Martina Samiotaki

Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1α

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1α, involves several post-translational modifications. To study HIF-1α phosphorylation, we have used human full-length recombinant HIF-1α as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1α. Analysis of in vitro phosphorylated HIF-1α by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1α. When these mutant forms of HIF-1α were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1α mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1α. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1α mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1α. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1α by blocking its CRM1-dependent nuclear export.

Characterization of Flavonoid Subgroups and Hydroxy Substitution by HPLC-MS/MS

HPLC-DAD coupled with mass spectrometry in the positive ionization mode was applied to study the fragmentation of twelve selected flavonoids. Compounds belonging to all the major subgroups found in common plants, i.e. flavonols, flavones, dihydroflavonols, flavanones and flavanols were studied. Compound standards were injected into the spectrometer and produced characteristic mass spectra. The fragmentation of each compound was studied and it was shown that the dehydration and carbon monoxide losses from the [M+H]+ ion by the members of each subgroup produced specific fragments, thus allowing the characterization of the flavonoid subgroups. Moreover, fragments resulting from fission of the C-rings are specific of each subgroup and revealed the substitution pattern of A- and B-rings. In order to verify the identifying efficiency of the positive ionization mode through these characteristic fragmentations, the unknown flavonoids of an Origanum vulgare diethyl ether extract were separated with the HPLC system and the major peaks were successfully identified with the mass spectrometer.

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.

A Common Single Nucleotide Polymorphism in Endoplasmic Reticulum Aminopeptidase 2 Induces a Specificity Switch That Leads to Altered Antigen Processing

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.

RNA-binding Motif Protein 15 Binds to the RNA Transport Element RTE and Provides a Direct Link to the NXF1 Export Pathway

Retroviruses/retroelements provide tools enabling the identification and dissection of basic steps for post-transcriptional regulation of cellular mRNAs. The RNA transport element (RTE) identified in mouse retrotransposons is functionally equivalent to constitutive transport element of Type D retroviruses, yet does not bind directly to the mRNA export receptor NXF1. Here, we report that the RNA-binding motif protein 15 (RBM15) recognizes RTE directly and specifically in vitro and stimulates export and expression of RTE-containing reporter mRNAs in vivo. Tethering of RBM15 to a reporter mRNA showed that RBM15 acts by promoting mRNA export from the nucleus. We also found that RBM15 binds to NXF1 and the two proteins cooperate in stimulating RTE-mediated mRNA export and expression. Thus, RBM15 is a novel mRNA export factor and is part of the NXF1 pathway. We propose that RTE evolved as a high affinity RBM15 ligand to provide a splicing-independent link to NXF1, thereby ensuring efficient nuclear export and expression of retrotransposon transcripts.

Analysis of secreted proteins for the study of bladder cancer cell aggressiveness.

Secreted proteins play a key role in cell signaling, communication, and migration. We recently described the development of an aggressive variant (T24M) of the bladder cancer cell line T24. Using this cell line model, the objective of our work was the identification of secreted proteins involved in the acquisition of the aggressive phenotype. Using in vitro assays, we demonstrate that conditioned media of the T24M cells promote motility of the parental less aggressive T24 cells. Proteomic analysis of cell culture conditioned media by the use of 2-dimensional gel electrophoresis coupled to MALDI TOF MS and LC-MS approaches resulted in enrichment and detection of multiple classical extracellular and secreted proteins such as fibronectin, cystatin, fibrillin, fibulin, interleukin 6, etc. Comparison of the secretome of the T24 and T24M cells indicated differences in proteins with potential involvement in the mechanisms of cell aggressiveness including SPARC, tPA, and clusterin. These findings were further confirmed by Western blot analysis. In the case of SPARC, further studies involving transwell assays indicated that blockage of the protein in the presence of SPARC-specific Abs results in decreased cell motility. Collectively, our study provides a 2DE-based comprehensive analysis of bladder cancer cell secretome. The results indicate various secreted proteins with potential involvement in bladder cancer cell aggressiveness and more specifically provide initial evidence for special role of SPARC in bladder cancer cell motility and invasiveness.

Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris

Hepcidin is a circulating cysteine-rich peptide with antimicrobial properties. It functions as a hormonal regulator of iron homeostasis by controlling iron efflux from target cells via ferroportin (FPN1), which is internalized and degraded upon hepcidin binding. Because of its profound biomedical significance, hepcidin has become the target of intense biochemical studies. The aim of this study was to produce functional recombinant hepcidin in sufficient quantities for advanced research or potential clinical use, as the native hepcidin can be isolated from urine in very low yield. We report the expression, purification and functional characterization of hepcidin variants in yeast P. pastoris. The yield of untagged hepcidin 20- and 25-mer peptides was too low for complete functional characterization. By contrast, Hep20 and Hep25 tagged with either single 6xHis or double Myc-6xHis epitopes were expressed at high quantities (5-7mg/l of culture), yet mostly in oligomeric forms. Purification of monomeric tagged hepcidins was achieved by size exclusion chromatography, with a yield of 0.5-1mg/l of culture. All recombinant hepcidins exhibited bacteriostatic activity and the ability to control cellular iron homeostasis, with Hep25-His being the most potent. Thus, Hep25-His promoted an increase in the levels of the labile iron pool (LIP) in macrophages and consistently bound to ferroportin (FPN1) causing its internalization and the subsequent downregulation of transferrin receptor 1 (TfR1) expression. Analysis by mass-spectrometry suggested that all eight cysteines participated in disulfide bond formation. Our results suggest that only the recombinant Hep25-His monomer was a fully active peptide. As Hep25-His faithfully recapitulates the functional properties of native Hep25, it represents a powerful tool for biochemical studies and potential diagnostic and therapeutic applications.

Comparative proteomic analysis of Arthrobacter phenanthrenivorans Sphe3 on phenanthrene, phthalate and glucose.

UNLABELLED In the present study, by applying comparative quantitative proteomics, we investigated the metabolic adaptation of Arthrobacter phenanthrenivorans Sphe3 when using phenanthrene, phthalate, glucose or glucose plus phenanthrene as sole carbon and energy sources. More than a third of the total Sphe3 proteins, with function prediction within the genome, were identified with confidence. Proteomic analysis data and annotated genomic information coincide, allowing us to clarify the phenanthrene catabolic pathway. We confirmed the implication of several proteins in aromatic substrate degradation by identifying those mediating the initial ring-hydroxylation and ring cleavage of phenanthrene to phthalate, phthalate degradation, as well as ortho- and meta-protocatechuate catabolism. Repression of catabolic genes by glucose was observed by both proteomic and transcriptional analyses. The presence of aromatic substrates resulted in changes in the abundance of proteins involved in substrate and amino acid metabolism, stress response, detoxification and membrane and cell wall metabolism. Uptake and transport associated proteins differ in the substrates used, indicating the use of different uptake mechanisms for transport of each compound in the Sphe3 cells. Our results also suggest the activation of a glyoxylate shunt in the presence of aromatic compounds, based on the up-regulation of the key enzymes of this pathway. BIOLOGICAL SIGNIFICANCE A. phenanthrenivorans Sphe3, isolated from a creosote contaminated soil in Greece, can grow on phenanthrene as the sole source of carbon and energy. To explore the phenanthrene catabolic pathway by determining the key proteins involved in this pathway, as well as the global changes in proteins due to the adaptive response of Sphe3 cells grown on different substrates, we applied a gel-free quantitative proteomic analysis using nanoLC-MS/MS. To our knowledge this is the first study of comparative global proteomic changes occurring in the Sphe3 cells under exposure in different nutritional environments. The extended proteomic changes observed in Sphe3 grown on different substrates provide an insight in the complex interactions occurring in the presence of aromatic compounds and could serve as a basis for further investigations intended to elucidate the general regulatory mechanism by which Sphe3 adapts to such xenobiotic environments. This may light the way for more efficient engineering of bacteria towards more effective bioremediation applications.

Proteomic methodologies and their application in colorectal cancer research.

Colorectal cancer usually develops in clearly defined stages, with distinct molecular alterations characterizing each transition. This often slow process makes colorectal cancer an ideal target for early detection programs. The blossoming of global, -omics approaches in recent years has led to greatly increased expectations for novel diagnostic and prognostic tools. Despite many early disappointments and the resulting skepticism, real progress has been made with exciting new prospects for cancer research. This review summarizes currently available proteomic tools for identifying novel biomarkers and drug targets, as well as an overview of their application in research on molecular mechanisms of carcinogenesis. Emphasis is given to novel sample preparation methods, protein separation and identification techniques, and advanced mass spectrometry tools for quantitative proteomic. The most important applications of these technologies in colorectal cancer research are discussed.

Detection and isolation of antiatherogenic and antioxidant substances present in olive mill wastes by a novel filtration system.

Olive mill waste water (OMWW) is a major environmental issue in the Mediterranean. We address this problem by investigating the wastes for the presence of biologically active compounds already detected in both olive oil and pomace. Two initial OMWW samples were filtered using two microporous filtering media: (a) clayey diatomite and (b) zeolitic volcanic tuffs, obtaining three filtered samples from each. All initial and filtrated samples were tested for their activity on platelet activating factor (PAF)-induced aggregation. The results showed that the initial samples contain biologically active compounds (PAF inhibitors) and that in their respective last-eluted filtered samples these compounds are purified. These eluted samples, along with their corresponding initial OMWW, were further separated with HPLC and the purified fractions responsible for the aforementioned biological activity, were further studied using chemical determinations and MS analysis. It was confirmed that the PAF inhibitor present in these fractions resembles the one isolated from olive oil. These results offer a new approach on the OMWW handling by offering an alternative use of this waste as starting material for nutritional and/or pharmaceutical purposes in the future.