Paracrine Signaling Mediated by the Cytosolic Tryparedoxin Peroxidase of Trypanosoma cruzi.

Peroxiredoxins are abundant and ubiquitous proteins that participate in different cellular functions, such as oxidant detoxification, protein folding, and intracellular signaling. Under different cellular conditions, peroxiredoxins can be secreted by different parasites, promoting the induction of immune responses in hosts. In this work, we demonstrated that the cytosolic tryparedoxin peroxidase of Trypanosoma cruzi (cTXNPx) is secreted by epimastigotes and trypomastigotes associated with extracellular vesicles and also as a vesicle-free protein. By confocal microscopy, we show that cTXNPx can enter host cells by an active mechanism both through vesicles and as a recombinant protein. Transcriptomic analysis revealed that cTXNPx induces endoplasmic reticulum stress and interleukin-8 expression in epithelial cells. This analysis also suggested alterations in cholesterol metabolism in cTXNPx-treated cells, which was confirmed by immunofluorescence showing the accumulation of LDL and the induction of LDL receptors in both epithelial cells and macrophages. BrdU incorporation assays and qPCR showed that cTXNPx has a mitogenic, proliferative, and proinflammatory effect on these cells in a dose-dependent manner. Importantly, we also demonstrated that cTXNPx acts as a paracrine virulence factor, increasing the susceptibility to infection in cTXNPx-pretreated epithelial cells by approximately 40%. Although the results presented in this work are from in vitro studies and likely underestimate the complexity of parasite-host interactions, our work suggests a relevant role for this protein in establishing infection.

DiagnoMass: A proteomics hub for pinpointing discriminative spectral clusters.

MOTIVATION: There are several well-established paradigms for identifying and pinpointing discriminative peptides/proteins using shotgun proteomic data; examples are peptide-spectrum matching, de novo sequencing, open searches, and even hybrid approaches. Such an arsenal of complementary paradigms can provide deep data coverage, albeit some unidentified discriminative peptides remain. RESULTS: We present DiagnoMass, software tool that groups similar spectra into spectral clusters and then shortlists those clusters that are discriminative for biological conditions. DiagnoMass then communicates with proteomic tools to attempt the identification of such clusters. We demonstrate the effectiveness of DiagnoMass by analyzing proteomic data from Escherichia coli, Salmonella, and Shigella, listing many high-quality discriminative spectral clusters that had thus far remained unidentified by widely adopted proteomic tools. DiagnoMass can also classify proteomic profiles. We anticipate the use of DiagnoMass as a vital tool for pinpointing biomarkers. AVAILABILITY: DiagnoMass and related documentation, including a usage protocol, are available at http://www.diagnomass.com.

PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: Molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.

Cardiolipin interactions with cytochrome c increase tyrosine nitration yields and site-specificity.

The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.

A Nitroalkene Benzoic Acid Derivative Targets Reactive Microglia and Prolongs Survival in an Inherited Model of ALS via NF-κB Inhibition.

Motor neuron degeneration and neuroinflammation are the most striking pathological features of amyotrophic lateral sclerosis (ALS). ALS currently has no cure and approved drugs have only a modest clinically therapeutic effect in patients. Drugs targeting different deleterious inflammatory pathways in ALS appear as promising therapeutic alternatives. Here, we have assessed the potential therapeutic effect of an electrophilic nitroalkene benzoic acid derivative, (E)-4-(2-nitrovinyl) benzoic acid (BANA), to slow down paralysis progression when administered after overt disease onset in SOD1G93A rats. BANA exerted a significant inhibition of NF-κB activation in NF-κB reporter transgenic mice and microglial cell cultures. Systemic daily oral administration of BANA to SOD1G93A rats after paralysis onset significantly decreased microgliosis and astrocytosis, and significantly reduced the number of NF-κB-p65-positive microglial nuclei surrounding spinal motor neurons. Numerous microglia bearing nuclear NF-κB-p65 were observed in the surrounding of motor neurons in autopsy spinal cords from ALS patients but not in controls, suggesting ALS-associated microglia could be targeted by BANA. In addition, BANA-treated SOD1G93A rats after paralysis onset showed significantly ameliorated spinal motor neuron pathology as well as conserved neuromuscular junction innervation in the skeletal muscle, as compared to controls. Notably, BANA prolonged post-paralysis survival by ~30%, compared to vehicle-treated littermates. These data provide a rationale to therapeutically slow paralysis progression in ALS using small electrophilic compounds such as BANA, through a mechanism involving microglial NF-κB inhibition.

A novel nitroalkene vitamin E analogue inhibits the NLRP3 inflammasome and protects against inflammation and glucose intolerance triggered by obesity.

Chronic metabolic diseases, like obesity, type II diabetes and atherosclerosis often involve a low-grade and sterile systemic inflammatory state, in which activation of the pro-inflammatory transcription factor NF-kB and the NLRP3 inflammasome play a major role. It is well established that genetic inhibition of the NLRP3 inflammasome ameliorates acute and chronic inflammation. Indeed, accumulating experimental evidences in murine models and also in humans suggest that inhibition of the NLRP3 inflammasome might be a suitable approach to tackle the deleterious effects of chronic metabolic diseases. In this work, we explored our previously synthesized nitroalkene-Trolox™ derivative named NATx0, as a non-conventional anti-inflammatory strategy to treat chronic inflammatory diseases, such as obesity-induced glucose intolerance. We found that NATx0 inhibited NF-kB nuclear translocation and pro-inflammatory gene expression in macrophages in vitro. In addition, treatment with NATx0 prevented NLRP3 inflammasome activation after LPS/ATP stimulation in macrophages in vitro. When tested acutely in vivo, NATx0 inhibited neutrophil recruitment in zebrafish larvae, and also diminished IL-1ß production after LPS challenge in mice. Finally, when NATx0 was administered chronically to diet-induced obese mice, it decreased muscle tissue inflammation and glucose intolerance, leading to improved glucose homeostasis. In conclusion, we propose that this novel nitroalkene-Trolox derivative is a suitable tool to tackle acute and chronic inflammation in vitro and in vivo mainly due to inhibition of NF-kB/NLRP3 activation.

The protein Deleted in Breast Cancer-1 (DBC1) regulates vascular response and formation of aortic dissection during Angiotensin II infusion.

Cardiovascular diseases are among the main causes of morbimortality in the adult population. Among them, hypertension is a leading cause for stroke, heart disease and kidney failure. Also, as a result of arterial wall weakness, hypertension can lead to the development of dissecting aortic aneurysms, a rare but often fatal condition if not readily treated. In this work, we investigated the role of DBC1 in the regulation of vascular function in an ANGII-induced hypertension mouse model. We found that WT and DBC1 KO mice developed hypertension in response to ANGII infusion. However, DBC1 KO mice showed increased susceptibility to develop aortic dissections. The effect was accompanied by upregulation of vascular remodeling factors, including MMP9 and also VEGF. Consistent with this, we found decreased collagen deposition and elastic fiber fragmentation, suggesting that increased expression of MMPs in DBC1 KO mice weakens the arterial wall, promoting the formation of aortic dissections during treatment with ANGII. Finally, DBC1 KO mice had reduced cell proliferation in the intima-media layer in response to ANGII, paralleled with an impairment to increase wall thickness in response to hypertension. Furthermore, VSMC purified from DBC1 KO mice showed impaired capacity to leave quiescence, confirming the in vivo results. Altogether, our results show for the first time that DBC1 regulates vascular response and function during hypertension and protects against vascular injury. This work also brings novel insights into the molecular mechanisms of the development of aortic dissections.

Proteomic analysis of Saccharomyces cerevisiae to study the effects of red wine polyphenols on oxidative stress.

Understanding the molecular mechanisms underlying the "French paradox" has contributed to a growing interest in the investigation of the biological activity of red wine polyphenols (RWP). The main goal of this research is to provide valuable information on how RWP could exert their biological action at the cellular level. So, we report a proteomic analysis of S. cerevisiae exposed to both pro-oxidant (H2O2) and antioxidant (wine) agents. Cellular proteome analysis shows that RWP modify the level of certain proteins. Under both normal conditions (Wine treatment) and oxidative stress situations (Wine + H2O2 treatment), the proteins involved in the metabolism and biosynthesis of biomolecules were down-regulated, while one ribosomal protein was up-regulated, probably performing its ribosome-independent functions, and so contributing to the stress defense system. Considering this action mechanism, we suggest that RWP may be acting as mild pro-oxidants and, therefore, exerting a hormetic effect that leads to the strengthening of cells' antioxidant capacity.

A Focused Library of NO-Donor Compounds with Potent Antiproliferative Activity Based on Green Multicomponent Reactions.

Cancer is the second leading cause of death worldwide. Herein, a strategy to quickly and efficiently identify novel lead compounds to develop anticancer agents, using green multicomponent reactions followed by antiproliferative activity and structure-activity relationship studies, is described. A second-generation focused library of nitric oxide-releasing compounds was prepared by microwave-assisted Passerini and Ugi reactions. Nearly all compounds displayed potent antiproliferative activities against a panel of human solid tumor cell lines, with 1-phenyl-1-[(tert-butylamino)carbonyl]methyl 3-[(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N2 -oxide)oxy]benzoate (4 k) and N-[1-(tert-butylaminocarbonyl)-1-phenylmethyl]-N-(4-methylphenyl)-3-(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N2 -oxide)oxyphenyl carboxamide (6 d) exhibiting the strongest activity on SW1573 lung cell line (GI50 =110 and 21 nm) with selectivity indices of 70 and 470, respectively. Preliminary mechanistic studies suggest a relationship between NO release and antiproliferative activity. Our strategy allowed the rapid identification of at least two molecules as future candidates for the development of potent antitumor drugs.

Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.

New substrates and interactors of the mycobacterial Serine/Threonine protein kinase PknG identified by a tailored interactomic approach.

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.

A novel nitroalkene-α-tocopherol analogue inhibits inflammation and ameliorates atherosclerosis in Apo E knockout mice.

BACKGROUND AND PURPOSE: Atherosclerosis is characterized by chronic low-grade inflammation with concomitant lipid accumulation in the arterial wall. Anti-inflammatory and anti-atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated-unsaturated fatty acids), formed during inflammation and digestion/absorption processes. The lipid-associated antioxidant α-tocopherol is transported systemically by LDL particles including to the atheroma lesions. To capitalize on the overlapping and complementary salutary properties of endogenous nitroalkenes and α-tocopherol, we designed and synthesized a novel nitroalkene-α-tocopherol analogue (NATOH) to address chronic inflammation and atherosclerosis, particularly at the lesion sites. EXPERIMENTAL APPROACH: We synthesized NATOH, determined its electrophilicity and antioxidant capacity and studied its effects over pro-inflammatory and cytoprotective pathways in macrophages in vitro. Moreover, we demonstrated its incorporation into lipoproteins and tissue both in vitro and in vivo, and determined its effect on atherosclerosis and inflammatory responses in vivo using the Apo E knockout mice model. KEY RESULTS: NATOH exhibited similar antioxidant capacity to α-tocopherol and, due to the presence of the nitroalkenyl group, like endogenous nitroalkenes, it exerted electrophilic reactivity. NATOH was incorporated in vivo into the VLDL/LDL lipoproteins particles to reach the atheroma lesions. Furthermore, oral administration of NATOH down-regulated NF-κB-dependent expression of pro-inflammatory markers (including IL-1ß and adhesion molecules) and ameliorated atherosclerosis in Apo E knockout mice. CONCLUSIONS AND IMPLICATIONS: In toto, the data demonstrate a novel pharmacological strategy for the prevention of atherosclerosis based on a creative, natural and safe drug delivery system of a non-conventional anti-inflammatory compound (NATOH) with significant potential for clinical application.

Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses.

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.

A green multicomponent synthesis of tocopherol analogues with antiproliferative activities.

A one-pot efficient, practical and eco-friendly synthesis of tocopherol analogues has been developed using water or solvent free conditions via Passerini and Ugi multicomponent reactions. These reactions can be optimized using microwave irradiation or ultrasound as the energy source. Accordingly, a small library of 30 compounds was prepared for biological tests. The evaluation of the antiproliferative activity in the human solid tumor cell lines A549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (lung), T-47D (breast), and WiDr (colon) provided lead compounds with GI50 values between 1 and 5 µM. A structure-activity relationship is also discussed. One of the studied compounds comes up as a future candidate for the development of potent tocopherol-mimetic therapeutic agents for cancer.

DiagnoProt: a tool for discovery of new molecules by mass spectrometry.

MOTIVATION: Around 75% of all mass spectra remain unidentified by widely adopted proteomic strategies. We present DiagnoProt, an integrated computational environment that can efficiently cluster millions of spectra and use machine learning to shortlist high-quality unidentified mass spectra that are discriminative of different biological conditions. RESULTS: We exemplify the use of DiagnoProt by shortlisting 4366 high-quality unidentified tandem mass spectra that are discriminative of different types of the Aspergillus fungus. AVAILABILITY AND IMPLEMENTATION: DiagnoProt, a demonstration video and a user tutorial are available at http://patternlabforproteomics.org/diagnoprot . CONTACT: andrerfsilva@gmail.com or paulo@pcarvalho.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

SIGNIFICANCE: "Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. CRITICAL ISSUES: Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. FUTURE DIRECTIONS: The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.