Analyse protéomique de l'inhibition de la télomérase par des ligands spécifiques des télomères

Les télomères sont des structures nucléoprotéiques nécessaires à la protection des extrémités des chromosomes contre les dégradations ou fusions induites par les processus de réparation de l’ADN. Ils sont constitués de complexes protéiques associés à des répétitions en tandem d’un motif 5’-(TTAGGG)-3’ sous forme double brin de plusieurs kilobases, et finalisés par une extrémité 3’simple brin de la même séquence de quelques centaines de bases. On observe in vitro un raccourcissement des télomères à chaque division cellulaire, ce même fait est corrélé in vivo avec le vieillissement. Lorsque les télomères se raccourcissent et atteignent une taille critique, les cellules entrent en sénescence réplicative qui se définit par un arrêt de croissance définitif et viable des cellules.La télomérase est une ADN polymerase ARN-dépendante qui allonge le télomère en lui ajoutant des séquences répétitives ‘TTAGGG’. Elle comprend une composante ARN (hTR) qui sert de matrice et une composante catalytique à activité transcriptase inverse (hTERT). L’expression seule d’hTERT suffit à immortaliser différents types cellulaires.La télomérase est fortement exprimée dans la majorité des cellules tumorales alors que son activité est difficilement détectable dans la plupart des cellules somatiques. Ces observations font de la télomérase une cible d’intérêt pour des nouvelles thérapies anticancéreuses. Une de ces nouvelles stratégies consiste en l’utilisation de molécules capables de stabiliser les structures en G-quadruplexe de l’ADN. La stabilisation des G-quadruplexes télomériques rend les télomères inaccessibles pour la télomérase et inhibe son activité par la séquestration de son substrat.L’objectif de cette thèse est d’évaluer la réponse cellulaire induite par le traitement cellulaire de deux ligands des G-quadruplexes au niveau du protéome des cellules WI38 transfectées pour exprimer hTERT. Les deux ligands, TMPyP4 et la télomestatine, inhibent la télomérase mais ont une spécificité différente pour les diverses structures G-quadruplexes.En premier lieu, nous avons étudié l’effet de la transfection d’hTERT sur des cellules fibroblastiques humaines (WI38). Cette première étude a été conduite afin de caractériser l’adaptation cellulaire résultante de l’immortalisation des cellules WI38. Par la suite, celle-ci permettra de comparer ces résultats avec ceux obtenus lors de l’étude protéomique de l’effet des ligands des G-quadruplexes. Nous avons montré que hTERT induit une augmentation de la capacité fonctionnelle du réticulum endoplasmique ainsi qu’une modulation des signaux cellulaire Ca2+-dépendant. Nous proposons que cette adaptation cellulaire est responsable d’une résistance accrue vis-à-vis de différents stress environnementaux. D’autres protéines impliquées dans des mécanismes d’oncogenèse ont été identifiées et sont différentiellement exprimées entre les cellules parentales et les cellules transfectées.L’analyse protéomique des traitements cellulaires indique que TMPyP4 induit une altération du protéome beaucoup plus prononcée que celle induite par la télomestatine. Ceci est probablement dû au manque de spécificité de TMPyP4 pour les G-quadruplexes télomériques. TMPyP4 induit, entre autres, une sous-expression massive des hnRNPs, une modulation de la voie protéasomale, une diminution probable de la traduction et une surexpression de plusieurs chaperonnes moléculaires. La télomestatine induit notamment une surexpression de la protéine BCL2A1 qui est impliquée dans les processus de résistance aux agents anticancéreux et une probable augmentation de la traduction. Les deux ligands ont des effets communs sur la variation d’expression des chaperons CCT (sou-expression), de l’HSP90 alpha (surexpression) et de l’hnRNP D (sous-expression). L’HSP90 est également surexprimée dans les cellules hTERT-WI38 par rapport aux cellules parentales. Cette protéine fait actuellement l’objet de nombreuses recherches visant à inhiber son activité du fait de son implication en oncogenèse ainsi que dans la modulation de l’activité de la télomérase.Enfin, nous avons montré l’intérêt de ce type d’étude protéomique dans l’évaluation d’agents à vocation thérapeutique préalablement aux études cliniques

Infinitely many periodic orbits of exact magnetic flows on surfaces for almost every subcritical energy level

We consider an exact magnetic flow on the tangent bundle of a closed surface. We prove that for almost every energy level $\kappa$ below the Ma\~n\'e critical value of the universal cover there are infinitely many periodic orbits with energy $\kappa$

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

Identification of stromal proteins overexpressed in nodular sclerosis Hodgkin lymphoma

Hodgkin lymphoma (HL) represents a category of lymphoid neoplasms with unique features, notably the usual scarcity of tumour cells in involved tissues. The most common subtype of classical HL, nodular sclerosis HL, characteristically comprises abundant fibrous tissue stroma. Little information is available about the protein composition of the stromal environment from HL. Moreover, the identification of valid protein targets, specifically and abundantly expressed in HL, would be of utmost importance for targeted therapies and imaging, yet the biomarkers must necessarily be accessible from the bloodstream. To characterize HL stroma and to identify potentially accessible proteins, we used a chemical proteomic approach, consisting in the labelling of accessible proteins and their subsequent purification and identification by mass spectrometry. We performed an analysis of potentially accessible proteins in lymph node biopsies from HL and reactive lymphoid tissues, and in total, more than 1400 proteins were identified in 7 samples. We have identified several extracellular matrix proteins overexpressed in HL, such as versican, fibulin-1, periostin, and other proteins such as S100-A8. These proteins were validated by immunohistochemistry on a larger series of biopsy samples, and bear the potential to become targets for antibody-based anti-cancer therapies

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

On the Risks of Phylogeny-Based Strain Prioritization for Drug Discovery: Streptomyces lunaelactis as a Case Study

peer reviewedStrain prioritization for drug discovery aims at excluding redundant strains of a collection in order to limit the repetitive identification of the same molecules. In this work, we wanted to estimate what can be unexploited in terms of the amount, diversity, and novelty of compounds if the search is focused on only one single representative strain of a species, taking Streptomyces lunaelactis as a model. For this purpose, we selected 18 S. lunaelactis strains taxonomically clustered with the archetype strain S. lunaelactis MM109T. Genome mining of all S. lunaelactis isolated from the same cave revealed that 54% of the 42 biosynthetic gene clusters (BGCs) are strain specific, and five BGCs are not present in the reference strain MM109T. In addition, even when a BGC is conserved in all strains such as the bag/fev cluster involved in bagremycin and ferroverdin production, the compounds produced highly differ between the strains and previously unreported compounds are not produced by the archetype MM109T. Moreover, metabolomic pattern analysis uncovered important profile heterogeneity, confirming that identical BGC predisposition between two strains does not automatically imply chemical uniformity. In conclusion, trying to avoid strain redundancy based on phylogeny and genome mining information alone can compromise the discovery of new natural products and might prevent the exploitation of the best naturally engineered producers of specific molecules

L’analyse protéomique du sécrétome des ostéoblastes fournis de nouvelles indications sur les mécanismes de la sclérose sous-chondrale dans l’arthrose

peer reviewe

Up-Regulated Salivary Proteins of Brown Marmorated Stink Bug Halyomorpha halys on Plant Growth-Promoting Rhizobacteria-Treated Plants

Plant Growth-Promoting Rhizobacteria (PGPR) induce systemic resistance (SR) in plants, decreasing the development of phytopathogens. The FZB42 strain of Bacillus velezensis is known to induce an SR against pathogens in various plant species. Previous studies suggested that it could also influence the interactions between plants and associated pests. However, insects have developed several strategies to counteract plant defenses, including salivary proteins that allow the insect escaping detection, manipulating defensive pathways to its advantage, deactivating early signaling processes, or detoxifying secondary metabolites. Because Brown Marmorated Stink Bug (BMSB) Halyomorpha halys is highly invasive and polyphagous, we hypothesized that it could detect the PGPR-induced systemic defenses in the plant, and efficiently adapt its salivary compounds to counteract them. Therefore, we inoculated a beneficial rhizobacterium on Vicia faba roots and soil, previous to plant infestation with BMSB. Salivary gland proteome of BMSB was analyzed by LC–MS/MS and a label-free quantitative proteomic method. Among the differentially expressed proteins, most were up-regulated in salivary glands of insects exposed to PGPR-treated plants for 24 h. We could confirm that BMSB was confronted with a stress during feeding on PGPR-treated plants. The to-be-confirmed defensive state of the plant would have been rapidly detected by the invasive H. halys pest, which consequently modified its salivary proteins. Among the up-regulated proteins, many could be associated with a role in plant defense counteraction, and more especially in allelochemicals detoxification or sequestration