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Highlights • SILAC proteomics approach yielded important cisplatin resistance biomarkers in NSCLC. • Cisplatin resistance upregulated NER-based repair and downregulated mRNA splicing-related pathways. • STAT3 KO conferred epithelial morphology and sensitized to mTOR inhibition. • Cisplatin responsiveness is dependent on STAT3 and mTOR crosstalk in NSCLC cells. • Synergistically inhibition of STAT3 and mTOR is possibly a potential approach against NSCLC., Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

Lung cancer is the leading cause of cancer‑associated death worldwide and exhibits intrinsic and acquired therapeutic resistance to cisplatin (CIS). The present study investigated the role of mTOR signaling and other signaling pathways after metformin (MET) treatment in control and cisplatin‑resistant A549 cells, mapping pathways and possible targets involved in CIS sensitivity. MTT, flow cytometry, clonogenic assay, western blotting, proteomic analysis using the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) approach and reverse transcription‑quantitative PCR were performed. The results revealed that CIS treatment induced mTOR signaling pathway overactivation, and the mTOR status was restored by MET. MET and the mTOR inhibitor rapamycin (RAPA) decreased the viability in control and resistant cells, and decreased the cell size increase induced by CIS. In control cells, MET and RAPA decreased colony formation after 72 h and decreased IC50 values, potentiating the effects of CIS. Proteomics analysis revealed important pathways regulated by MET, including transcription, RNA processing and IL‑12‑mediated signaling. In CIS‑resistant cells, MET regulated the apoptotic process, oxidative stress and G2/M transition. Annexin 4 (ANXA4) and superoxide dismutase 2 (SOD2), involved in apoptosis and oxidative stress, respectively, were chosen to validate the SILAC analysis and may represent potential therapeutic targets for lung cancer treatment. In conclusion, the chemosensitizing and antiproliferative effects of MET were associated with mTOR signaling and with potential novel targets, such as ANXA4 and SOD2, in human lung cancer cells.

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods.Several disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.

Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely associated with lymph node metastasis. However, the molecular mechanisms that drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are membrane-bound particles that play a role in intercellular communication and impact cancer development and progression. Thus, profiling EVs would be of great significance to decipher their role in OSCC metastasis. For that purpose, we used a reductionist approach to map the proteomic, miRNA, metabolomic, and lipidomic profiles of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Distinct omics profiles were associated with the metastatic phenotype, including 670 proteins, 217 miRNAs, 26 metabolites, and 63 lipids differentially abundant between LN1 cell– and SCC-9 cell–derived EVs. A multi-omics integration identified 11 ‘hub proteins’ significantly decreased at the metastatic site compared with primary tumor–derived EVs. We confirmed the validity of these findings with analysis of data from multiple public databases and found that low abundance of seven ‘hub proteins’ in EVs from metastatic lymph nodes (ALDH7A1, CAD, CANT1, GOT1, MTHFD1, PYGB, and SARS) is correlated with reduced survival and tumor aggressiveness in patients with cancer. In summary, this multi-omics approach identified proteins transported by EVs that are associated with metastasis and which may potentially serve as prognostic markers in OSCC., Graphical Abstract, Highlights • Proteomic, miRNA, metabolomic, and lipidomic profiles were mapped in oral cancer EVs. • The molecular profile of EVs was associated with the lymph node metastatic phenotype. • A multi-omics integrative analysis revealed 11 highly connected ‘hub proteins.’ • ‘Hub proteins’ from EVs are candidates as prognostic markers in oral cancer., In Brief A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 ‘hub proteins’ that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor−node−metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis., Oral cancer has region-specific histopathological and molecular characteristics, complicating its classification by the standard tumor-node-metastasis system. Here, the authors combine discovery and targeted proteomics with IHC to identify region-specific and saliva biomarkers for oral cancer prognosis.

Made available in DSpace on 2019-10-06T16:02:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01 National Physical Laboratory Paracoccidioidomycosis (PCM) is a systemic disease caused by thermodymorphic fungi of the Paracoccidioides brasiliensis complex, (Paracoccidioides spp.). Patients with PCM reveal specific cellular immune impairment. Despite the effective treatment, quiescent fungi can lead to relapse, usually late, the serological diagnosis of which has been deficient. The present study was carried out with the objective of investigating a biomarker for the identification of PCM relapse and another molecule behaving as an immunological recovery biomarker; therefore, it may be used as a cure criterion. In the evolutionary analysis of the proteins identified in PCM patients, comparing those that presented with those that did not reveal relapse, 29 proteins were identified. The interactions observed between the proteins, using transferrin and haptoglobin, as the main binding protein, were strong with all the others. Patient follow-up suggests that cerulosplamin may be a marker of relapse and that transferrin and apolipoprotein A-II may contribute to the evaluation of the treatment efficacy and avoiding a premature decision. Universidade Estadual Paulista (UNESP) Faculdade de Medicina de Botucatu Universidade Federal de Mato Grosso do Sul (UFMS) Faculdade de Medicina Instituto de Ciências Exatas e Tecnologia (ICET) Universidade Federal do Amazonas (UFAM) Laboratório Nacional de Biociências–CNPEM Universidade Estadual Paulista (UNESP) Instituto de Biociência de Botucatu Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) UNESP Universidade Estadual Paulista (UNESP) Faculdade de Medicina de Botucatu Universidade Estadual Paulista (UNESP) Instituto de Biociência de Botucatu Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) UNESP National Physical Laboratory: 13083–970

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

To cope with toxic levels of H2S, the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens employ the bigR operon to oxidize H2S into sulfite. The bigR operon is regulated by the transcriptional repressor BigR and it encodes a bifunctional sulfur transferase (ST) and sulfur dioxygenase (SDO) enzyme, Blh, required for H2S oxidation and bacterial growth under hypoxia. However, how Blh operates to enhance bacterial survival under hypoxia and how BigR is deactivated to derepress operon transcription is unknown. Here, we show that the ST and SDO activities of Blh are in vitro coupled and necessary to oxidize sulfide into sulfite, and that Blh is critical to maintain the oxygen flux during A. tumefaciens respiration when oxygen becomes limited to cells. We also show that H2S and polysulfides inactivate BigR leading to operon transcription. Moreover, we show that sulfite, which is produced by Blh in the ST and SDO reactions, is toxic to Citrus sinensis and that X. fastidiosa-infected plants accumulate sulfite and higher transcript levels of sulfite detoxification enzymes, suggesting that they are under sulfite stress. These results indicate that BigR acts as a sulfide sensor in the H2S oxidation mechanism that allows pathogens to colonize plant tissues where oxygen is a limiting factor.

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17.Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases.This unexpected Trx-1

Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO) recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms’ quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating—and discarding—poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms.

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.

The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5–9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes., Author summary Previously thought to produce a harmless bout of fever associated with skin rash and muscle, or joint pain, the Zika virus (ZIKV) is an important cause of morbidity/mortality to human embryos/fetuses. Different from other models, here we report data from wild-type immunocompetent mice, rather than transgenic animals with suppressed immune responses. We found that intravascular ZIKV produced infection of maternal tissues, placentas and conceptuses, but that embryos/fetuses were comparatively much more affected than pregnant females, which seemed to tolerate well the viral challenge with no signs of encephalopathy. Importantly, 10.5 days post-coitum (dpc) embryos exposed to ZIKV at embryonic day 5 (second week in humans) displayed dysraphia, which is a regional failure of neural tube closure, and hydrocephalus, which is a symptom recently shown to precede microcephaly in humans. Characteristic phenotypes in more developed embryos/fetuses included abnormal articular postures analogous to arthrogryposis, which are typical human congenital contractures, gross and generalized malformations and intra uterine growth restriction (IUGR), rather than isolated microcephaly. Some developmental abnormalities and IUGR correlated with placental damage, suggesting that loss of placental function may play an important role in the disease. We believe our model is an asset in the search for useful concepts and prospective therapies for ZIKV because it better reproduces the human condition.

Baculoviruses infect insects, producing two distinct phenotypes during the viral life cycle: the budded virus (BV) and the occlusion-derived virus (ODV) for intra- and inter-host spread, respectively. Since the 1980s, several countries have been using Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) as a biological control agent against the velvet bean caterpillar, A. gemmatalis. The genome of AgMNPV isolate 2D (AgMNPV-2D) carries at least 152 potential genes, with 24 that possibly code for structural proteins. Proteomic studies have been carried out on a few baculoviruses, with six ODV and two BV proteomes completed so far. Moreover, there are limited data on virion proteins carried by AgMNPV-2D. Therefore, structural proteins of AgMNPV-2D were analysed by MALDI- quadrupole-TOF and liquid chromatography MS/MS. A total of 44 proteins were associated with the ODV and 33 with the BV of AgMNPV-2D. Although 38 structural proteins were already known, we found six new proteins in the ODV and seven new proteins carried by the AgMNPV-2D BV. Eleven cellular proteins that were found on several other enveloped viruses were also identified, which are possibly carried with the virion. These findings may provide novel insights into baculovirus biology and their host interaction. Moreover, our data may be helpful in subsequent applied studies aiming to improve AgMNPV use as a biopesticide and a biotechnology tool for gene expression or delivery.

ADAM17 has been initially identified as the main sheddase responsible for releasing the soluble form of a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules, and receptors, most of which are associated with pathological processes, including cancer and inflammation. However, the function and composition of the ADAM17-dependent secretome on a proteome-wide scale is poorly understood. In this study, we observed that the ADAM17-dependent secretome plays an important role in promoting cell proliferation and migration. To further demonstrate the repertoire of proteins involved in this cross-talk, we employed mass-spectrometry-based proteomics using nonmetabolic and metabolic labeling approaches to explore the secretome composition of wild-type and ADAM17(-/-) knockout mouse embryonic fibroblast (mEF) cells. Bioinformatic analyses indicated the differential regulation of 277 soluble proteins in the ADAM17-dependent secretome as well as novel direct ADAM17 cleavage substrates, such as mimecan and perlecan. Furthermore, we found that the ADAM17-dependent secretome promoted an opposite regulation of ERK and FAK pathways as well as PPARγ downstream activation. These findings demonstrated fine-tuning of cell signaling rendered by the soluble molecules mediated by ADAM17.

The mechanism of alternative splicing in the transcriptome may increase the proteome diversity in eukaryotes. In proteomics, several studies aim to use protein sequence repositories to annotate MS experiments or to detect differentially expressed proteins. However, the available protein sequence repositories are not designed to fully detect protein isoforms derived from mRNA splice variants. To foster knowledge for the field, here we introduce SpliceProt, a new protein sequence repository of transcriptome experimental data used to investigate for putative splice variants in human proteomes. Current version of SpliceProt contains 159 719 non-redundant putative polypeptide sequences. The assessment of the potential of SpliceProt in detecting new protein isoforms resulting from alternative splicing was performed by using publicly available proteomics data. We detected 173 peptides hypothetically derived from splice variants, which 54 of them are not present in UniprotKB/TrEMBL sequence repository. In comparison to other protein sequence repositories, SpliceProt contains a greater number of unique peptides and is able to detect more splice variants. Therefore, SpliceProt provides a solution for the annotation of proteomics experiments regarding splice isofoms. The repository files containing the translated sequences of the predicted splice variants and a visualization tool are freely available at http://lbbc.inca.gov.br/spliceprot.

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.

To evaluate whether protein changes in extracellular matrix of dental biofilm are a unique property of sucrose, this in situ study was conducted using as active control glucose and fructose, the sucrose monosaccharide constituents. Proteins were analyzed by two-dimensional electrophoresis followed by LC-MS/MS after trypsin digestion. Absence or lower abundance of calcium-binding proteins and higher abundance of prolactin-induced proteins were found in biofilm formed in the presence of sucrose or its monosaccharide constituents compared with water, the negative control group. The data suggest that besides sucrose, other dietary carbohydrates may also provoke a change in the protein profile of extracellular matrix of dental biofilm formed.

Patago-calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms. Fil: Peichoto, María Elisa. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina. Instituto Butantan. Laboratório de Fisiopatologia; Brasil. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Paes Leme, Adriana F.. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Pauletti, Bianca A.. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Correia Batista, Isabel. Instituto Butantan. Laboratório de Bioquímica e Biofísica; Brasil Fil: Mackessy, Stephen P.. University of Northern Colorado; Estados Unidos Fil: Acosta, Ofelia Cristina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias. Departamento de Clinica. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina Fil: Santoro, Marcelo Larami. Instituto Butantan. Laboratório de Fisiopatologia; Brasil

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.

Oral cancer disease represents a significant fraction of all human cancer types and its poor early diagnosis contributes to reduced individual survival rate. The identification of proteins modulated in tumorigenic cells and its post-translational modifications may improve our understanding of tumor development in epithelial cells. We have analyzed the phosphoproteome of tumorigenic (SCC-9) and non-tumorigenic (HaCaT) cell lines using MS-based approach in order to identify phosphopeptides with differing patterns of modifications and/or abundance. Our results revealed the identity of 4,206 protein phosphorylation sites with sixty-two sites showing to be significantly modulated between the two cell lines. The phosphoproteome data showed an overrepresentation of proteins with a possible role in nuclear regulatory functions. Pathway analysis was further performed on the phosphoproteome dataset and differences and commonalities of the functional pathways present in tumorigenic and non-tumorigenic cells were identified. Phosphopeptides that belong to the proteins lamina-associated polypeptide 2 isoform alpha and serine–arginine repetitive matrix protein 2 were identified with differential abundance and they appear as promising tumor-related phosphopeptides. These two proteins may be related to the structural alterations generally found in the nucleus of tumorigenic cells. The identification of phosphorylation sites in tumorigenic cells may contribute to disclose novel signaling mechanisms associated with OSCC. Significance Oral Squamous Cell Carcinoma (OSCC) is an important cancer disease affecting thousands of people worldwide. Many cellular processes related to the development of oral cancer remain unknown; however, the studies performed in vitro with cancer cells have contributed to guide more specific research which may be further performed by using in vivo approaches or clinical samples. To our knowledge, only few studies have been published showing the results of phosphoproteome profiling of squamous cell carcinoma models, and many signaling proteins must be identified and functionally characterized in order to increase the knowledge available about the complexity of the signaling networks responsible for oral cancer development and its progression. Furthermore, our knowledge regarding proteins exclusive or very low abundant in cancer cells remains limited. A better understanding of the differences between signaling pathways present in epithelial cell lines may contribute to reveal the processes underlying the OSCC.

Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

The extracellular milieu is comprised in part by products of cellular secretion and cell surface shedding. The presence of such molecules of the sheddome and secretome in the context of the extracellular milieu may have important clinical implications. In cancer they have been hypothesized to play a role in tumor growth and metastasis. The objective of this study was to evaluate whether the sheddome/secretome from two cell lines could be correlated with their potential for tumor development. Two epithelial cell lines, HaCaT and SCC-9, were chosen based on their differing abilities to form tumors in animal models of tumorigenesis. These cell lines when stimulated with phorbol-ester (PMA) showed different characteristics as assessed by cell migration, adhesion and higher gelatinase activity. Proteomic analysis of the media from these treated cells identified interesting, functionally relevant differences in their sheddome/secretome. Among the shed proteins, soluble syndecan-1 was found only in media from stimulated tumorigenic cells (SCC-9) and its fragments were observed in higher amount in the stimulated tumorigenic cells than stimulated non-tumorigenic cells (HaCaT). The increase in soluble syndecan-1 was associated with a decrease in membrane-bound syndecan-1 of SCC-9 cells after PMA stimuli. To support a functional role for soluble syndecan-1 fragments we demonstrated that the synthetic syndecan-1 peptide was able to induce cell migration in both cell lines. Taken together, these results suggested that PMA stimulation alters the sheddome/secretome of the tumorigenic cell line SCC-9 and one such component, the syndecan-1 peptide identified in this study, was revealed to promote migration in these epithelial cell lines.

Oral squamous cell carcinoma (OSCC) is the eighth most prevalent malignant neoplasm and accounts for 2% of all deaths by cancer worldwide. OSCCs have a highly variable clinical course, and because it is often diagnosed only after it has reached an advanced stage, the overall survival rate is less than 50% in 5 years. A better knowledge of the mechanisms that influence OSCC development and its progression is still needed. Particularly, most of the attention has been driven to mechanisms associated with tumor cell invasion and how the surrounding tumoral stroma may influence it. The myoma organotypic model is a novel fully human organotypic invasion model that mimics the tumor microenvironment (TME) (Nurmenniemi et al. 2009). Aiming to investigate the molecular mechanisms related to OSCC development and search for prognostic markers for this tumor, we used the myoma organotypic assay, followed by MS-ESI technique of laser microdissected invaded tissue, to analyze several features related to OSCC invasive vs. non-invasive cells. Our results showed that the HSC-3, an aggressive and invasive oral squamous cell carcinoma cell line, invaded as small islands and cell chords and penetrated deeply in the tissue. Unlike, the less aggressive LN2 cells, formed a well structured epithelium layer, but they invaded less in myoma. The proteomic analysis was able to characterize the differential expressed proteins of these, and revealed a group of proteins exclusively expressed in the cells that deeply invade the myoma, such as HMGA2 (high mobility group), JUP (junction plakoglobin), PLEC (plectin), HIST1H2B (Histone H2B), IQGAP1 (Ras GTPase-activating-like protein), EEF1A1 (elongation factor 1-alpha), HSPA9 (Stress-70 protein, mitochondrial), HADHA (Trifunctional enzyme subunit alpha). Additionally, the TME is a unique ambient created and shaped by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. Interestingly, we found a group of high abundant proteins expressed in the TME surrounding the invasive island. Among them are PALLD (palladin), TNC (tenascin C), LMNA (laminin), VIM (vimentin), and COL1A (collagen-type I-alpha I), which have been described to be involved in cell migration and proliferation. Our preliminary data suggests that the myoma organotypic tumor invasion assay is successful in determining new molecules and potential pathways associated with OSCC tumor invasion. We also present here a new insight for head and neck cancer biomarkers research that can, ultimately, contribute to more individualized treatment of patients affected by OSCC. However, validation of such results in clinical sample cohorts is important for a better understanding of the biological events associate with tumor invasion and disease spread (metastasis) in those patients. Citation Format: Nilva K. Cervigne, Carolina Bitu, Bianca A. Pauletti, Adriana F. Paes Leme, Tuula Salo, Ricardo Della Coletta. In-depth analysis of myoma organotypic model can determine potential markers of oral cancer invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2014-4998

Background Multiple Myeloma (MM) is a malignant proliferation of B-cells with plasma cell differentiation. Interactions of plasma cells with stromal cells and other cells in bone marrow (BM) microenvironment are responsible for MM progression. Screening of proteins expressed on the membrane surface of plasma cells, stromal cells and mononuclear cells from MM patients by mass spectrometry (MS) may identify new prognostic and therapeutic targets in this incurable disease.