Untargeted metabolomics approach to discriminate mistletoe commercial products.

Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe's anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure-activity relationships based on LC-MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia.

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.

Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

A large scale proteome analysis of the gefitinib primary resistance overcome by KDAC inhibition in KRAS mutated adenocarcinoma cells overexpressing amphiregulin.

KDAC inhibitors (KDACi) overcome gefitinib primary resistance in non-small cell lung cancer (NSCLC) including mutant-KRAS lung adenocarcinoma. To identify which proteins are involved in the restoration of this sensitivity and to provide new therapeutic targets for mutant-KRAS lung adenocarcinoma, we performed an iTRAQ quantitative proteomic analysis after subcellular fractionation of H358-NSCLC treated with gefitinib and KDACi (TSA/NAM) versus gefitinib alone. The 86 proteins found to have been significantly dysregulated between the two conditions, were mainly involved in cellular metabolism and cell transcription processes. As expected, the pathway related to histone modifications was affected by the KDACi. Pathways known for controlling tumor development and (chemo)-resistance (miRNA biogenesis/glutathione metabolism) were affected by the KDACi/gefitinib treatment. Moreover, 57 dysregulated proteins were upstream of apoptosis (such as eEF1A2 and STAT1) and hence provide potential therapeutic targets. The inhibition by siRNA of eEF1A2 expression resulted in a slight decrease in H358-NSCLC viability. In addition, eEF1A2 and STAT1 siRNA transfections suggested that both STAT1 and eEF1A2 prevent AKT phosphorylation known for enhancing gefitinib resistance in NSCLC. Therefore, altogether our data provide new insights into proteome regulations in the context of overcoming the NSCLC resistance to gefitinib through KDACi in H358 KRAS mutated and amphiregulin-overexpressing NSCLC cells.

Proteomic Identification of Allergenic Proteins of Morus alba L. Pollenexacerbation.

BACKGROUND: Tree pollens are well-known aeroallergens all over the world. Little is known about the allergenicity of Morus alba (white mulberry) pollen. OBJECIVE: We aimed to explore the potential allergens of this pollen and its clinical relevance in tree pollen allergic patients living in Istanbul, Turkey. METHODS: Twenty three seasonal allergic rhinitis patients with a confirmed tree pollen allergy and 5 healthy control subjects underwent skin prick and nasal provocation tests with M.alba pollen extract. The pollen extract was then resolved by gel electrophoresis, and immunoblotted with sera from patients/control individuals to detect the potential allergenic proteins. The prevalent IgE binding proteins from 1D-gel were analyzed by MALDI-TOF/TOF. RESULTS: Eleven out of 23 patients were reactive to the extract with skin prick tests. Seven of those patients also reacted positively to the nasal provocation tests. The most common IgE-binding pollen proteins were detected between 55-100 kDa, and also at molecular weights lower than 30 kDa for some patients. Mass spectrometry analyses revealed that the principal IgE-binding protein was methionine synthase (5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase), which is then proposed as a novel allergen in M.alba pollen. CONCLUSION: This study provides the first detailed information for the potential allergens of Morus alba pollen of Istanbul. Methionine synthase with an apparent molecular weight of 80 to 85 kDa has been recognized as one of the allergens in Morus alba pollen for the first time.

Current trends in protein acetylation analysis.

INTRODUCTION: Acetylation is a widely occurring post-translational modification (PTM) of proteins that plays a crucial role in many cellular physiological and pathological processes. Over the last decade, acetylation analyses required the development of multiple methods to target individual acetylated proteins, as well as to cover a broader description of acetylated proteins that comprise the acetylome. Areas covered: This review discusses the different types of acetylation (N-ter/K-/O-acetylation) and then describes some major strategies that have been reported in the literature to detect, enrich, identify and quantify protein acetylation. The review highlights the advantages and limitations of these strategies, to guide researchers in designing their experimental investigations and analysis of protein acetylation. Finally, this review highlights the main applications of acetylomics (proteomics based on mass spectrometry) for understanding physiological and pathological conditions. Expert opinion: Recent advances in acetylomics have enhanced knowledge of the biological and pathological roles of protein acetylation and the acetylome. Besides, radiolabeling and western blotting remain also techniques-of-choice for targeted protein acetylation. Future challenges in acetylomics to analyze the N-ter and K-acetylome will most likely require enrichment/fractionation, MS instrumentation and bioinformatics. Challenges also remain to identify the potential biological roles of O-acetylation and cross-talk with other PTMs.

Low-molecular-weight color pI markers to monitor on-line the peptide focusing process in OFFGEL fractionation.

High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling.

Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK ß-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

Cardiac phosphoproteome reveals cell signaling events involved in doxorubicin cardiotoxicity.

The successful use of anthracyclines like doxorubicin in chemotherapy is limited by their severe cardiotoxicity. Despite decades of clinical application, a satisfying description of the molecular mechanisms involved and a preventive treatment have not yet been achieved. Here we address doxorubicin-induced changes in cell signaling as a novel potential mediator of doxorubicin toxicity by applying a non-biased screen of the cardiac phosphoproteome. Two-dimensional gel electrophoresis, phosphospecific staining, quantitative image analysis, and MALDI-TOF/TOF mass spectrometry were combined to identify (de)phosphorylation events occurring in the isolated rat heart upon Langendorff-perfusion with clinically relevant (5 µM) and supraclinical concentrations (25 µM) of doxorubicin. This approach identified 22 proteins with a significantly changed phosphorylation status and these results were validated by immunoblotting for selected phosphosites. Overrepresentation of mitochondrial proteins (>40%) identified this compartment as a prime target of doxorubicin. Identified proteins were mainly involved in energy metabolism (e.g. pyruvate dehydrogenase and acyl-CoA dehydrogenase), sarcomere structure and function (e.g. desmin) or chaperone-like activities (e.g. α-crystallin B chain and prohibitin). Changes in phosphorylation of pyruvate dehydrogenase, regulating pyruvate entry into the Krebs cycle, and desmin, maintaining myofibrillar array, are relevant for main symptoms of cardiac dysfunction related to doxorubicin treatment, namely energy imbalance and myofibrillar disorganization. This article is part of a Special Issue entitled: Translational Proteomics.

The cell line secretome, a suitable tool for investigating proteins released in vivo by tumors: application to the study of p53-modulated proteins secreted in lung cancer cells.

Malignant processes such as metastasis, invasion, or angiogenesis are tightly dependent on the composition of the extracellular medium, which is itself affected by the release of proteins by the tumor cells. p53, a major tumor suppressor protein very frequently mutated and/or inactivated in cancer cells, is known to modulate the release of proteins by the tumor cells; however, while p53-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. Here, we characterized the p53-dependent secretome of a lung tumor model in vitro (H358 human nonsmall cell lung adenocarcinoma cell line with a homozygous deletion of p53) and demonstrate that the modulation of exported proteins can also be detected in vivo in the plasma of tumor-bearing mice. We used a clone of H358, stably transfected with a tetracycline-inducible wild-type p53-expressing vector. With the use of iTRAQ labeling and LC-MALDI-MS/MS analysis, we identified 909 proteins released in vitro by the cells, among which 91 are p53-modulated. Three proteins (GDF-15, FGF-19, and VEGF) were also investigated in H358/TetOn/p53 xenograft mice. The ELISA dosage on total tumor protein extracts confirmed the influence of p53 on the release of these proteins in vivo. Moreover, the GDF-15 concentration was measured in the plasma and its p53-dependent modulation was confirmed. To our knowledge, this is the first report establishing that the in vitro cell line secretome is reliable and reflects the extracellular release of proteins from tumor cells in vivo and could be used to identify putative tumor markers.

Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling.

BACKGROUND: The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes. RESULTS: We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3-10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow. CONCLUSION: The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.

Methylation of tumor-suppressor genes in neuroblastoma: The RASSF1A gene is almost always methylated in primary tumors.

BACKGROUND: Currently, the best characterized genetic aberration in neuroblastoma (NB) is MYCN amplification, which has been clearly related to prognosis. In the present study, we investigated whether specific epigenetic alterations are associated with stage of disease. PROCEDURE: Sixty-two NBs (45 primary tumors and 17 NBs at relapse) were studied in terms of the methylation status of 19 genes (p15INK4a, p16INK4a, p14ARF, APC, RB1, RASSF1A, BLU, FHIT, RARbeta, INI1, TIMP3, NF2, MGMT, DAPK, FLIP, ECAD, CASP8, and the receptors DcR1 and DcR2). RESULTS: At diagnosis, we found hypermethylation of RASSF1A in 93% of these tumors, hypermethylation of TIMP3 in 51%, of CASP8 in 38%, of BLU in 34%, of DcR2 in 25%, and of DcR1 in 11%. All 17 tumors tested at relapse showed hypermethylation of RASSF1A (100%), while 10 showed hypermethylation of TIMP3 (59%), six of CASP8 (35%), five of DcR2 (29%), four of BLU (24%), and three of DcR1 (18%). Hypermethylation was related to clinical stage; NBs at stages 1, 2, and 4s were less frequently methylated than stages 3 and 4 disease (P = 0.002). CONCLUSION: These results from our series indicate that hypermethylation of tumor-suppressor genes may be important in the development and evolution of NB. These epigenetic alterations could be used as a marker of the disease and genes regulating methylation should be considered as possible therapeutic targets in NB.

Methylation of RASSF1A and TRAIL pathway-related genes is frequent in childhood intracranial ependymomas and benign choroid plexus papilloma.

Ependymomas (EP) represent the third most frequent type of central nervous system (CNS) tumor of childhood, after astrocytomas and medulloblastomas. No prognostic biological markers are available, and differentiation from choroid plexus papilloma (CPP) is difficult. The present objective was, for a sample of 27 children with intracranial EP and 7 with CPP, to describe and compare the methylation status of 19 genes (with current HUGO symbol, if any): p15INK4a (CDKN2B), p16INK4a and p14ARF (both CDKN2A), APC, RB1, RASSF1A (RASSF1), BLU (ZMYND10) FHIT, RARB, MGMT, DAPK (DAPK1), ECAD (CDH1), CASP8, TNFRSF10C, TNFRSF10D, FLIP (CFLAR), INI1 (SMARCB1), TIMP3, and NF2. Three adult corteses were used as a control. We detected a similar percentage of methylated tumors in both groups (71% in CPP and 77% in EP). No gene was methylated in that control group. RASSF1A was the most frequently methylated gene in both benign tumors (66%) and EP (56%). The genes associated with apoptosis were methylated in both groups of tumors. The percentages of TRAIL pathway genes (CASP8, TFRSF10C, and TFRSF10D) methylated were 30, 9.5, and 36.4%, respectively, in ependymomas and 50, 50, and 16.7%, respectively, in choroid plexus papillomas. No other gene was methylated in the benign tumors, whereas FHIT was methylated in 22%, RARB in 14.8%, BLU in 13.6%, p16INK4a in 11.1%, TNFRSF10C in 9.5%, and DAPK in 7.4% of ependymomas. Although we did not observe a statistical relationship between methylation and clinical outcome, the methylation pattern does not appear to be randomly distributed in ependymoma and may represent a mechanism of tumor development and evolution.