RESUMEN
We present RawVegetable 2.0, a software tailored for assessing mass spectrometry data quality and fine-tuned for cross-linking mass spectrometry (XL-MS) applications. Building upon the capabilities of its predecessor, RawVegetable 2.0 introduces four main modules, each providing distinct and new functionalities: 1) Pair Finder, which identifies ion doublets characteristic of cleavable cross-linking experiments; 2) Diagnostic Peak Finder, which locates potential reporter ions associated with a specific cross-linker; 3) Precursor Signal Ratio, which computes the ratio between precursor intensity and the total signal in an MS/MS scan; and 4) Xrea, which evaluates spectral quality by analyzing the heterogeneity of peak intensities within a spectrum. These modules collectively streamline the process of optimizing mass spectrometry data acquisition for both Proteomics and XL-MS experiments. RawVegetable 2.0, along with a comprehensive tutorial is freely accessible for academic use at: http://patternlabforproteomics.org/rawvegetable2.
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Proteómica , Control de Calidad , Programas Informáticos , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas , Proteómica/métodos , Proteómica/normasRESUMEN
SpliceProt 2.0 is a public proteogenomics database that aims to list the sequence of known proteins and potential new proteoforms in human, mouse, and rat proteomes. This updated repository provides an even broader range of computationally translated proteins and serves, for example, to aid with proteomic validation of splice variants absent from the reference UniProtKB/SwissProt database. We demonstrate the value of SpliceProt 2.0 to predict orthologous proteins between humans and murines based on transcript reconstruction, sequence annotation and detection at the transcriptome and proteome levels. In this release, the annotation data used in the reconstruction of transcripts based on the methodology of ternary matrices were acquired from new databases such as Ensembl, UniProt, and APPRIS. Another innovation implemented in the pipeline is the exclusion of transcripts predicted to be susceptible to degradation through the NMD pathway. Taken together, our repository and its applications represent a valuable resource for the proteogenomics community.
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Proteogenómica , Proteómica , Ratas , Ratones , Humanos , Animales , Bases de Datos de Proteínas , Bases del Conocimiento , Proteoma/genéticaRESUMEN
Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.
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Caspasas/química , Replegamiento Proteico , Proteínas Protozoarias/química , Trypanosoma cruzi/enzimología , Caspasas/genética , Dominios Proteicos , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trypanosoma cruzi/genéticaRESUMEN
BACKGROUND: Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES: Investigate LbGS structure and generate structural models of the protein. METHODS: We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS: 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS: In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.
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Glutamato-Amoníaco Ligasa/química , Leishmania braziliensis , Proteínas Protozoarias/química , Leishmania braziliensis/enzimología , Espectrometría de Masas , PielRESUMEN
In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap-specific messenger RNA (mRNA) (nucleoside-2'-O-)-methyltransferase 1 (hMTr1), which catalyzes methylation of the 2'-O -ribose of the first nucleotide of capped mRNAs. Pre-mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5'-end of the whole cellular pre-mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre-mRNA maturation was identified, including splicing factors, spliceosome-associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA-binding proteins and proteins involved in mRNA 5'- and 3'-end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre-mRNA synthesis.
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Metiltransferasas/metabolismo , Mapas de Interacción de Proteínas , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Células HEK293 , Humanos , NucleofosminaRESUMEN
Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.
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Plaquetas/inmunología , Virus del Dengue/fisiología , Dengue/inmunología , Proteoma/inmunología , Adulto , Plaquetas/química , Estudios de Cohortes , Dengue/sangre , Dengue/genética , Dengue/virología , Virus del Dengue/inmunología , Femenino , Humanos , Masculino , Activación Plaquetaria , Proteoma/genéticaRESUMEN
BACKGROUND: RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite. RESULTS: We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress. CONCLUSIONS: The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.
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Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/metabolismo , Regulación de la Expresión Génica , Unión Proteica , Dominios Proteicos , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/genéticaRESUMEN
BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method. RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Líquido Aspirado del Pezón/metabolismo , Proteoma/análisis , Proteómica/métodos , Microambiente Tumoral , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Pronóstico , Flujo de TrabajoRESUMEN
Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.
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Ácido Aspártico/análisis , Biología Computacional , Reactivos de Enlaces Cruzados/química , Ácido Glutámico/análisis , Lisina/análisis , Serina/análisis , Algoritmos , Ésteres/química , Espectrometría de Masas , Proteínas/química , Succinimidas/química , TemperaturaRESUMEN
Gene expression regulation in trypanosomes differs from other eukaryotes due to absence of transcriptional regulation for most of their genes. RNA-binding proteins (RBPs) associate with mRNAs and other regulatory proteins to form ribonucleoprotein complexes (mRNPs), which play a major role in post-transcriptional regulation. Here, we show that RBP9 is a cytoplasmic RBP in Trypanosoma cruzi with one RNA-recognition motif (RRM). The RBP9 sedimentation profile in a sucrose gradient indicated its presence in cytoplasmic translational complexes, suggesting its involvement in translation regulation. Taking this result as a motivation, we used shotgun proteomics and RNA-seq approaches to assess the core of the RBP9-mRNP complex. In epimastigotes in exponential growth, the complex was composed mostly by RBPs involved in RNA metabolism, such as ZC3H39, UBP1/2, NRBD1, and ALBA3/4. When parasites were subjected to nutritional stress, our analysis identified regulatory RBPs and the translation initiation factors eIF4E5, eIF4G5, eIF4G1, and eIF4G4. The RNA-seq results showed that RBP9-mRNP complex regulates transcripts encoding some RBPs - e.g. RBP5, RBP6, and RBP10 -, and proteins involved in metabolic processes. Therefore, we argue that RBP9 is part of cytoplasmic mRNPs complexes associated with mRNA metabolism and translation regulation in T. cruzi.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Regulación de la Expresión Génica , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Homología de Secuencia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle-free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with the identifier PXD004895.
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Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Proteómica/métodos , Toxoplasma/metabolismo , Toxoplasmosis/parasitología , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/parasitología , Prepucio/citología , Prepucio/metabolismo , Prepucio/parasitología , Humanos , Masculino , Toxoplasmosis/metabolismoRESUMEN
BACKGROUND: DNA methylation is commonly linked with the silencing of the gene expression for many tumor suppressor genes. As such, determining DNA methylation patterns should aid, in times to come, in the diagnosis and personal treatment for various types of cancers. Here, we analyzed the methylation pattern from five colorectal cancer patients from the Amazon state in Brazil for four tumor suppressor genes, viz.: DAPK, CDH1, CDKN2A, and TIMP2 by employing a polymerase chain reaction (PCR) specific to methylation. Efforts in the study of colorectal cancer are fundamental as it is the third most of highest incidence in the world. RESULTS: Tumor biopsies were methylated in 1/5 (20%), 2/5 (40%), 4/5 (80%), and 4/5 (80%) for CDH1, CDKN2A, DAPK, and TIMP2 genes, respectively. The margin biopsies were methylated in 3/7 (43%), 2/7 (28%), 7/7 (100%), and 6/7 (86%) for CDH1, CDKN2A, DAPK, and TIMP2, respectively. CONCLUSIONS: Our findings showed DAPK and TIMP2 to be methylated in most samples from both tumor tissues and adjacent non-neoplastic margins; thus presenting distinct methylation patterns. This emphasizes the importance of better understanding of the relation of these patterns with cancer in the context of different populations.
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Neoplasias Colorrectales/genética , Metilación de ADN/genética , Genes Supresores de Tumor , Adulto , Anciano , Brasil , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
Acute kidney injury (AKI) is a critical systemic complication caused by Bothrops envenoming, a neglected health problem in the Brazilian Amazon. Understanding the underlying mechanisms leading to AKI is crucial for effectively mitigating the burden of this complication. This study aimed to characterize the urinary protein profile of Bothrops atrox snakebite victims who developed AKI. We analyzed three groups of samples collected on admission: healthy subjects (controls, n = 10), snakebite victims who developed AKI (AKI, n = 10), and those who did not evolve to AKI (No-AKI, n = 10). Using liquid-chromatography tandem mass spectrometry, we identified and quantified (label-free) 1190 proteins. A panel of 65 proteins was identified exclusively in the urine of snakebite victims, with 32 exclusives to the AKI condition. Proteins more abundant or exclusive in AKI's urine were associated with acute phase response, endopeptidase inhibition, complement cascade, and inflammation. Notable proteins include serotransferrin, SERPINA-1, alpha-1B-glycoprotein, and NHL repeat-containing protein 3. Furthermore, evaluating previously reported biomarkers candidates for AKI and renal injury, we found retinol-binding protein, beta-2-microglobulin, cystatin-C, and hepcidin to be significant in cases of AKI induced by Bothrops envenoming. This work sheds light on physiological disturbances caused by Bothrops envenoming, highlighting potential biological processes contributing to AKI. Such insights may aid in better understanding and managing this life-threatening complication.
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Lesión Renal Aguda , Fenómenos Biológicos , Bothrops , Mordeduras de Serpientes , Animales , Humanos , Mordeduras de Serpientes/complicaciones , Bothrops atrox , Proteómica , Lesión Renal Aguda/etiologíaRESUMEN
Peptide sequence matching algorithms used for peptide identification by tandem mass spectrometry (MS/MS) enumerate theoretical peptides from the database, predict their fragment ions, and match them to the experimental MS/MS spectra. Here, we present an approach for scoring MS/MS identifications based on the high mass accuracy matching of precursor ions, the identification of a high intensity b1 fragment ion, and partial sequence tags from phenylthiocarbamoyl-derivatized peptides. This derivatization process boosts the b1 fragment ion signal, which turns it into a powerful feature for peptide identification. We demonstrate the effectiveness of our scoring system by implementing it on a computational tool called "HI-bone" and by identifying mass spectra of an Escherichia coli sample acquired on an Orbitrap Velos instrument using Higher-energy C-trap dissociation. Following this strategy, we identified 1614 peptide spectrum matches with a peptide false discovery rate (FDR) below 1%. These results were significantly higher than those from Mascot and SEQUEST using a similar FDR.
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Isotiocianatos/química , Péptidos/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Secuencia de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/química , Humanos , Iones/química , Datos de Secuencia Molecular , Programas InformáticosRESUMEN
This work discloses a unique, comprehensive proteomic dataset of Acinetobacter baumannii strains, both resistant and non-resistant to polymyxin B, isolated in Brazil generated using Orbitrap Fusion Lumos. From nearly 4 million tandem mass spectra, the software DiagnoMass produced 240,685 quality-filtered mass spectral clusters, of which PatternLab for proteomics identified 44,553 peptides mapping to 3479 proteins. Crucially, DiagnoMass shortlisted 3550 and 1408 unique mass spectral clusters for the resistant and non-resistant strains, respectively, with only about a third with sequences (and PTMs) identified by PatternLab. Further open-search attempts via FragPipe yielded an additional â¼20% identifications, suggesting the remaining unidentified spectra likely arise from complex combinations of post-translational modifications and amino-acid substitutions. This highlights the untapped potential of the dataset for future discoveries, particularly given the importance of PTMs, which remain elusive to nucleotide sequencing approaches but are crucial for understanding biological mechanisms. Our innovative approach extends beyond the identifications that are typically subjected to the bias of a search engine; we discern which spectral clusters are differential and subject them to increased scrutiny, akin to spectral library matching by comparing captured spectra to themselves. Our analysis reveals adaptations in the resistant strain, including enhanced detoxification, altered protein synthesis, and metabolic adjustments. SIGNIFICANCE: We present comprehensive proteomic profiles of non-resistant and resistant Acinetobacter baumannii from Brazilian Hospitals strains, and highlight the presence of discriminative and yet unidentified mass spectral clusters. Our work emphasizes the importance of exploring this overlooked data, as it could hold the key to understanding the complex dynamics of antibiotic resistance. This approach not only informs antimicrobial stewardship efforts but also paves the way for the development of innovative diagnostic tools. Thus, our findings have profound implications for the field, as far as methods for providing a new perspective on diagnosing antibiotic resistance as well as classifying proteomes in general.
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Acinetobacter baumannii , Polimixinas , Polimixinas/metabolismo , Antibacterianos/farmacología , Acinetobacter baumannii/metabolismo , Proteómica/métodos , Proteoma/metabolismo , Brasil , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad MicrobianaRESUMEN
Our current knowledge in biology has been mostly derived from studying model organisms and cell lines in which only a small fraction of all described species have been extensively studied. Although these model organisms are amenable to genetic manipulations, this blinds researchers to the true variability of life. Groundbreaking discoveries are often achieved by analyzing "noncanonical" species; for example, the characterization of Taq polymerase from Thermus aquaticus ultimately led to a revolution in the field of molecular biology. Brazil possesses a rich biodiversity and a considerable fraction of Brazilian groups use current proteomic techniques to explore this natural treasure-trove. However, in our opinion, much more than the widely adopted peptide spectrum match approach is required to explore this rich "proteomosphere." Here, we provide a critical overview of the available strategies for the analysis of proteomic data from "noncanonical" biological samples (e.g. proteins from unsequenced genomes or genomes with high levels of polymorphisms), and demonstrate some limitations of existing approaches for large-scale protein identification and quantitation. An understanding of the premises behind these computational tools is necessary to properly deal with their limitations and draw accurate conclusions.
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Proteínas/química , Proteómica/métodos , Animales , Brasil , Humanos , Péptidos/química , Análisis de Secuencia/métodosRESUMEN
Iron is essential and ubiquitous in living organisms. The competition for this micronutrient between the host and its pathogens has been related to disease establishment. Cryptococcus gattii is an encapsulated yeast that causes cryptococcosis mainly in immunocompetent individuals. In this study, we analyzed the proteomic profile of the C. gattii R265 Vancouver Island isolate under iron-depleted and -repleted conditions by multidimensional protein identification technology (MudPIT) and by 2D-GE. Proteins and key mechanisms affected by alteration of iron levels such as capsule production, cAMP-signaling pathway, response to stress, and metabolic pathways related to mitochondrial function were identified. Our results also show both proteomic methodologies employed to be complementary.
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Cryptococcus gattii/metabolismo , Proteínas Fúngicas/metabolismo , Hierro/fisiología , Proteoma/metabolismo , Vías Biosintéticas , Cryptococcus gattii/genética , Cryptococcus gattii/crecimiento & desarrollo , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Expresión Génica , Regulación Fúngica de la Expresión Génica , Hierro/metabolismo , Anotación de Secuencia Molecular , Proteoma/clasificación , Proteoma/genética , ProteómicaRESUMEN
Bothrops spp. is responsible for about 70% of snakebites in Brazil, causing a diverse and complex pathophysiological condition. Bothrops leucurus is the main species of medical relevance found in the Atlantic coast in the Brazilian Northeast region. The pathophysiological effects involved B. leucurus snakebite as well as the organism's reaction in response to this envenoming, it has not been explored yet. Thus, edema was induced in mice paw using 1.2, 2.5, and 5.0 µg of B. leucurus venom, the percentage of edema was measured 30 min after injection and the blood plasma was collected and analyzed by shotgun proteomic strategy. We identified 80 common plasma proteins with differential abundance among the experimental groups and we can understand the early aspects of this snake envenomation, regardless of the suggestive severity of an ophidian accident. The results showed B. leucurus venom triggers a thromboinflammation scenario where family's proteins of the Serpins, Apolipoproteins, Complement factors and Component subunits, Cathepsins, Kinases, Oxidoreductases, Proteases inhibitors, Proteases, Collagens, Growth factors are related to inflammation, complement and coagulation systems, modulators platelets and neutrophils, lipid and retinoid metabolism, oxidative stress and tissue repair. Our findings set precedents for future studies in the area of early diagnosis and/or treatment of snakebites. SIGNIFICANCE: The physiopathological effects that the snake venoms can cause have been investigated through classical and reductionist tools, which allowed, so far, the identification of action mechanisms of individual components associated with specific tissue damage. The currently incomplete limitations of this knowledge must be expanded through new approaches, such as proteomics, which may represent a big leap in understanding the venom-modulated pathological process. The exploration of the complete protein set that suffer modifications by the simultaneous action of multiple toxins, provides a map of the establishment of physiopathological phenotypes, which favors the identification of multiple toxin targets, that may or may not act in synergy, as well as favoring the discovery of biomarkers and therapeutic targets for manifestations that are not neutralized by the antivenom.
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Bothrops , Venenos de Crotálidos , Mordeduras de Serpientes , Trombosis , Animales , Antivenenos/metabolismo , Bothrops/metabolismo , Venenos de Crotálidos/toxicidad , Inflamación , Ratones , Plasma/metabolismo , Proteoma , Proteómica , Venenos de Serpiente/toxicidadRESUMEN
Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.
RESUMEN
Pathogen identification is crucial to confirm bacterial infections and guide antimicrobial therapy. Although MALDI-TOF mass spectrometry (MS) serves as foundation for tools that enable rapid microbial identification, some bacteria remain challenging to identify. We recently showed that top-down proteomics (TDP) could be used to discriminate closely related enterobacterial pathogens (Escherichia coli, Shigella, and Salmonella) that are indistinguishable with tools rooted in the MALDI-TOF MS approach. Current TDP diagnostic relies on the identification of specific proteoforms for each species through a database search. However, microbial proteomes are often poorly annotated, which complicates the large-scale identification of proteoforms and leads to many unidentified high-quality mass spectra. Here, we describe a new computational pipeline called DiagnoTop that lists discriminative spectral clusters found in TDP data sets that can be used for microbial diagnostics without database search. Applied to our enterobacterial TDP data sets, DiagnoTop could easily shortlist high-quality discriminative spectral clusters, leading to increased diagnostic power. This pipeline opens new perspectives in clinical microbiology and biomarker discovery using TDP.