Proteomics and Phospho-Proteomics Profiling of the Co-Formulation of Type I and II Interferons, HeberFERON, in the Glioblastoma-Derived Cell Line U-87 MG.

HeberFERON, a co-formulation of Interferon (IFN)-α2b and IFN-γ, has effects on skin cancer and other solid tumors. It has antiproliferative effects over glioblastoma multiform (GBM) clones and cultured cell lines, including U-87 MG. Here, we report the first label-free quantitative proteomic and phospho-proteomic analyses to evaluate changes induced by HeberFERON after 72 h incubation of U-87 MG that can explain the effect on cellular proliferation. LC-MS/MS, functional enrichment and networking analysis were performed. We identified 7627 proteins; 122 and 211 were down- and up-regulated by HeberFERON (fold change > 2; p < 0.05), respectively. We identified 23,549 peptides (5692 proteins) and 8900 phospho-peptides; 523 of these phospho-peptides (359 proteins) were differentially modified. Proteomic enrichment showed IFN signaling and its control, direct and indirect antiviral mechanisms were the main modulated processes. Phospho-proteome enrichment displayed the cell cycle as one of the most commonly targeted events together with cytoskeleton organization; translation/RNA splicing, autophagy and DNA repair, as represented biological processes. There is a high interconnection of phosphoproteins in a molecular network; mTOR occupies a centric hub with interactions with translation machinery, cytoskeleton and autophagy components. Novel phosphosites and others with unknown biological functionality in key players in the aforementioned processes were regulated by HeberFERON and involved CDK and ERK kinases. These findings open new experimental hypotheses regarding HeberFERON action. The results obtained contribute to a better understanding of HeberFERON effector mechanisms in the context of GBM treatment.

CIGB-300-Regulated Proteome Reveals Common and Tailored Response Patterns of AML Cells to CK2 Inhibition.

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis.

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.

In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Phosphoproteomic Landscape of AML Cells Treated with the ATP-Competitive CK2 Inhibitor CX-4945.

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells.

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

CIGB-300 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome.

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha' (CK2α/CK2α') and two regulatory beta subunits (CK2ß), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2ß also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.

Targeting the hydrophilic regions of recombinant proteins by MS via in-solution buffer-free trypsin digestion.

A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with "high-mannose" N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation.

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

Promiscuous T cell epitopes boosts specific IgM immune response against a P0 peptide antigen from sea lice in different teleost species.

The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.

An "on-matrix" digestion procedure for AP-MS experiments dissects the interplay between complex-conserved and serotype-specific reactivities in Dengue virus-human plasma interactome.

The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. SIGNIFICANCE: False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix ("on-matrix" digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by the domain III of the viral envelope glycoprotein. Fifty nine proteins were identified as putative interaction partners of Dengue virus (IPs) either due to direct binding or by co-isolation with interacting proteins. Collectively the IPs identified from the pull-down with the recombinant domain III proteins representing the four viral serotypes, 29% were identified only after "on-matrix" digestion which demonstrate the usefulness of this method of recovering bait-bound proteins. Results highlight a particular importance of "on-matrix" digestion procedure for comparative studies where a stronger interaction with one of the interest baits could prevent a bound protein to elute under standard conditions thus leading to misinterpretation as absent in the interactome of this particular bait. The analysis of the Interaction Network indicates that Dengue virus interactome mediated by the domain III of the envelope protein is rather conserved in the viral complex suggesting a key role of these interactions for viral infection thus making candidates to explore for potential biomarkers of clinical outcome in DENV-caused disease. Interestingly, some particular IPs exhibit significant differences in the strength of the interaction with the viral serotypes representing interactions that involve more variable regions in the surface of the domain III. Since such variable regions are the consequence of the interaction with antibodies generated by human immune response; this result relates the interaction with proteins from human plasma with the interplay of the virus and the human immune system.

Development and validation of a bioanalytical method based on LC-MS/MS analysis for the quantitation of CIGB-814 peptide in plasma from Rheumatoid Arthritis patients.

CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel CD4+ T-cell epitope of human heat shock protein 60 (83-109, MW 2988.38g/mol) with a mutation (D100→L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical method, based on LC-MS/MS analysis, in the SRM mode was developed and fully validated to quantify this peptide in human plasma. An internal standard with the same amino acid sequence but labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear range from 1.5 to 48ng/mL (without matrix effect and carry over) and an accuracy and precision good enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The peptide was administered subcutaneously in three dose levels (1, 2.5 and 5mg) not normalized to the body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of 1.5ng/mL enabled the PK analysis. The Cmax, reached at 0.5h, showed a great variability, that was most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the variability between patients. A rapid clearance of this peptide (4-6h) is advantageous for an immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore peripheral tolerance.

Novel interactions of domain III from the envelope glycoprotein of dengue 2 virus with human plasma proteins.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. BIOLOGICAL SIGNIFICANCE: Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

Accurate estimation of isoelectric point of protein and peptide based on amino acid sequences.

MOTIVATION: In any macromolecular polyprotic system-for example protein, DNA or RNA-the isoelectric point-commonly referred to as the pI-can be defined as the point of singularity in a titration curve, corresponding to the solution pH value at which the net overall surface charge-and thus the electrophoretic mobility-of the ampholyte sums to zero. Different modern analytical biochemistry and proteomics methods depend on the isoelectric point as a principal feature for protein and peptide characterization. Protein separation by isoelectric point is a critical part of 2-D gel electrophoresis, a key precursor of proteomics, where discrete spots can be digested in-gel, and proteins subsequently identified by analytical mass spectrometry. Peptide fractionation according to their pI is also widely used in current proteomics sample preparation procedures previous to the LC-MS/MS analysis. Therefore accurate theoretical prediction of pI would expedite such analysis. While such pI calculation is widely used, it remains largely untested, motivating our efforts to benchmark pI prediction methods. RESULTS: Using data from the database PIP-DB and one publically available dataset as our reference gold standard, we have undertaken the benchmarking of pI calculation methods. We find that methods vary in their accuracy and are highly sensitive to the choice of basis set. The machine-learning algorithms, especially the SVM-based algorithm, showed a superior performance when studying peptide mixtures. In general, learning-based pI prediction methods (such as Cofactor, SVM and Branca) require a large training dataset and their resulting performance will strongly depend of the quality of that data. In contrast with Iterative methods, machine-learning algorithms have the advantage of being able to add new features to improve the accuracy of prediction. CONTACT: yperez@ebi.ac.uk AVAILABILITY AND IMPLEMENTATION: The software and data are freely available at https://github.com/ypriverol/pIRSupplementary information: Supplementary data are available at Bioinformatics online.

Proteomic Study to Survey the CIGB-552 Antitumor Effect.

CIGB-552 is a cell-penetrating peptide that exerts in vitro and in vivo antitumor effect on cancer cells. In the present work, the mechanism involved in such anticancer activity was studied using chemical proteomics and expression-based proteomics in culture cancer cell lines. CIGB-552 interacts with at least 55 proteins, as determined by chemical proteomics. A temporal differential proteomics based on iTRAQ quantification method was performed to identify CIGB-552 modulated proteins. The proteomic profile includes 72 differentially expressed proteins in response to CIGB-552 treatment. Proteins related to cell proliferation and apoptosis were identified by both approaches. In line with previous findings, proteomic data revealed that CIGB-552 triggers the inhibition of NF-κB signaling pathway. Furthermore, proteins related to cell invasion were differentially modulated by CIGB-552 treatment suggesting new potentialities of CIGB-552 as anticancer agent. Overall, the current study contributes to a better understanding of the antitumor action mechanism of CIGB-552.

Receptor-activated human α2-macroglobulin interacts with the envelope protein of dengue virus and protects virions from temperature-induced inactivation through multivalent binding.

Based on the hypothesis that interactions between virions and serum components may influence the outcome of dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from the envelope (E) protein of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and α2-macroglobulin (α2M) as novel viral interactors. After confirming the specific binding of both SAP and α2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species was actually the receptor-activated form of α2M (α2M*), that α2M* could bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4>DENV1~DENV2>DENV3 and that this interaction exhibited a strong avidity effect when multivalent binding was favoured (KD 8 × 10(-8) M for DIIIE2). We also showed that α2M* bound to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibited an ED50 of 2.9 × 10(-8) M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factor interaction network during human DENV infection.

Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.

HI-bone: a scoring system for identifying phenylisothiocyanate-derivatized peptides based on precursor mass and high intensity fragment ions.

Peptide sequence matching algorithms used for peptide identification by tandem mass spectrometry (MS/MS) enumerate theoretical peptides from the database, predict their fragment ions, and match them to the experimental MS/MS spectra. Here, we present an approach for scoring MS/MS identifications based on the high mass accuracy matching of precursor ions, the identification of a high intensity b1 fragment ion, and partial sequence tags from phenylthiocarbamoyl-derivatized peptides. This derivatization process boosts the b1 fragment ion signal, which turns it into a powerful feature for peptide identification. We demonstrate the effectiveness of our scoring system by implementing it on a computational tool called "HI-bone" and by identifying mass spectra of an Escherichia coli sample acquired on an Orbitrap Velos instrument using Higher-energy C-trap dissociation. Following this strategy, we identified 1614 peptide spectrum matches with a peptide false discovery rate (FDR) below 1%. These results were significantly higher than those from Mascot and SEQUEST using a similar FDR.