In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

Proteomic Mapping of Multifunctional Complexes Within Triatomine Saliva.

Triatomines are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent of Chagas disease. This neglected tropical disease represents a global health issue as it is spreading worldwide. The saliva of Triatominae contains miscellaneous proteins crucial for blood feeding acquisition, counteracting host's hemostasis while performing vasodilatory, anti-platelet and anti-coagulant activities, besides modulating inflammation and immune responses. Since a set of biological processes are mediated by protein complexes, here, the sialocomplexomes (salivary protein complexes) of five species of Triatominae were studied to explore the protein-protein interaction networks. Salivary multiprotein complexes from Triatoma infestans, Triatoma dimidiata, Dipetalogaster maxima, Rhodnius prolixus, and Rhodnius neglectus were investigated by Blue-Native- polyacrylamide gel electrophoresis coupled with liquid chromatography tandem mass spectrometry. More than 70 protein groups, uncovering the landscape of the Triatominae salivary interactome, were revealed. Triabin, actin, thioredoxin peroxidase and an uncharacterized protein were identified in sialocomplexes of the five species, while hexamerin, heat shock protein and histone were identified in sialocomplexes of four species. Salivary proteins related to triatomine immunity as well as those required during blood feeding process such as apyrases, antigen 5, procalins, and nitrophorins compose different complexes. Furthermore, unique proteins for each triatomine species were revealed. This study represents the first Triatominae sialocomplexome reference to date and shows that the approach used is a reliable tool for the analysis of Triatominae salivary proteins assembled into complexes.

Paternal Resistance Training Induced Modifications in the Left Ventricle Proteome Independent of Offspring Diet.

Ancestral obesogenic exposure is able to trigger harmful effects in the offspring left ventricle (LV) which could lead to cardiovascular diseases. However, the impact of the father's lifestyle on the offspring LV is largely unexplored. The aim of this study was to investigate the effects of 8 weeks of paternal resistance training (RT) on the offspring left ventricle (LV) proteome exposed to control or high-fat (HF) diet. Wistar rats were randomly divided into two groups: sedentary fathers and trained fathers (8 weeks, 3 times per week with weights secured to the animals' tails). The offspring were obtained by mating with sedentary females. Upon weaning, male offspring were divided into 4 groups (5 animals per group): offspring from sedentary fathers, exposed to control diet (SFO-C); offspring from trained fathers, exposed to control diet (TFO-C); offspring from sedentary fathers, exposed to high-fat diet (SFO-HF); and offspring from trained fathers, exposed to high-fat diet (TFO-HF). The LC-MS/MS analysis revealed 537 regulated proteins among groups. Offspring exposure to HF diet caused reduction in the abundance levels of proteins related to cell component organization, metabolic processes, and transport. Proteins related to antioxidant activity, transport, and transcription regulation were increased in TFO-C and TFO-HF as compared with the SFO-C and SFO-HF groups. Paternal RT demonstrated to be an important intervention capable of inducing significant effects on the LV proteome regardless of offspring diet due to the increase of proteins involved into LV homeostasis maintenance. This study contributes to a better understanding of the molecular aspects involved in transgenerational inheritance.

Hylin a1, the first cytolytic peptide isolated from the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog").

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog") led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH(2)). The alpha-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC(50)=18 microM). Similarity analysis using PSI-BLAST revealed 50-44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).

Exoproteome profiling of Trypanosoma cruzi during amastigogenesis early stages.

Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2-4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.

Label-free quantitative proteomics of rat hypothalamus under fever induced by LPS and PGE2.

Fever is a brain-mediated increase in body temperature mainly during inflammatory or infectious challenges. Although there is considerable data regarding the inflammation pathways involved in fever, metabolic alterations necessary to orchestrate the complex inflammatory response are not totally understood. We performed proteomic analysis of rat hypothalamus using label-free LC-MS/MS in a model of fever induced by lipopolysaccharide (LPS) or prostaglandin E2 (PGE2). In total, 7021 proteins were identified. As far as we know, this is the largest rat hypothalamus proteome dataset available to date. Pathway analysis showed proteins from both stimuli associated with inflammatory and metabolic pathways. Concerning metabolic pathways, rats exposed to LPS or PGE2 presented lower relative abundance of proteins involved in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle. Mitochondrial function may also be altered by both stimuli because significant downregulation of several proteins was found, mainly in complexes I and IV. LPS was able to induce downregulation of important proteins in the enzymatic antioxidant system, thereby contributing to oxidative stress. The results offered comprehensive information about fever responses and helped to reveal new insights into proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during systemic LPS and central PGE2 administration. SIGNIFICANCE: The evolutionary persistence of fever, despite the elevated cost for maintenance of this response, suggests that elevation in core temperature may represent an interesting strategy for survival. Fever response is achieved through the integrated behavioral, physiological, immunological and biochemical processes that determine the balance between heat generation and elimination. The development of such complex response arouses interest in studying how the cell metabolism responds or even contributes to promote fever. Our results offered comprehensive information about fever responses, including metabolic and inflammatory pathways, providing new insights into candidate proteins potentially involved in inflammatory signaling and metabolic changes in the hypothalamus during fever induced by systemic LPS and central PGE2 perturbation.

Comparative Two-Dimensional Gel Electrophoresis of Trypanosoma cruzi Mammalian-Stage Forms in an Alkaline pH Range.

It is estimated that several million people are currently infected worldwide by the protozoan parasite, Trypanosoma cruzi, which causes Chagas disease. After mammalian host infection, a fundamental event is the differentiation from infective trypomastigotes into replicative amastigotes (amastigogenesis) inside host-cells. To unravel the particularities of both forms, it is essential to identify molecules presented in each form. Since T. cruzi gene expression regulation occurs mainly at posttranscriptional level, a proteomic approach is appropriate. Due to intrinsic difficulties with performing 2-DE in the alkaline pH range, there are no reports on 2-DE-based comparative proteome analysis of T. cruzi mammalianstage forms that focus on alkaline polypeptides. Here, we performed a comparative proteome analysis between tissue culture- derived trypomastigotes and extracellular amastigote-like cells using conditions optimized for the 6-11 pH range followed by identification by MALDI-TOF/TOF technology. The alkaline 2-DE maps from both forms show that proteins with a pI above 7.0 were not underrepresented (= 65% of proteins detected). Moreover the differences in protein expression between the Human-hosted T. cruzi forms corroborated previous proteomic studies and corresponded to their biological traits.

Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest.

A comparative study of venoms from juvenile, sub-adult and adult wild Bothrops atrox specimens captured in Manaus region (Brazil) was performed. All venoms tested had acidic pH (5.5) and the human plasma coagulant activity was higher in venoms from juvenile and sub-adult specimens than in adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the most intense bands in adult venoms corresponded to polypeptides of 23 and 50kDa. The 23kDa protein was not detected in juvenile venoms. The 23 and 50kDa proteins were purified by two steps of reversed phase-HPLC followed by size exclusion HPLC. Partial amino acid sequence of the 23kDa protein showed homology to metalloproteinases from other snake venoms. Electrospray ionization mass spectrometric analysis (ESI-MS) showed that the 23kDa band contained at least three isoforms of 23030, 23300 and 23645Da. The 50kDa polypeptide was N-terminally blocked for Edman degradation and presented molecular masses ranging from 46.8 to 49.4kDa by ESI-MS. Both proteins were detected by anti-mutalysin II antibodies in immunoblotting assay indicating that they belong to the metalloproteinase family. Immunoblotting analysis also showed that the 23kDa band increased in intensity from juvenile to adult specimens.SDS-PAGE analysis of juvenile and adult venoms following autoproteolysis in pH 7.4 suggested that endogenous venom metalloproteinases can digest the 50kDa metalloproteinase, originating a new protein band of 27kDa. It was also demonstrated in juvenile venoms that the 23kDa band was not the result of proteolytic processing of the 50kDa metalloproteinase.

2-DE-based proteomic investigation of the saliva of the Amazonian triatomine vectors of Chagas disease: Rhodnius brethesi and Rhodnius robustus.

The triatomine bugs are obligatory haematophagous organisms that act as vectors of Chagas disease by transmitting the protozoan Trypanosoma cruzi. Their feeding success is strongly related to salivary proteins that allow these insects to access blood by counteracting host haemostatic mechanisms. Proteomic studies were performed on saliva from the Amazonian triatomine bugs: Rhodnius brethesi and R. robustus, species epidemiologically relevant in the transmission of T. cruzi. Initially, salivary proteins were separated by two-dimensional gel electrophoresis (2-DE). The average number of spots of the R. brethesi and R. robustus saliva samples were 129 and 135, respectively. The 2-DE profiles were very similar between the two species. Identification of spots by peptide mass fingerprinting afforded limited efficiency, since very few species-specific salivary protein sequences are available in public sequence databases. Therefore, peptide fragmentation and de novo sequencing using a MALDI-TOF/TOF mass spectrometer were applied for similarity-driven identifications which generated very positive results. The data revealed mainly lipocalin-like proteins which promote blood feeding of these insects. The redundancy of saliva sequence identification suggested multiple isoforms caused by gene duplication followed by gene modification and/or post-translational modifications. In the first experimental assay, these proteins were predominantly phosphorylated, suggesting functional phosphoregulation of the lipocalins.

Identification, characterization and regulation studies of the aconitase of Paracoccidioides brasiliensis.

A protein species preferentially expressed in yeast cells with a molecular mass of 80 kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of Paracoccidioides brasiliensis and characterized as an aconitase (ACO) (E.C. 4.2.1.3). ACO is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle and the glyoxylate cycle. We report the cloning and characterization of the cDNA encoding the ACO of P. brasiliensis (PbACO). The cDNA showed a 2361 bp open reading frame (ORF) and encoded a predicted protein with 787 amino acids. Polyclonal antibodies against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall enriched fraction, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed by quantitative real-time RT-PCR and results demonstrated an increased expression in yeast cells compared to mycelia. Real-time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in high-iron conditions.