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1.
J Biol Chem ; 295(18): 6165-6176, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32179648

RESUMEN

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+ Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+ This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.


Asunto(s)
Bacterias/citología , Bacterias/metabolismo , Carbono/metabolismo , NAD/biosíntesis , Nitrógeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Transducción de Señal , Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína
2.
J Biol Chem ; 294(22): 8717-8731, 2019 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-30967474

RESUMEN

Costameres are signaling hubs at the sarcolemma and important contact points between the extracellular matrix and cell interior, sensing and transducing biomechanical signals into a cellular response. The transmembrane proteoglycan syndecan-4 localizes to these attachment points and has been shown to be important in the initial stages of cardiac remodeling, but its mechanistic function in the heart remains insufficiently understood. Here, we sought to map the cardiac interactome of syndecan-4 to better understand its function and downstream signaling mechanisms. By combining two different affinity purification methods with MS analysis, we found that the cardiac syndecan-4 interactome consists of 21 novel and 29 previously described interaction partners. Nine of the novel partners were further validated to bind syndecan-4 in HEK293 cells (i.e. CAVIN1/PTRF, CCT5, CDK9, EIF2S1, EIF4B, MPP7, PARVB, PFKM, and RASIP). We also found that 19 of the 50 interactome partners bind differently to syndecan-4 in the left ventricle lysate from aortic-banded heart failure (ABHF) rats compared with SHAM-operated animals. One of these partners was the well-known mechanotransducer muscle LIM protein (MLP), which showed direct and increased binding to syndecan-4 in ABHF. Nuclear translocation is important in MLP-mediated signaling, and we found less MLP in the nuclear-enriched fractions from syndecan-4-/- mouse left ventricles but increased nuclear MLP when syndecan-4 was overexpressed in a cardiomyocyte cell line. In the presence of a cell-permeable syndecan-4-MLP disruptor peptide, the nuclear MLP level was reduced. These findings suggest that syndecan-4 mediates nuclear translocation of MLP in the heart.


Asunto(s)
Núcleo Celular/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas Musculares/metabolismo , Sindecano-4/metabolismo , Animales , Línea Celular , Células HEK293 , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Proteínas con Dominio LIM/química , Ratones , Ratones Noqueados , Proteínas Musculares/química , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Dominios PDZ , Mapas de Interacción de Proteínas , Transporte de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Sindecano-4/química , Sindecano-4/genética
3.
Mol Cell Proteomics ; 17(5): 850-870, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29371290

RESUMEN

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.


Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Factores de Crecimiento de Fibroblastos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Osteosarcoma/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteómica , Reproducibilidad de los Resultados
4.
Nat Methods ; 13(10): 837-40, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27571551

RESUMEN

We report a tool for the analysis of subcellular proteomics data, called MetaMass, based on the use of standardized lists of subcellular markers. We analyzed data from 11 studies using MetaMass, mapping the subcellular location of 5,970 proteins. Our analysis revealed large variations in the performance of subcellular fractionation protocols as well as systematic biases in protein annotation databases. The Excel and R versions of MetaMass should enhance transparency and reproducibility in subcellular proteomics.


Asunto(s)
Metaanálisis como Asunto , Proteínas/metabolismo , Proteómica/métodos , Fracciones Subcelulares/metabolismo , Algoritmos , Animales , Biomarcadores/metabolismo , Células Cultivadas , Células Madre Embrionarias/metabolismo , Ratones , Proteómica/estadística & datos numéricos
5.
Bioessays ; 39(7)2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28582591

RESUMEN

In this manuscript we describe Proteogenomics Viewer, a web-based tool that collects MS peptide identification, indexes to genomic sequence and structure, assigns exon usage, reports the identified protein isoforms with genomic alignments and, most importantly, allows the inspection of MS2 information for proper peptide identification. It also provides all performed indexing to facilitate global analysis of the data. The relevance of such tool is that there has been an increase in the number of proteogenomic efforts to improve the annotation of both genomics and proteomics data, culminating with the release of the two human proteome drafts. It is now clear that mass spectrometry-based peptide identification of uncharacterized sequences, such as those resulting from unpredicted exon joints or non-coding regions, is still prone to a higher than expected false discovery rate. Therefore, proper visualization of the raw data and the corresponding genome alignments are fundamental for further data validation and interpretation. Also see the video abstract here: http://youtu.be/5NzyRvuk4Ac.


Asunto(s)
Genoma/genética , Espectrometría de Masas/métodos , Péptidos/genética , Proteogenómica/métodos , Genómica/métodos , Humanos , Proteoma/genética , Proteómica/métodos
6.
Mol Cell Proteomics ; 15(3): 1007-16, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26637539

RESUMEN

T-helper cells are differentiated from CD4+ T cells and are traditionally characterized by inflammatory or immunosuppressive responses in contrast to cytotoxic CD8+ T cells. Mass-spectrometry studies on T-helper cells are rare. In this study, we aimed to identify the proteomes of human Th1 and Th1/Th17 clones derived from intestinal biopsies of Crohn's disease patients and to identify differentially expressed proteins between the two phenotypes. Crohn's disease is an inflammatory bowel disease, with predominantly Th1- and Th17-mediated response where cells of the "mixed" phenotype Th1/Th17 have also been commonly found. High-resolution mass spectrometry was used for protein identification and quantitation. In total, we identified 7401 proteins from Th1 and Th1/Th17 clones, where 334 proteins were differentially expressed. Major differences were observed in cytotoxic proteins that were overrepresented in the Th1 clones. The findings were validated by flow cytometry analyses using staining with anti-granzyme B and anti-perforin and by a degranulation assay, confirming higher cytotoxic features of Th1 compared with Th1/Th17 clones. By testing a larger panel of T-helper cell clones from seven different Crohn's disease patients, we concluded that only a subgroup of the Th1 cell clones had cytotoxic features, and these expressed the surface markers T-cell-specific surface glycoprotein CD28 and were negative for expression of natural killer group 2 member D.


Asunto(s)
Antígenos CD28/metabolismo , Enfermedad de Crohn/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/deficiencia , Proteómica/métodos , Células TH1/metabolismo , Células Th17/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Enfermedad de Crohn/patología , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Mucosa Intestinal/metabolismo , Espectrometría de Masas , Proteoma/inmunología , Proteoma/aislamiento & purificación , Linfocitos T Citotóxicos/metabolismo
7.
Proteomics ; 17(17-18)2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28755400

RESUMEN

The sodium (Na+ )-calcium (Ca2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca2+ homeostasis, serving as the primary mechanism for Ca2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti-NCX1 against endogenous NCX1 and (2) anti-His (where His is histidine) with His-trigger factor-NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where "cell communication" and "signal transduction" formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in "cardiovascular disease" which can be explored as novel drug targets in future research.


Asunto(s)
Ventrículos Cardíacos/metabolismo , Corazón/fisiología , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Intercambiador de Sodio-Calcio/metabolismo , Animales , Calcio/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Transducción de Señal , Sodio/metabolismo
8.
J Proteome Res ; 15(10): 3841-3855, 2016 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-27615514

RESUMEN

The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.


Asunto(s)
Endosomas/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Biotinilación , Línea Celular Tumoral , Clatrina/metabolismo , Endocitosis , Humanos , Microscopía/métodos , Transporte de Proteínas , Transducción de Señal , Coloración y Etiquetado , Red trans-Golgi/metabolismo
9.
Anal Chem ; 85(4): 2478-85, 2013 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-23316706

RESUMEN

Isobaric peptide termini labeling (IPTL) is based on labeling of both peptide termini with complementary isotopic labels resulting in isobaric peptides. MS/MS analysis after IPTL derivatization produces peptide-specific fragment ions which are distributed throughout the MS/MS spectrum. Thus, several quantification points can be obtained per peptide. In this report, we present triplex-IPTL, a chemical labeling strategy for IPTL allowing the simultaneous quantification of three states within one MS run. For this purpose, dimethylation of the N-terminal amino group followed by dimethylation of lysines was used with different stable isotopes of formaldehyde and cyanoborohydride. Upon LC-MS/MS analysis, the combined samples revealed three corresponding isotopic fragment ion series reflecting quantitatively the peptide ratios. To support this multiplexing labeling strategy, we have further developed the data analysis tool IsobariQ and included multidimensional VSN normalization, statistical inference, and graphical visualization of triplex-IPTL data and clustering of protein profiling patterns. The power of the triplex-IPTL approach in combination with IsobariQ was demonstrated through temporal profiling of HeLa cells incubated with the kinesin Eg5 inhibitor S-Trityl-l-cysteine (STLC). As a result, clusters of quantified proteins were found by their ratio profiles which corresponded well to their gene ontology association in mitotic arrest and cell death, respectively.


Asunto(s)
Péptidos/análisis , Espectrometría de Masas en Tándem , Apoptosis/efectos de los fármacos , Isótopos de Carbono/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Cisteína/farmacología , Deuterio/química , Células HeLa , Humanos , Marcaje Isotópico , Péptidos/química
10.
Neurosurgery ; 2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-38084989

RESUMEN

BACKGROUND AND OBJECTIVES: A bacterial brain abscess is an emergency and should be drained of pus within 24 hours of diagnosis, as recently recommended. In this cross-sectional study, we investigated whether delaying pus drainage entails brain abscess expansion and what the underlying mechanism might be. METHODS: Repeated brain MRI of 47 patients who did not undergo immediate pus drainage, pus osmolarity measurements, immunocytochemistry, proteomics, and 18F-fluorodeoxyglucose positron emission tomography. RESULTS: Time from first to last MRI before neurosurgery was 1 to 14 days. Abscesses expanded in all but 2 patients: The median average increase was 23% per day (range 0%-176%). Abscesses expanded during antibiotic therapy and even if the pus did not contain viable bacteria. In a separate patient cohort, we found that brain abscess pus tended to be hyperosmolar (median value 360 mOsm; range 266-497; n = 14; normal cerebrospinal fluid osmolarity is ∼290 mOsm). Hyperosmolarity would draw water into the abscess cavity, causing abscess expansion in a ballooning manner through increased pressure in the abscess cavity. A mechanism likely underlying pus hyperosmolarity was the recruitment of neutrophils to the abscess cavity with ensuing neutrophil cell death and decomposition of neutrophil proteins and other macromolecules to osmolytes: Pus analysis showed the presence of neutrophil proteins (protein-arginine deiminases, citrullinated histone, myeloperoxidase, elastase, cathelicidin). Previous studies have shown very high levels of osmolytes (ammonia, amino acids) in brain abscess pus. 18F-fluorodeoxyglucose positron emission tomography showed focal neocortical hypometabolism 1 to 8 years after brain abscess, indicating long-lasting damage to brain tissue. CONCLUSION: Brain abscesses expand despite effective antibiotic treatment. Furthermore, brain abscesses cause lasting damage to surrounding brain tissue. These findings support drainage of brain abscesses within 24 hours of diagnosis.

11.
Fluids Barriers CNS ; 19(1): 45, 2022 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-35659255

RESUMEN

BACKGROUND: The growth of malignant tumors is influenced by their microenvironment. Glioblastoma, an aggressive primary brain tumor, may have cysts containing fluid that represents the tumor microenvironment. The aim of this study was to investigate whether the cyst fluid of cystic glioblastomas contains growth-stimulating factors. Identification of such growth factors may pave the way for the development of targeted anti-glioblastoma therapies. METHODS: We performed hormone analysis of cyst fluid from 25 cystic glioblastomas and proteomics analysis of cyst fluid from another 12 cystic glioblastomas. RESULTS: Glioblastoma cyst fluid contained hormones within wide concentration ranges: Insulin-like growth factor 1 (0-13.7 nmol/L), insulin (1.4-133 pmol/L), erythropoietin (4.7-402 IU/L), growth hormone (0-0.93 µg/L), testosterone (0.2-10.1 nmol/L), estradiol (0-1.0 nmol/L), triiodothyronine (1.0-11.5). Tumor volume correlated with cyst fluid concentrations of growth hormone and testosterone. Survival correlated inversely with cyst fluid concentration of erythropoietin. Several hormones were present at concentrations that have been shown to stimulate glioblastoma growth in vitro. Concentrations of erythropoietin and estradiol (in men) were higher in cyst fluid than in serum, suggesting formation by tumor or brain tissue. Quantitatively, glioblastoma cyst fluid was dominated by serum proteins, illustrating blood-brain barrier leakage. Proteomics identified several proteins that stimulate tumor cell proliferation and invasiveness, others that inhibit apoptosis or mediate adaption to hypoxia and some that induce neovascularization or blood-brain barrier leakage. CONCLUSION: The microenvironment of glioblastomas is rich in growth-stimulating factors that may originate from the circulation, the tumor, or the brain. The wide variation in cyst fluid hormone concentrations may differentially influence tumor growth.


Asunto(s)
Eritropoyetina , Glioblastoma , Estradiol/análisis , Estradiol/farmacología , Glioblastoma/metabolismo , Hormona del Crecimiento , Humanos , Masculino , Testosterona , Microambiente Tumoral
12.
Cancers (Basel) ; 14(20)2022 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-36291793

RESUMEN

BACKGROUND: Uterine leiomyosarcoma (uLMS) are rare and malignant tumors that arise in the myometrium cells and whose diagnosis is based on histopathological features. Identifying diagnostic biomarkers for uLMS is a challenge due to molecular heterogeneity and the scarcity of samples. In vivo and in vitro models for uLMS are urgently needed. Knockout female mice for the catalytic subunit of the immunoproteasome PSMB9 (MIM:177045) develop spontaneous uLMS. This study aimed to analyze the role of PSMB9 in uLMS tumorigenesis and patient outcome. METHODS: Molecular data from 3 non-related uLMS cohorts were integrated and analyzed by proteotranscriptomic using gene expression and protein abundance levels in 68 normal adjacent myometrium (MM), 66 uterine leiomyoma (LM), and 67 uLMS. RESULTS: the immunoproteasome pathway is upregulated and the gene PMSB9 shows heterogeneous expression values in uLMS. Quartile group analysis showed no significant difference between groups high and low PSMB9 expression groups at 3-years overall survival (OS). Using CYBERSORTx analysis we observed 9 out of 17 samples in the high group clustering together due to high M2 macrophages and CD4 memory resting, and high CD8+/PSMB9 ratio was associated with better OS. The main pathway regulated in the high group is IFNγ and in the low is the ECM pathway dependent on the proto-oncogene SRC. CONCLUSION: these findings suggest 2 subtypes of uLMS (immune-related and ECM-related) with different candidate mechanisms of malignancy.

13.
J Neuroimmunol ; 333: 576966, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31153015

RESUMEN

In multiple sclerosis (MS), B cells are trafficking across the blood-brain barrier, but it is not known how this relates to the synthesis of oligoclonal IgG. We used quantitative mass spectrometry of oligoclonal bands and high-throughput sequencing of immunoglobulin heavy-chain variable transcripts to study the longitudinal B cell response in the cerebrospinal fluid (CSF) and blood of two MS patients. Twenty of 22 (91%) and 25 of 28 (89%) of oligoclonal band peptides persisted in samples collected 18 months apart, in spite of a dynamic exchange across the blood-CSF barrier of B lineage cells connecting to oligoclonal IgG.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Inmunoglobulina G/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Bandas Oligoclonales/líquido cefalorraquídeo , Adulto , Secuencia de Aminoácidos , Barrera Hematoencefálica , Linaje de la Célula , Femenino , Estudios de Seguimiento , Reordenamiento Génico de Linfocito B , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Factores Inmunológicos/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcriptoma , Adulto Joven
14.
Front Microbiol ; 10: 1410, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31281302

RESUMEN

In proteomics, peptide information within mass spectrometry (MS) data from a specific organism sample is routinely matched against a protein sequence database that best represent such organism. However, if the species/strain in the sample is unknown or genetically poorly characterized, it becomes challenging to determine a database which can represent such sample. Building customized protein sequence databases merging multiple strains for a given species has become a strategy to overcome such restrictions. However, as more genetic information is publicly available and interesting genetic features such as the existence of pan- and core genes within a species are revealed, we questioned how efficient such merging strategies are to report relevant information. To test this assumption, we constructed databases containing conserved and unique sequences for 10 different species. Features that are relevant for probabilistic-based protein identification by proteomics were then monitored. As expected, increase in database complexity correlates with pangenomic complexity. However, Mycobacterium tuberculosis and Bordetella pertussis generated very complex databases even having low pangenomic complexity. We further tested database performance by using MS data from eight clinical strains from M. tuberculosis, and from two published datasets from Staphylococcus aureus. We show that by using an approach where database size is controlled by removing repeated identical tryptic sequences across strains/species, computational time can be reduced drastically as database complexity increases.

15.
Proteomics ; 8(9): 1859-70, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18442171

RESUMEN

Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.


Asunto(s)
Membrana Celular/metabolismo , Detergentes/farmacología , Proteínas de la Membrana/química , Mycobacterium bovis/metabolismo , Polietilenglicoles/farmacología , Proteómica/métodos , Complejos de ATP Sintetasa/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida/métodos , Complejo I de Transporte de Electrón/química , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , NADP Transhidrogenasas/química , Octoxinol , Estructura Terciaria de Proteína , Fracciones Subcelulares
16.
J Neurosurg ; 129(3): 829-837, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29053067

RESUMEN

OBJECTIVE What determines the extent of tissue destruction during brain abscess formation is not known. Pyogenic brain infections cause destruction of brain tissue that greatly exceeds the area occupied by microbes, as seen in experimental studies, pointing to cytotoxic factors other than microbes in pus. This study examined whether brain abscess pus contains cytotoxic proteins that might explain the extent of tissue destruction. METHODS Pus proteins from 20 human brain abscesses and, for comparison, 7 subdural empyemas were analyzed by proteomics mass spectrometry. Tissue destruction was determined from brain abscess volumes as measured by MRI. RESULTS Brain abscess volume correlated with extracellular pus levels of antibacterial proteins from neutrophils and macrophages: myeloperoxidase (r = 0.64), azurocidin (r = 0.61), lactotransferrin (r = 0.57), and cathelicidin (r = 0.52) (p values 0.002-0.018), suggesting an association between leukocytic activity and tissue damage. In contrast, perfringolysin O, a cytotoxic protein from Streptococcus intermedius that was detected in 16 patients, did not correlate with abscess volume (r = 0.12, p = 0.66). The median number of proteins identified in each pus sample was 870 (range 643-1094). Antibiotic or steroid treatment prior to pus evacuation did not reduce the number or levels of pus proteins. Some of the identified proteins have well-known neurotoxic effects, e.g., eosinophil cationic protein and nonsecretory ribonuclease (also known as eosinophil-derived neurotoxin). The cellular response to brain infection was highly complex, as reflected by the presence of proteins that were specific for neutrophils, eosinophils, macrophages, platelets, fibroblasts, or mast cells in addition to plasma and erythrocytic proteins. Other proteins (neurofilaments, myelin basic protein, and glial fibrillary acidic protein) were specific for brain cells and reflected damage to neurons, oligodendrocytes, and astrocytes, respectively. Pus from subdural empyemas had significantly higher levels of plasma proteins and lower levels of leukocytic proteins than pus from intracerebral abscesses, suggesting greater turnover of the extracellular fluid of empyemas and washout of pus constituents. CONCLUSIONS Brain abscess pus contains leukocytic proteins that are neurotoxic and likely participate actively in the excessive tissue destruction inherent in brain abscess formation. These findings underscore the importance of rapid evacuation of brain abscess pus.


Asunto(s)
Absceso Encefálico/genética , Neurotoxinas/genética , Proteoma/genética , Supuración/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Péptidos Catiónicos Antimicrobianos/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Sanguíneas/metabolismo , Encéfalo/patología , Absceso Encefálico/patología , Proteínas Portadoras/metabolismo , Niño , Preescolar , Empiema Subdural/genética , Empiema Subdural/patología , Eosinófilos/patología , Femenino , Proteínas Hemolisinas/metabolismo , Humanos , Lactoferrina/metabolismo , Macrófagos/patología , Masculino , Mastocitos/patología , Persona de Mediana Edad , Neutrófilos/patología , Peroxidasa/metabolismo , Supuración/patología , Adulto Joven , Catelicidinas
17.
Oncotarget ; 8(26): 43114-43129, 2017 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-28562344

RESUMEN

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.


Asunto(s)
Antineoplásicos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Proteínas Represoras/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Melanoma/genética , Melanoma/patología , Prohibitinas , Proteómica , Proteínas Represoras/genética , Tunicamicina/farmacología
18.
Oncotarget ; 8(54): 92966-92977, 2017 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-29190970

RESUMEN

Cancer/testis (CT) genes are excellent candidates for cancer immunotherapies because of their restrict expression in normal tissues and the capacity to elicit an immune response when expressed in tumor cells. In this study, we provide a genome-wide screen for CT genes with the identification of 745 putative CT genes. Comparison with a set of known CT genes shows that 201 new CT genes were identified. Integration of gene expression and clinical data led us to identify dozens of CT genes associated with either good or poor prognosis. For the CT genes related to good prognosis, we show that there is a direct relationship between CT gene expression and a signal for CD8+ cells infiltration for some tumor types, especially melanoma.

19.
Rev Assoc Med Bras (1992) ; 52(4): 203-7, 2006.
Artículo en Portugués | MEDLINE | ID: mdl-16967135

RESUMEN

OBJECTIVES: To analyze the occurrence of adverse effects in the first 24-hour postoperative/postanesthetic period in women undergoing minor gynecologic or minor breast surgeries and to identify main associated factors. METHODS: A cross-sectional study was conducted with 159 women who underwent minor gynecologic or breast surgeries. The women were admitted to the hospital one day before surgery and remained hospitalized for at least 24 hours after surgery. The anesthetic techniques performed were intercostal nerve blockade, spinal anesthesia, and general anesthesia. RESULTS: The most frequent adverse effects were vomiting, nausea and pain that occurred in 40.3% of women. Of these effects, 60% were observed in the first four hours and 80% were observed in up to six hours after surgical intervention. Women submitted to intercostal blockade received earlier postanesthetic release. Spinal anesthesia was most frequently associated with postoperative pain, although with a lower incidence of nausea and vomiting when compared to general anesthesia and intercostal blockade. The incidence of pain was higher in women who smoked. CONCLUSIONS: A six-hour period of postoperative observation appeared to be adequate for assessment of most complications and adverse effects occurring in women who undergo minor gynecologic or minor breast surgeries.


Asunto(s)
Anestesia/efectos adversos , Procedimientos Quirúrgicos Ginecológicos/efectos adversos , Mastectomía/efectos adversos , Dolor Postoperatorio/epidemiología , Náusea y Vómito Posoperatorios/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Comorbilidad , Estudios Transversales , Femenino , Humanos , Persona de Mediana Edad , Dolor Postoperatorio/etiología , Náusea y Vómito Posoperatorios/etiología , Estadísticas no Paramétricas , Factores de Tiempo
20.
Mol Ther Nucleic Acids ; 5(10): e373, 2016 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-27727249

RESUMEN

Osteoarthritis is a serious disease of articular cartilage. The pathogenic factors contributing to this disorder are inflammation, extracellular matrix degradation and failure to rebuild the articular cartilage. Preclinical studies suggest that microRNA-140 may play a protective role in osteoarthritis development, but little is known about the mechanism by which this occurs. Here we present the results of forced expression of microRNA-140 in an in vitro model of osteoarthritis, evaluated by global proteomics analysis. We show that inflammation was reduced through the altered levels of multiple proteins involved in the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 pathway. microRNA-140 upregulated many of the components involved in the synthesis of hyaline extracellular matrix and reduced the levels of aggrecanases and syndecan 4, thus potentially both increasing cartilage repair and reducing cartilage breakdown. These results show how forced expression of microRNA-140 is likely to counteract all three pathogenic processes, and support the idea that intra-articular injection of microRNA-140 may benefit patients suffering from early osteoarthritis.

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