Les topoisomérases I et II contrôlent l'épissage alternatif (exemple de la caspase-2)

L épissage alternatif permet un contrôle de l activité génique, à partir d une même matrice d ARN, en générant des protéines structurellement et fonctionnellement distinctes. Il est impliqué au niveau de nombreux gènes de l apoptose, dont le gène CASP-2 pour lequel deux épissages alternatifs sont connus : un épissage en 5 conduisant à l inclusion d un premier exon non codant spécifique de chaque transcrit, et un deuxième épissage alternatif dans la région 3 conduisant à l inclusion de l exon 9 dans l ARN Casp-2S. Le pré-ARNm permet en effet l assemblage de deux ARNm principaux : Casp-2L, qui contient 11 exons et code la procaspase-2L, forme d environ 48 kDa, et Casp-2S qui contient 12 exons et code la procaspase-2S de 35 kDa. Les deux isoformes de la procaspase-2 ont des rôles distincts : l isoforme longue est pro-apoptotique et l isoforme courte, anti-apoptotique lorsqu elle est surexprimée. Dans ce travail, nous avons observé que le traitement de cellules leucémiques humaines U-937 par un inhibiteur de la topoisomérase II, l étoposide (VP16) à 50 M pendant 4h, entraîne une augmentation de l inclusion de l exon 9, une diminution du transcrit Casp-2L ainsi qu une diminution de la procaspase-2L. En outre, la majorité des inhibiteurs spécifiques des topoisomérases I ou II induisent l inclusion de l exon 9, un effet post-transcriptionnel, dépendant en partie de la PKCzêta, prédominant en phase G1/S du cycle cellulaire, et dissociable de l apoptose. Les autres agents anticancéreux, dont les cibles ne sont pas des topoisomérases, sont sans effet. Dans leur ensemble, nos résultats permettent de penser que l activité des topoisomérases est responsable du contrôle de l épissage alternatif du pré-ARNm Casp-2, ce qui pourrait permettre une reprogrammation du potentiel apoptotique associé aux procaspases-2, en addition du mécanisme de NMD ( Nonsense-Mediated mRNA Decay ) qui dégrade Casp-2S. De plus les inhibiteurs de topoisomérases modifient le profil d épissage d autres transcrits de caspases et contrôlent le répresseur d épissage SRp38. Au total, les inhibiteurs de topoisomérases, et vraisemblablement les topoisomérases elles-mêmes, ont un impact étendu sur l épissage alternatif et donc, entre autres, sur la modulation de l apoptose.Alternative splicing allows a control of genetic activity, starting from the same pre-mRNA, by generating structurally and functionally distinct proteins. It is involved in the control of expression of many apoptotic genes, such as the CASP-2 gene for which two alternative splicing events are known : a first one occurs in the 5' region, leading to the inclusion of a specific non coding first exon, and a second event occurs in the 3' region, leading to the inclusion of exon 9 in Casp-2S RNA. The pre-mRNA allows the assembly of two main mRNA species : Casp-2L, which contains 11 exons and encodes the 48 kDa procaspase-2L form, and Casp-2S, which contains 12 exons and encodes the 35 kDa procaspase-2S protein. These two procaspases-2 isoforms have distinct roles: the long isoform is pro-apoptotic and the short is anti-apoptotic when overexpressed. In this work, we observed that the treatment of human leukemic cells U-937 by an inhibitor of topoisomerase II, etoposide (VP16) at 50 M during 4h, leads to an increase in the inclusion of exon 9, a decrease in Casp-2L transcript as well as a decrease in procaspase-2L. Moreover, the majority of topoisomerases I or II inhibitors induce the inclusion of the exon 9, a post-transcriptional effect, partly dependent on PKCzeta, predominant in the G1/S phase of the cellular cycle, and independent of apoptosis. The other anticancer drugs, whose targets are not topoisomerases, have no effect. Hence, our results strongly suggest that the activity of topoisomerases controls the alternative splicing of Casp-2 pre-mRNA, which could allow a modulation of procaspase-2-dependent apoptosis. In addition, we have obtained evidence suggesting that the short isoform of caspase-2 is likely not to be expressed or expressed only at very low levels since it s transcript is subjected to Nonsense-Mediated mRNA Decay. Finally, topoisomerase inhibitors modify the splicing of others caspases transcripts and control the SRp38 repressor protein. Consequently, topoisomerase inhibitors, and probably topoisomerases themselves, have an extended impact on alternative splicing, and consequently on apoptotic modulation.DIJON-BU Médecine Pharmacie (212312103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Épissage alternatif, pathologie et thérapeutique moléculaire

L’épissage des pré-ARN messagers constitue une étape obligatoire pour la très grande majorité des transcrits primaires chez les eucaryotes. Il est sujet à de nombreuses variantes, ou épissages alternatifs, qui permettent l’assemblage de transcrits codant pour des protéines requises de manière transitoire ou traduisant une adaptation pathologique. Cette diversification du réservoir de transcrits augmente sensiblement la variabilité protéique. L’identification de mutations dans les séquences nécessaires à l’épissage ou à son contrôle a permis de déterminer les modifications d’épissage en relation avec le développement de maladies héréditaires ou cancéreuses. Rapidement, des approches ont été développées pour tenter de contourner de façon expérimentale l’effet de ces mutations, afin de restaurer un niveau d’épissage suffisant à une compensation fonctionnelle efficace. Les espoirs suscités par ces nouvelles approches méritent que l’on en prenne toute la dimension et que les perspectives thérapeutiques qu’elles offrent soient évaluées.Pre-mRNA splicing operates towards at least 95 % of the transcript pool. It is subjected to a large number of variations, collectively regrouped under the term of alternative mRNA splicing, which occurs, on average, 6 to 8 times per pre-mRNA molecule. Consequently, many more proteins may be encoded from a single gene, which may satisfy a physiological need, or mark a pathological adaptation. The identification of mutations in sequences required for splicing, both constitutive and alternative, or for their control, has permitted to determine the causes of qualitative or quantitative variations in transcript levels associated with inherited diseases or cancer development. A number of molecular approaches have been undertaken to try to compensate for the effect of deleterious splicing mutations and to restore, at least in part, sufficient amounts of either the normal or a surrogate transcript. These include overexpression of splicing proteins, improvement of their activity by post-translational modification, splice-site increased or decreased usage, and RNA-mediated trans-splicing. Using such approaches, phenotypic improvements have been obtained in animal models, carrying new hopes for the development of therapeutic strategies aimed at correcting both inherited and acquired diseases that involve pre-mRNA splicing defects

Implication des topoisomérases dans le mécanisme de l'épissage alternatif du pré-ARNm de la caspase-2

DIJON-BU Médecine Pharmacie (212312103) / SudocSudocFranceF

Le cuivre contrôle la plasticité cellulaire et déclenche la réponse inflammatoire

International audienc

Targeting M2 Macrophages with a Novel NADPH Oxidase Inhibitor

ROS in cancer cells play a key role in pathways regulating cell death, stemness maintenance, and metabolic reprogramming, all of which have been implicated in resistance to chemo/ immunotherapy. Adjusting ROS levels to reverse the resistance of cancer cells without impairing normal cell functions is a new therapeutic avenue. In this paper, we describe new inhibitors of NADPH oxidase (NOX), a key enzyme in many cells of the tumor microenvironment. The first inhibitor, called Nanoshutter-1, NS1, decreased the level of tumor-promoting “M2” macrophages differentiated from human blood monocytes. NS1 disrupted the active NADPH oxidase-2 (NOX2) complex at the membrane and in the mitochondria of the macrophages, as shown by confocal microscopy. As one of the characteristics of tumor invasion is hypoxia, we tested whether NS1 would affect vascular reactivity by reducing ROS or NO levels in wire and pressure myograph experiments on isolated blood vessels. The results show that NS1 vasodilated blood vessels and would likely reduce hypoxia. Finally, as both NOX2 and NOX4 are key proteins in tumors and their microenvironment, we investigated whether NS1 would probe these proteins differently. Models of NOX2 and NOX4 were generated by homology modeling, showing structural differences at their C-terminal NADPH site, in particular in their last Phe. Thus, the NADPH site presents an unexploited chemical space for addressing ligand specificity, which we exploited to design a novel NOX2-specific inhibitor targeting variable NOX2 residues. With the proper smart vehicle to target specific cells of the microenvironment as TAMs, NOX2-specific inhibitors could open the way to new precision therapies

Targeting M2 Macrophages with a Novel NADPH Oxidase Inhibitor

ROS in cancer cells play a key role in pathways regulating cell death, stemness maintenance, and metabolic reprogramming, all of which have been implicated in resistance to chemo/ immunotherapy. Adjusting ROS levels to reverse the resistance of cancer cells without impairing normal cell functions is a new therapeutic avenue. In this paper, we describe new inhibitors of NADPH oxidase (NOX), a key enzyme in many cells of the tumor microenvironment. The first inhibitor, called Nanoshutter-1, NS1, decreased the level of tumor-promoting “M2” macrophages differentiated from human blood monocytes. NS1 disrupted the active NADPH oxidase-2 (NOX2) complex at the membrane and in the mitochondria of the macrophages, as shown by confocal microscopy. As one of the characteristics of tumor invasion is hypoxia, we tested whether NS1 would affect vascular reactivity by reducing ROS or NO levels in wire and pressure myograph experiments on isolated blood vessels. The results show that NS1 vasodilated blood vessels and would likely reduce hypoxia. Finally, as both NOX2 and NOX4 are key proteins in tumors and their microenvironment, we investigated whether NS1 would probe these proteins differently. Models of NOX2 and NOX4 were generated by homology modeling, showing structural differences at their C-terminal NADPH site, in particular in their last Phe. Thus, the NADPH site presents an unexploited chemical space for addressing ligand specificity, which we exploited to design a novel NOX2-specific inhibitor targeting variable NOX2 residues. With the proper smart vehicle to target specific cells of the microenvironment as TAMs, NOX2-specific inhibitors could open the way to new precision therapies