Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus-vector interaction does not disturb the mosquito's fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.

Proteomics reveals disturbances in the immune response and energy metabolism of monocytes from patients with septic shock.

Sepsis results from a dyshomeostatic response to infection, which may lead to hyper or hypoimmune states. Monocytes are central regulators of the inflammatory response, but our understanding of their role in the genesis and resolution of sepsis is still limited. Here, we report a comprehensive exploration of monocyte molecular responses in a cohort of patients with septic shock via proteomic profiling. The acute stage of septic shock was associated with an impaired inflammatory phenotype, indicated by the down-regulation of MHC class II molecules and proinflammatory cytokine pathways. Simultaneously, there was an up-regulation of glycolysis enzymes and a decrease in proteins related to the citric acid cycle and oxidative phosphorylation. On the other hand, the restoration of immunocompetence was the hallmark of recovering patients, in which an upregulation of interferon signaling pathways was a notable feature. Our results provide insights into the immunopathology of sepsis and propose that, pending future studies, immunometabolism pathway components could serve as therapeutic targets in septic patients.

Beyond Melanin: Proteomics Reveals Virulence-Related Proteins in Paracoccidioides brasiliensis and Paracoccidioides lutzii Yeast Cells Grown in the Presence of L-Dihydroxyphenylalanine.

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen's survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

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.

Potential correlation between tumor aggressiveness and protein expression patterns of nipple aspirate fluid (NAF) revealed by gel-based proteomic analysis.

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64.

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.

Estudo da relação estrutura-função da proteína antiofídica DM43 / Study of structure-function relationship of antiophidic protein DM43

A proteína antiofídica DM43 isolada do soro do gambá Didelphis aurita inibe o efeito hemorrágico e a atividade proteolítica de metaloproteases de venenos de serpentes (SVMPs). Ao se ligar às SVMPs, o homodímero de DM43 se dissocia e cada monômero se liga a uma molécula de metaloprotease. O monômero é composto de três domínios tipo-imunoglobulina, dois dos quais são glicosilados. Para investigar a relação estrutura-função de DM43, os genes codificantes para esta proteína e para seus domínios foram clonados nos vetores pET101D/TOPO ou pET102D/TOPO; neste último, a proteína recombinante foi expressa em fusão com a tiorredoxina. Expressamos as construções obtidas em bactérias competentes da linhagem BL21 Star (DE3) de E. coli, na forma de corpúsculos de inclusão. Após purificação e reenovelamento por diálise, os espectros de dicroísmo circular (CD) na região do ultravioleta distante indicaram uma mistura de alfa-hélices e folhas beta pregueadas para os domínios recombinantes expressos em fusão com a tiorredoxina. A identidade das proteínas recombinantes foi confirmada, tanto por immunoblotting com anticorpo policlonal contra DM43, quanto por espectrometria de massas (MS/MS). Entretanto, nenhum dos domínios recombinantes, isolados ou combinados, foi capaz de inibir a atividade proteolítica da jararagina, uma metaloprotease de Bothrops jararaca. Ao contrário, os domínios fusionados com a tiorredoxina foram degradados quando incubados com esta SVMP. Estes resultados podem indicar que a glicosilação é funcionalmente importante e/ou que a conformação nativa multidomínio é necessária para a atividade biológica deste inibidor. Para melhor entender seus requisitos estruturais, estudamos a DM43 nativa após degradação proteolítica. Sob condições nativas, a digestão limitada de DM43 com tripsina ou quimotripsina gerou vários fragmentos, mas nenhum peptídeo ativo contra a SVMP foi obtido. Sob condições desnaturantes, a digestão extensiva de DM43 pela endoproteinase Lys-C produziu peptídeos menores. Mesmo sem atividade biológica contra a jararagina, três destes peptídeos foram capazes de interagir com esta SVMP. Após sequenciamento por MS/MS, cada peptídeo foi localizado em um domínio diferente de DM43. Estes resultados parecem sugerir a participação conjunta dos três domínios na interação com as SVMPs.