UMR9187 / U1196 – Chimie et Modélisation pour la Biologie du Cancer (CMBC)

Publications de l’unité

Année de publication : 2020

Rahima Chennoufi, Ngoc-Duong Trinh, Françoise Simon, Guillaume Bordeau, Delphine Naud-Martin, Albert Moussaron, Bertrand Cinquin, Houcine Bougherara, Béatrice Rambaud, Patrick Tauc, Céline Frochot, Marie-Paule Teulade-Fichou, Florence Mahuteau-Betzer & Eric Deprez (2020 Apr 23)

Interplay between cellular uptake, intracellular localization and the cell death mechanism in triphenylamine-mediated photoinduced cell death

Scientific Reports : 10 : 6881 : DOI : 10.1038/s41598-020-63991-9 En savoir plus
Résumé

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

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Zuffo M., Gandolfini A., Heddi B., Granzhan A. (2020 Apr 20)

Harnessing intrinsic fluorescence for typing of secondary structures of DNA

Nucleic Acids Research : Epub ahead of print : DOI : 10.1093/nar/gkaa257 En savoir plus
Résumé

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

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Ehlen A., Martin C., Miron S., Julien M., Theillet F.X., Boucherit V., Ropars V., Duchambon P., El Marjou A., Zinn Justin S., Carreira A. (2020 Apr 14)

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

Nature Communications : 11 : 1819 : DOI : 10.1038/s41467-020-15689-9 En savoir plus
Résumé

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

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Abhijit Saha, Patricia Duchambon, Vanessa Masson, Damarys Loew, Sophie Bombard, Marie-Paule Teulade-Fichou (2020 Mar 20)

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.

Biochemistry : 59 : 1261-1272 : DOI : 10.1021/acs.biochem.9b01094 En savoir plus
Résumé

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2′-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL-G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop-quartet interaction.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

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Paudel B.P., Moye A.L., Assi H.A., El-Khoury R., Cohen S.B., Birrento M.L., Samosorn S., Intharapichai K., Tomlinson C.G., Teulade-Fichou M.P., Gonz'alez C., Beck J.L., Damha M.J., van Oijen A.M., Bryan T.M. (2020 Feb 27)

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

bioRxiv : DOI : 10.1101/2020.02.26.965269 En savoir plus
Résumé

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

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Année de publication : 2019

Stéphanie Lemaître, Florent Poyer, Paul Fréneaux, Sophie Leboucher, François Doz, Nathalie Cassoux, Carole D Thomas (2019 Dec 25)

Low retinal toxicity of intravitreal carboplatin associated with good retinal tumor control in transgenic murine retinoblastoma.

Clinical & experimental ophthalmology : DOI : 10.1111/ceo.13711 En savoir plus
Résumé

Purpose

Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice.

Methods

Mice were divided into 9 groups: control, carboplatin 1.5 and 4μg, melphalan 0.1 and 1μg, topotecan 0.1 and 1μg, carboplatin 4μg/ topotecan 0.1μg and melphalan 1μg/ topotecan 0.1μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumor and presence of calcified tumors were the criteria for therapeutic response assessment. Ocular complications were assessed after 4 weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers.

Results

Topotecan was inactive on retinal tumors. Melphalan (1μg) led to a complete tumor control in 91.7% of eyes. Carboplatin strongly decreased the tumor burden (85.7‐93.8% of eyes without retinal tumor). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumors with a reduced retinal toxicity.

Conclusions

This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.

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Mouawad L., Beswick V., Jamin N., Montigny C., Quiniou E., Barbot T. (2019 Dec 18)

Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulations

bioRxiv : DOI : 10.1101/2019.12.17.879825 En savoir plus
Résumé

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well understood. To decipher this mechanism, we have performed normal modes analysis in the all-atom model, with the SERCA1a-SLN complex or the isolated SERCA1a embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304 and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 r A away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+-deprived E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN, although the SERCA1a-SLN complex was crystallized in an E1-like state. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight in the conformational transition between the E2 and E1 states.

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Katerina Duskova, Pauline Lejault, Élie Benchimol, Régis Guillot, Sébastien Britton, Anton Granzhan, David Monchaud (2019 Dec 13)

DNA junction ligands trigger DNA damage and are synthetic lethal with DNA repair inhibitors in cancer cells

Journal of the American Chemical Society : 142 : 424-435 : DOI : 10.1021/jacs.9b11150 En savoir plus
Résumé

Translocation of DNA and RNA polymerases along their duplex substrates results in DNA supercoiling. This torsional stress promotes the formation of plectonemic structures, including three-way DNA junction (TWJ), which can block DNA transactions and lead to DNA damage. While cells have evolved multiple mechanisms to prevent the accumulation of such structures, stabilizing TWJ through ad hoc ligands offer an opportunity to trigger DNA damage in cells with high level of transcription and replication, such as cancer cells. Here, we develop a series of azacryptand-based TWJ ligands, we thoroughly characterize their TWJ-interacting properties in vitro and demonstrate their capacity to trigger DNA damage in rapidly dividing human cancer cells. We also demonstrate that TWJ ligands are amenable to chemically induced synthetic lethality strategies upon association with inhibitors of DNA repair, thus paving the way towards innovative drug combinations to fight cancers.

DNA Junction Ligands Trigger DNA Damage

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Nathalie Grandin, Bruno Pereira, Camille Cohen, Pauline Billard, Caroline Dehais, Catherine Carpentier, Ahmed Idbaih, Franck Bielle, François Ducray, Dominique Figarella-Branger, Jean-Yves Delattre, Marc Sanson, Patrick Lomonte, Delphine Poncet, Pierre Verrelle, Michel Charbonneau, (2019 Nov 11)

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.

Acta neuropathologica communications : 7 : 175 : DOI : 10.1186/s40478-019-0833-0 En savoir plus
Résumé

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative « C-circle » assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

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Priyanka Toshniwal, Michelle Nguyen, Aurore Guédin, Helena Viola, Diwei Ho, Yongeun Kim, Uditi Bhatt, Charles Bond, Livia Hool, Laurence H Hurley, Jean-Louis Mergny, Mark Fear, Fiona Wood, K Swaminathan Iyer, Nicole M Smith (2019 Nov 2)

TGF-β-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.

FEBS letters : 593 : 3149-3161 : DOI : 10.1002/1873-3468.13658 En savoir plus
Résumé

Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-β- induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.

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Chen J., Zhang Y., Cheng M., Mergny J.L., Lin Q., Zhou J., Ju H. (2019 Nov 1)

Highly active G-quadruplex/hemin DNAzyme for sensitive colorimetric determination of lead(II)

Microchimica Acta : 186 : 786 : DOI : 10.1007/s00604-019-3950-3 En savoir plus
Résumé

A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3extasciiacutex end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2extasciiacutex-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 muM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples.

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Franck Court, Elisa Le Boiteux, Anne Fogli, Mélanie Müller-Barthélémy, Catherine Vaurs-Barrière, Emmanuel Chautard, Bruno Pereira, Julian Biau, Jean-Louis Kemeny, Toufic Khalil, Lucie Karayan-Tapon, Pierre Verrelle, Philippe Arnaud (2019 Sep 20)

Transcriptional alterations in glioma result primarily from DNA methylation-independent mechanisms.

Genome research : 29 : 1605-1621 : DOI : 10.1101/gr.249219.119 En savoir plus
Résumé

In cancer cells, aberrant DNA methylation is commonly associated with transcriptional alterations, including silencing of tumor suppressor genes. However, multiple epigenetic mechanisms, including polycomb repressive marks, contribute to gene deregulation in cancer. To dissect the relative contribution of DNA methylation-dependent and -independent mechanisms to transcriptional alterations at CpG island/promoter-associated genes in cancer, we studied 70 samples of adult glioma, a widespread type of brain tumor, classified according to their isocitrate dehydrogenase (IDH1) mutation status. We found that most transcriptional alterations in tumor samples were DNA methylation-independent. Instead, altered histone H3 trimethylation at lysine 27 (H3K27me3) was the predominant molecular defect at deregulated genes. Our results also suggest that the presence of a bivalent chromatin signature at CpG island promoters in stem cells predisposes not only to hypermethylation, as widely documented, but more generally to all types of transcriptional alterations in transformed cells. In addition, the gene expression strength in healthy brain cells influences the choice between DNA methylation- and H3K27me3-associated silencing in glioma. Highly expressed genes were more likely to be repressed by H3K27me3 than by DNA methylation. Our findings support a model in which altered H3K27me3 dynamics, more specifically defects in the interplay between polycomb protein complexes and the brain-specific transcriptional machinery, is the main cause of transcriptional alteration in glioma cells. Our study provides the first comprehensive description of epigenetic changes in glioma and their relative contribution to transcriptional changes. It may be useful for the design of drugs targeting cancer-related epigenetic defects.

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Angrand G., Quillévéré A., Loaëc N., Daskalogianni C., Granzhan A., Teulade-Fichou M.P., Fahraeus R., Prado Martins R., Blondel M. (2019 Sep 1)

Sneaking Out for Happy Hour: Yeast-Based Approaches to Explore and Modulate Immune Response and Immune Evasion

Genes : 10 : 667-689 : DOI : 10.3390/genes10090667 En savoir plus
Résumé

Many pathogens (virus, bacteria, fungi, or parasites) have developed a wide variety of mechanisms to evade their host immune system. The budding yeast Saccharomyces cerevisiae has successfully been used to decipher some of these immune evasion strategies. This includes the cis-acting mechanism that limits the expression of the oncogenic Epstein–Barr virus (EBV)-encoded EBNA1 and thus of antigenic peptides derived from this essential but highly antigenic viral protein. Studies based on budding yeast have also revealed the molecular bases of epigenetic switching or recombination underlying the silencing of all except one members of extended families of genes that encode closely related and highly antigenic surface proteins. This mechanism is exploited by several parasites (that include pathogens such as Plasmodium, Trypanosoma, Candida, or Pneumocystis) to alternate their surface antigens, thereby evading the immune system. Yeast can itself be a pathogen, and pathogenic fungi such as Candida albicans, which is phylogenetically very close to S. cerevisiae, have developed stealthiness strategies that include changes in their cell wall composition, or epitope-masking, to control production or exposure of highly antigenic but essential polysaccharides in their cell wall. Finally, due to the high antigenicity of its cell wall, yeast has been opportunistically exploited to create adjuvants and vectors for vaccination.

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Abegão L.M.G., Fonseca R.D., Santos F.A., Rodrigues J.J., Kamada K., Mendonça C.R., Piguel S., De Boni L. (2019 Aug 23)

First molecular electronic hyperpolarizability of series of π-conjugated oxazole dyes in solution: an experimental and theoretical study

RSC Adv. : 9 : 26476-26482 : DOI : 10.1039/C9RA05246A En savoir plus
Résumé

In this work, we report the experimental and theoretical first molecular electronic hyperpolarizability (βHRS) of eleven π-conjugated oxazoles compounds in toluene medium. The Hyper-Rayleigh Scattering (HRS) technique allowed the determination of the experimental dynamic βHRS values, by exciting the compounds with a picosecond pulse trains from a Q-switched and mode-locked Nd:YAG laser tuned at 1064 nm. Theoretical predictions based on time-dependent density functional theory level using the Gaussian 09 program package were performed with three different functionals (B3LYP, CAM-B3LYP, and M06-2X), to calculate both static and dynamic theoretical βHRS values. Good accordance was found between the experimental and theoretical values, in particular for the CAM-B3LYP and M06-2X functionals.

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Michaela Krafcikova, Simon Dzatko, Coralie Caron, Anton Granzhan, Radovan Fiala, Tomas Loja, Marie-Paule Teulade-Fichou, Tomas Fessl, Robert Hänsel-Hertsch, Jean-Louis Mergny, Silvie Foldynova-Trantirkova, Lukas Trantirek (2019 Aug 9)

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

Journal of the American Chemical Society : 141 : 13281-13285 : DOI : 10.1021/jacs.9b03031 En savoir plus
Résumé

Studies on DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA–ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

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