab resistance. Clin Cancer Res; 24(5); 1216-26. ©2017 AACR.

9.
J Biol Chem ; 293(1): 368-378, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29101228

RESUMO

Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B- and l-rhamnose-specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose-containing microorganisms.


Assuntos
Hemaglutininas/química , Hemaglutininas/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular/métodos , Galactose/metabolismo , Lectinas/metabolismo , Espectrometria de Massas/métodos , Peso Molecular , Polissacarídeos/metabolismo , Ramnose/metabolismo
10.
Methods Mol Biol ; 1625: 281-293, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28584997

RESUMO

Adaptive effector CD4+ T cells play essential roles in the defense against fungal infections, especially against invasive aspergillosis (IA). Such protective CD4+ T cells can be generated through immunization with specialized antifungal vaccines, as has been demonstrated for pulmonary Aspergillus fumigatus infections in mouse experiments. Adaptive transfer of fungal antigen-specific CD4+ T cells conferred protection onto non-immunized naive mice, an experimental approach that could potentially become a future treatment option for immunosuppressed IA patients, focusing on the ultimate goal to improve their otherwise dim chances for survival. Here, we describe the different techniques to analyze CD4+ T cell immune responses after immunization with a recombinant fungal protein. We present three major methods that are used to analyze the role of CD4+ T cells in protection against A. fumigatus challenge. They include (1) transplantation of CD4+ T cells from vaccinated mice into immunosuppressed naive mice, observing increasing protection of the cell recipients, (2) depletion of CD4+ T cells from vaccinated mice, which abolishes vaccine protection, and (3) T cell proliferation studies following stimulation with overlapping synthetic peptides or an intact protein vaccine. The latter can be used to validate immunization status and to identify protective T cell epitopes in vaccine antigens. In the methods detailed here, we used versions of the well-studied Asp f3 protein expressed in a bacterial host, either as the intact full length protein or its N-terminally truncated version, comprised of residues 15-168. However, these methods are generally applicable and can well be adapted to study other protein-based subunit vaccines.


Assuntos
Aspergilose/imunologia , Aspergilose/prevenção & controle , Aspergillus/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Fúngicas/imunologia , Transferência Adotiva , Animais , Anticorpos Antifúngicos/imunologia , Aspergilose/microbiologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/imunologia , Feminino , Vacinas Fúngicas/administração & dosagem , Imunização , Hospedeiro Imunocomprometido , Imunofenotipagem , Ativação Linfocitária , Depleção Linfocítica , Camundongos , Peptídeos/imunologia
11.
Sci Rep ; 7: 44321, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28287171

RESUMO

A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

12.
Oncotarget ; 8(8): 13818-13831, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28099154

RESUMO

Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Varfarina/farmacologia , Animais , Anticoagulantes/farmacologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Espectrometria de Massas , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos
13.
Behav Neurol ; 2017: 5238402, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29358844

RESUMO

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV's beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms.


Assuntos
Aciclovir/farmacologia , Encefalite por Herpes Simples/tratamento farmacológico , Imunoglobulinas Intravenosas/farmacologia , Animais , Feminino , Herpesvirus Humano 1/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
14.
Sci Rep ; 6: 33396, 2016 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-27624005

RESUMO

Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.


Assuntos
Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/química , Estresse Oxidativo , Peroxirredoxinas/química , Animais , Aspergilose/microbiologia , Cristalografia por Raios X , Feminino , Deleção de Genes , Cinética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Peroxidase/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Superóxidos/toxicidade , Virulência
15.
Microbiologyopen ; 5(5): 802-818, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27353144

RESUMO

Bacterial-derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host-microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human-derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10-methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10-methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti-inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid-induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild-type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial-derived immunoregulatory molecules may lead to improved anti-inflammatory strategies for digestive diseases.


Assuntos
Colite/terapia , Lactobacillus reuteri/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/metabolismo , Probióticos/uso terapêutico , Animais , Células Cultivadas , Colite/induzido quimicamente , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/terapia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , Tetra-Hidrofolatos/metabolismo , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa/biossíntese
16.
Nat Struct Mol Biol ; 23(7): 656-62, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27294781

RESUMO

Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan-which is conserved in all SV2 isoforms across vertebrates-is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.


Assuntos
Anticorpos Monoclonais/química , Antídotos/química , Toxinas Botulínicas Tipo A/química , Clostridium botulinum/química , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Toxinas Botulínicas Tipo A/metabolismo , Clonagem Molecular , Clostridium botulinum/patogenicidade , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Nat Commun ; 7: 10965, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26965827

RESUMO

Dysregulated expression of miR-219, a brain-specific microRNA, has been observed in neurodevelopmental disorders, such as schizophrenia (SCZ). However, its role in normal mammalian neural stem cells (NSCs) and in SCZ pathogenesis remains unknown. We show here that the nuclear receptor TLX, an essential regulator of NSC proliferation and self-renewal, inhibits miR-219 processing. miR-219 suppresses mouse NSC proliferation downstream of TLX. Moreover, we demonstrate upregulation of miR-219 and downregulation of TLX expression in NSCs derived from SCZ patient iPSCs and DISC1-mutant isogenic iPSCs. SCZ NSCs exhibit reduced cell proliferation. Overexpression of TLX or inhibition of miR-219 action rescues the proliferative defect in SCZ NSCs. Therefore, this study uncovers an important role for TLX and miR-219 in both normal neurodevelopment and in SCZ patient iPSC-derived NSCs. Moreover, this study reveals an unexpected role for TLX in regulating microRNA processing, independent of its well-characterized role in transcriptional regulation.


Assuntos
Proliferação de Células/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neurais/metabolismo , Processamento Pós-Transcricional do RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Esquizofrenia/genética , Animais , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Eletroporação , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Proteínas do Tecido Nervoso/genética , Neurogênese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esquizofrenia/metabolismo
18.
J Fungi (Basel) ; 2(1)2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26878023

RESUMO

We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry-Elevated Collision Energy). Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-ß-glucanosyltransferases (Bgt1, Gel1-4), Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

19.
Comp Med ; 63(6): 477-81, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24326222

RESUMO

Immunocompromised mice were infected intranasally with Aspergillus fumigatus as part of a vaccine efficacy study. Although body temperature was measured throughout the study, a formal evaluation of its usefulness as an endpoint criterion was not performed. We retrospectively evaluated survival data and temperature records to determine whether body temperature can be used as an objective predictor of death and included in the humane endpoint criteria for this mouse model. CF1 mice were immunosuppressed with either cortisone acetate or by treatment with antiGR1 (a neutrophil-depleting antibody) and then intranasally challenged with A. fumigatus. Body temperature was measured by using an infrared noncontact thermometer a maximum of 3 times daily until death or euthanasia. A surface body temperature below 29.0 °C was correlated with a poor chance of survival, and using this cutoff point with signs of morbidity (hunched, ruffled fur, respiratory distress) reliably indicates mice for euthanasia without negatively affecting data collection. Using 2 subsequent readings of less than 31.0 °C as an endpoint would have led to premature euthanasia of only one mouse (2.2%). As a single reading, a body temperature of 28.8 °C had a sensitivity of 92.2% and specificity of 90.9%. Hypothermia proved to be a useful addition to the humane endpoint criteria for this mouse model, and veterinary and research groups should discuss their study needs in relation to animal welfare to best determine the most appropriate means of including this parameter.


Assuntos
Modelos Animais de Doenças , Hipotermia/fisiopatologia , Aspergilose Pulmonar/fisiopatologia , Animais , Regulação da Temperatura Corporal , Feminino , Camundongos , Aspergilose Pulmonar/imunologia
20.
Anal Chem ; 85(11): 5569-76, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23656526

RESUMO

Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT "Assay with a Large Immunosorbent Surface Area" that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis-Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ~3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 µg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.


Assuntos
Bioensaio , Toxinas Botulínicas/análise , Botulismo/metabolismo , Endopeptidases/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/metabolismo , Cromatografia Líquida/métodos , Modelos Animais de Doenças , Feminino , Humanos , Cinética , Camundongos , Proteínas Recombinantes/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA