Cation Involvement in Telomestatin Binding to G-Quadruplex DNA

The binding mode of telomestatin to G-quadruplex DNA has been investigated using electrospray mass spectrometry, by detecting the intact complexes formed in ammonium acetate. The mass measurements show the incorporation of one extra ammonium ion in the telomestatin complexes. Experiments on telomestatin alone also show that the telomestatin alone is able to coordinate cations in a similar way as a crown ether. Finally, density functional theory calculations suggest that in the G-quadruplex-telomestatin complex, potassium or ammonium cations are located between the telomestatin and a G-quartet. This study underlines that monovalent cation coordination capabilities should be integrated in the rational design of G-quadruplex binding ligands

Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results. We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite’s major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable Gquadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusions. This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family

Proteome alteration induced by hTERT transfection of human fibroblast cells

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (W138). Cytosolic and nuclear fractions of W138 cells, empty vector transfected W138 (W138-HPV) and hTERT W138 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in W138 and W138-HPV, but were differentially expressed in W138 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control W138-HPV cells. The proteome alteration induced by hTERT W138 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions

Influence of the matrix on the In Source Decay of permethylated glycans during MALDI-TOF analysis

Introduction In source decay (ISD) is a common phenomenon occurring very rapidly during ionization process in the source of MALDI-MS instruments and resulting in the presence of well resolved peaks of fragments in mass spectrum. While they make interpretation of spectra more complex, these fragments were shown to be useful to sequence peptides and proteins. Concerning glycans, only a few reports were published, using different matrices on various samples and therefore making it difficult to compare. In this context, the goal of this work is to perform a systematic study allowing to define optimal conditions to induce ISD of glycans or, inversely, to minimize this phenomenon in the study of more complex mixtures. Methods Glycans were purchased from Sigma-Aldrich. Iodomethane was used in DMSO/NaOH to permethylate the glycans. This reaction was stopped by water and permethylated glycans were extracted by chloroform. Spectra were recorded on a Bruker Ultraflex II in positive ion mode. 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT) were prepared at 20 mg/ml in 50 % acetonitrile, 0.1 % formic acid solution. 9-aminoacridine (9-AA) was dissolved at saturation in a 50 % acetonitrile, 0.1 % formic acid and further diluted 4 times in the same solution. α-Cyano-4-hydroxycinnamic (HCCA) acid was prepared at 20 mg/ml in 97 % acetone, 0,1 % formic acid. In some spots, LiI was added to obtain Li+ adducts instead of Na+ adducts. Preliminary data In source fragmentation of permethylated Lacto-N-difucoHexaose I and LS tetrasaccharide B was first studied in DHB. While the MS/MS of the Na+ adducts of these compounds (performed by LID) produces intenses B and Y fragments, those resulting from in source fragmentation are mainly oxonium ions, resulting from the cleavage of a glycosylic bond without any exchange of hydrogen atoms. These oxonium fragments were also obtained for lithium adducts. It was previously described that these fragments are produced by the cleavage of a protonated glycosidic bond. These ions carry their positive charge on a trivalent oxygen atom and are therefore not present on the spectra as sodium adducts. Since the peaks of protonated glycans are very low in MALDI spectra, it would indicate that protonation of glycosidic bonds of permethylated glycans would strongly favor a fragmentation reaction. Different matrices were tested to compare their ability to induce in source fragmentation of permethylated glycans. Interestingly, ATT gave similar results comparing to DHB while HCCA showed a lesser ability to promote in source fragmentation. However, the most striking result came from the use of 9-AA. This matrix, which is usually used in negative ion mode, was able to produce easily sodium adducts ions of permethylated glycan with a satisfying signal to noise ratio in positive ion mode. Moreover, practically no in source fragmentation was observed with this matrix. The few produced fragments were B ions but no oxonium ions were detected. Presence of these B fragments was increased for Li+ adducts. As 9-AA is the most basic of tested matrices, the absence of oxonium ions could result from its inability to transfer protons to the glycosidic bond of permethylated glycans. 9-AA could therefore become a matrix of choice to study complex mixtures of glycans, by reducing artefact peaks produced by ISD. Novel aspect ISD of permethylated glycans is induced by DHB while 9-AA strongly favors the presence of molecular ions

Optimization of Matrix Conditions for the Control of MALDI In-Source Decay of Permethylated Glycans.

Due to its fastness and its easiness to use, MALDI-MS is currently an analytical tool widely used in glycomic applications. However, the MALDI ionization process could result in the so-called "in-source decay", or ISD, of analytes, leading to complex spectra. On the other hand, ISD opens the possibility to perform pseudo-MS(3) experiments. This phenomenon must therefore be controlled in order to be used on demand as a supplementary tool for the analysis of permethylated glycans by MALDI mass spectrometry. For this purpose, several matrices were tested and MALDI imaging was used to determine optimal conditions promoting or, inversely, avoiding ISD of permethylated glycans. 2,5-DHB was shown to be a versatile matrix allowing one to induce or prevent ISD according to the location of laser shots. Inversely, it was shown that 9-aminoacridine forms homogeneous spots and avoids completely ISD. This matrix would therefore be suitable for automatic analysis.GLYCO (FEDER

Identification and Relative-quantification of Glycans by Matrix-assisted Laser Desorption/Ionization In-Source Decay with Hydrogen Abstraction

The use of specific matrices allows enhancing the scope of in-source decay (ISD) applications in matrix-assisted laser desorption ionization (MALDI) thanks to the specificity of analyte-matrix chemistry. The use of an oxidizing matrix, 5-nitrosalicylic acid (5-NSA) for MALDI-ISD of glycans is shown to promote fragmentation pathways involving radical precursors. Both glycosidic and cross-ring cleavages are promoted by hydrogen abstraction from hydroxyl group of glycans by 5-NSA molecules. Cross-ring cleavage ions are potentially useful in linkage analysis, one of the most critical steps of glycan characterization. Moreover, we show here that isobaric glycans could be distinguished by structure specific ISD ions, and that the molar ratio of glycan isomers in the mixture can be estimated from their fragment ions abundance. The use of 5-NSA also opens the possibility to perform pseudo-MS3 analysis of glycans. Therefore, MALDI-ISD with 5-NSA is a useful method for identification of glycans and semi-quantitative analysis of mixture of glycan isomers