Publications by authors named "Monique R O Trugilho"

12 Publications

  • Page 1 of 1

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium.

Protist 2019 12 1;170(6):125698. Epub 2019 Nov 1.

Laboratory of Toxinology, IOC, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ 21040-900, Brazil. Electronic address:

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.
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http://dx.doi.org/10.1016/j.protis.2019.125698DOI Listing
December 2019

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

Sci Transl Med 2017 Sep;9(409)

Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.
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http://dx.doi.org/10.1126/scitranslmed.aan1589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612058PMC
September 2017

Dataset of proteins mapped on HepG2 cells and those differentially abundant after expression of the dengue non-structural 1 protein.

Data Brief 2017 Feb 6;10:248-263. Epub 2016 Dec 6.

Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.

The data supplied in this article are related to the research article entitled "The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach" (K. Rabelo, M.R. Trugillo, S.M. Costa, B.A. Pereira, O.C. Moreira, A.T. Ferreira et al., 2016) [1]. The present article provides the inventory of peptides and proteins mapped in a hepatocyte cell line (HepG2) by mass spectrometry in the presence of the non-structural protein 1 (NS1) of Dengue 2 virus (DENV2). Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) or pMAXGFP, encoding the green fluorescent protein (GFP), a protein unrelated to dengue. Differentially abundant protein lists were obtained by comparing cells transfected with pcENS1 and controls.
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http://dx.doi.org/10.1016/j.dib.2016.11.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5153426PMC
February 2017

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.

J Proteomics 2017 01 5;152:339-354. Epub 2016 Nov 5.

Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. Electronic address:

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines.

Biological Significance: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.
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http://dx.doi.org/10.1016/j.jprot.2016.11.001DOI Listing
January 2017

Proteomic analysis of the secretome of HepG2 cells indicates differential proteolytic processing after infection with dengue virus.

J Proteomics 2017 01 15;151:106-113. Epub 2016 Jul 15.

Rede Proteômica do Rio de Janeiro, Brazil; Programa de Biologia Estrutural, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil. Electronic address:

Secretome analysis can be described as a subset of proteomics studies consisting in the analysis of the molecules secreted by cells or tissues. Dengue virus (DENV) infection can lead to a broad spectrum of clinical manifestations, with the severe forms of the disease characterized by hemostasis abnormalities and liver injury. The hepatocytes are a relevant site of viral replication and a major source of plasma proteins. Until now, we had limited information on the small molecules secreted by hepatic cells after infection by DENV. In the present study, we analysed a fraction of the secretome of mock- and DENV-infected hepatic cells (HepG2 cells) containing molecules with <10kDa, using different proteomic approaches. We identified 175 proteins, with 57 detected only in the samples from mock-infected cells, 59 only in samples from DENV-infected cells, and 59 in both conditions. Most of the peptides identified were derived from proteins larger than 10kDa, suggesting a proteolytic processing of the secreted molecules. Using in silico analysis, we predicted consistent differences between the proteolytic processing occurring in mock and DENV-infected samples, raising, for the first time, the hypothesis that differential proteolysis of secreted molecules would be involved in the pathogenesis of dengue.

Biological Significance: Since the liver, one of the targets of DENV infection, is responsible for producing molecules involved in distinct biological processes, the identification of proteins and peptides secreted by hepatocytes after infection would help to a better understanding of the physiopathology of dengue. Proteomic analyses of molecules with <10kDa secreted by HepG2 cells after infection with DENV revealed differential proteolytic processing as an effect of DENV infection.
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http://dx.doi.org/10.1016/j.jprot.2016.07.011DOI Listing
January 2017

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

J Proteomics 2017 01 20;151:204-213. Epub 2016 May 20.

Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Toxinas, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil. Electronic address:

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA, a few spots corresponding to acidic PLA were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies.

Biological Significance: Local tissue damage induced by myotoxic PLA remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this important antivenom protein.
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http://dx.doi.org/10.1016/j.jprot.2016.05.020DOI Listing
January 2017

Alterations of the kidney cortex proteome in response to exercise training in normoglycemic and hyperglycemic conditions.

Curr Top Med Chem 2014 ;14(3):450-61

Laboratorio de Investigacao Cardiovascular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365 - Manguinhos, 21045-900, Rio de Janeiro, RJ, Brazil.

Hyperglycemia induces systemic vascular endothelial dysfunction and renal damage through the overproduction of reactive oxygen species (ROS). Regular aerobic exercise decreases the incidence of ROS-associated diseases and is involved in protection against systemic and renal vascular alterations. To investigate the impact of exercise training on renal protein expression in hyperglycemic conditions, we performed gel-based proteomic analyses of the rabbit kidney cortex from sedentary and exercised rabbits after exposure to normal or high glucose concentrations. Abundance of proteins in the renal cortex was determined by two-dimensional polyacrylamide gel electrophoresis followed by protein identification with mass spectrometry, using peptide mass and fragment fingerprintings. We identified the differential abundance of twenty seven proteins in exercise trained animals among the total of 324 spots, from which five proteins are related to the down-regulation of cellular oxidative stress (albumin, protein disulfide isomerase, heat shock protein 60-like chaperonin, DJ-1 and ubiquinol-cytochrome-c reductase), and three proteins are involved in energy metabolism (shortchain acyl-coenzyme A dehydrogenase, malate dehydrogenase and L-arginine-glycine amidinotransferase). We concluded that exercise training induces an increase in the abundance of five antioxidant proteins in the renal cortex, which could explain the well-known increase in endothelial-dependent vasodilation that results from exercise and the consequential protective effect against increased oxidative stress of the hyperglycemic milieu. Moreover, this protective effect could be important in the prevention of kidney vascular damage associated with diabetes pathophysiology.
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http://dx.doi.org/10.2174/1568026613666131204115656DOI Listing
October 2014

Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes.

J Proteome Res 2012 Feb 20;11(2):1152-62. Epub 2012 Jan 20.

Centro de Biotecnologia, Instituto Butantan, Av. Vital Brazil, 1500, São Paulo, SP, 05503-900, Brazil.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.
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http://dx.doi.org/10.1021/pr200876cDOI Listing
February 2012

Two-dimensional difference gel electrophoresis (DiGE) analysis of plasmas from dengue fever patients.

J Proteome Res 2009 Dec;8(12):5431-41

Lab Toxinologia and Lab. Imunofarmacologia, Pavilhao Ozorio de Almeida, Instituto Oswaldo Cruz and Instituto de Pesquisa Clinica Evandro Chagas, Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, Brazil.

Dengue fever is the world's most important arthropod-born viral disease affecting humans. To contribute to a better understanding of its pathogenesis, this study aims to identify proteins differentially expressed in plasmas from severe dengue fever patients relative to healthy donors. The use of 2-D Fluorescence Difference Gel Electrophoresis to analyze plasmas depleted of six high-abundance proteins (albumin, IgG, antitrypsin, IgA, transferrin and haptoglobin) allowed for the detection of 73 differentially expressed protein spots (n = 13, p < 0.01), of which 37 could be identified by mass spectrometry. These 37 spots comprised a total of 14 proteins, as follows: 7 had increased expression in plasmas from dengue fever patients (C1 inhibitor, alpha1-antichymotrypsin, vitamin D-binding protein, fibrinogen gamma-chain, alpha1-acid glycoprotein, apolipoprotein J and complement component C3c), while 7 others had decreased expression in the same samples (alpha-2 macroglobulin, prothrombin, histidine-rich glycoprotein, apolipoproteins A-IV and A-I, transthyretin and complement component C3b). The possible involvement of these proteins in the inflammatory process triggered by dengue virus infection and in the repair mechanisms of vascular damage occurring in this pathology is discussed in this study.
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http://dx.doi.org/10.1021/pr900236fDOI Listing
December 2009

Crotalid snake venom subproteomes unraveled by the antiophidic protein DM43.

J Proteome Res 2009 May;8(5):2351-60

Laboratorio de Toxinologia, Pavilhão Ozório de Almeida, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil, 4365, 21040-900 Rio de Janeiro, RJ, Brazil.

Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity.
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http://dx.doi.org/10.1021/pr800977sDOI Listing
May 2009

Bothrops insularis venomics: a proteomic analysis supported by transcriptomic-generated sequence data.

J Proteomics 2009 Mar 11;72(2):241-55. Epub 2009 Jan 11.

Oswaldo Cruz Foundation, IOC, Laboratory of Toxinology, Rio de Janeiro, Brazil.

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.
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http://dx.doi.org/10.1016/j.jprot.2009.01.001DOI Listing
March 2009

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

Eur J Biochem 2002 Dec;269(24):6052-62

Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.
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http://dx.doi.org/10.1046/j.1432-1033.2002.03308.xDOI Listing
December 2002