Allosteric communication within cancer therapeutic target PARP1 : mechanism of catalytic activation and modulation of allostery by inhibitors

L’ADN contient l’information génétique essentielle au développement et au bon fonctionnement de tout organisme vivant. Cependant, l’ADN peut être endommagé ou modifié par une exposition régulière à différents facteurs tels que la lumière du soleil, la pollution, la radiation, etc. La cellule a ainsi développé des mécanismes de réparation très efficaces puisqu’un ADN sain est essentiel pour la santé d’un organisme. La protéine humaine PARP1 est une enzyme clé de la réparation de l’ADN. PARP1 détecte rapidement les dommages à l’ADN en s’y liant, ce qui stimule son activité catalytique. PARP1 catalyse la formation de chaînes d’ADP-ribose qui sont ajoutées à PARP1, ainsi que d’autres protéines, en tant que modification post-traductionnelle. Les chaînes d’ADP- ribose permettent la décondensation de la chromatine ainsi que le recrutement de facteurs de réparation à l’ADN endommagé. PARP1 possède plusieurs domaines régulateurs en plus de son domaine catalytique, le domaine catalytique lui-même se divisant en un domaine hélicoïdal (HD) ainsi qu’un domaine ADP- ribosyltransférase. L’augmentation de l’activité catalytique de PARP1, à la suite de sa liaison à l’ADN endommagé, implique qu’un signal allostérique se transmette à travers ses différents domaines. Le HD joue un rôle essentiel dans le relais de cette communication allostérique puisque c’est ultimement un changement de conformation du HD (i.e « ouverture ») qui révèle le site actif et active l’enzyme. De plus, il a été démontré que les inhibiteurs de PARP1 peuvent moduler l’affinité de l’enzyme pour l’ADN endommagé. Certains inhibiteurs peuvent ainsi provoquer la « capture » de l’ADN par PARP1, un phénomène qui requiert la présence du HD et qui est particulièrement toxique pour les cellules cancéreuses présentant des défauts de réparation de l’ADN. Pour cette raison, la mort cellulaire induite par les inhibiteurs de PARP1 est un traitement prometteur et quatre inhibiteurs sont déjà utilisés pour traiter le cancer des ovaires et du sein. Cependant, le mécanisme précis derrière la capture de l’ADN par PARP1 est encore nébuleux et nécessite de plus amples recherches. Puisque la capture de l’ADN par PARP1 requiert le relais d’un signal allostérique par le HD, et que l’ouverture du HD participe à cette communication, il est donc essentiel de comprendre les changements de conformations effectués par ce domaine. Nous avons ainsi obtenu pour la première fois une structure atomique de PARP1 en conformation active. Celle-ci montre que l’ouverture du HD amène la formation d’une interface additionnelle entre ce domaine et les autres domaines régulateurs de PARP1. Ainsi, entraver la formation de cette nouvelle interaction, par des mutations ponctuelles, diminue grandement l’activité catalytique de PARP1 lié à l’ADN, ce qui suggère que l’interface participe à la communication allostérique de l’enzyme. Tel que mentionné plus haut, les inhibiteurs de PARP1 peuvent moduler de manières différentes l’affinité de PARP1 pour les dommages à l’ADN et ainsi influencer distinctement la communication allostérique de l’enzyme. Nous avons caractérisé une nouvelle série d’inhibiteurs de PARP1 et évalué leur capacité à moduler l’affinité de PARP1 pour l’ADN endommagé. Nos travaux démontrent qu’un inhibiteur volumineux occupant le site actif n’augmentera pas nécessairement l’affinité de PARP1 pour les dommages à l’ADN. Leur capacité à favoriser la capture de l’ADN dépend plutôt de leur interaction avec la région du HD voisine au site actif. En résumé, nos travaux participent à l’amélioration des connaissances concernant l’activation catalytique de PARP1 et la communication allostérique. Une meilleure compréhension de l’allostérie de PARP1 permettra la conception de médicaments ayant la toxicité désirée pour tuer les cellules cancéreuses.DNA contains the genetic instructions for the development and proper function of all living organisms. However, DNA can be broken and modified in harmful ways through daily exposures to exterior stresses such as sun light, pollution, radiation, etc. Since stable and undamaged DNA is essential for the health of an organism, cells have developed repair mechanisms to ensure that DNA damage is taken care of efficiently. The human protein PARP1 is a key enzyme that contributes to DNA repair. PARP1 rapidly detects DNA lesions which greatly stimulates its catalytic activity. PARP1 catalyzes the formation of chains of ADP-ribose that are attached covalently to PARP1 itself, or other target proteins, as a posttranslational modification. The chains of ADP-ribose allow for the recruitment of chromatin remodelling factors and repair factors to process the DNA lesions. PARP1 carries multiple regulatory domains in addition to its catalytic domain, with the catalytic domain itself composed of the helical domain (HD) and the ADP-ribosyltransferase fold. DNA damage binding greatly stimulates PARP1 catalytic activity, which requires that an allosteric signal is relayed across the enzyme’s domains. Interestingly, the HD has been found to play an essential role in the PARP1 allostery. The HD undergoes a change of conformation (i.e. opening) following PARP1 DNA damage binding which reveals the active site. Additionally, inhibitors binding the active site of the enzyme can modulate PARP1 DNA binding affinity. Some inhibitors can induce PARP1 DNA “trapping”, a phenomenon that requires the HD and appears particularly toxic to cancer cells bearing DNA repair deficiencies. Cell death induced by PARP1 inhibitors is a promising cancer treatment and four inhibitors have approval for clinical use against ovarian and breast cancers. However, the precise mechanism underlying PARP1 trapping on DNA is still unclear and requires further research. Since PARP1 trapping requires the presence of the HD, and that the HD opening is involved in relaying the allosteric signal, it remains essential to characterize its change of conformation. We have obtained for the first time atomic structures of PARP1 in a catalytically active state. Crystal structures show that the HD in open conformation forms an additional interdomain interface. Mutating this interface prevents PARP1 strong catalytic activation following DNA damage binding, suggesting that the allosteric communication is impaired. Additionally, these structures reveal how the HD active conformation leads to the reveal of the active site in the ART domain. As mentioned above, PARP1 inhibitors can modulate the enzyme’s DNA binding affinity and therefore impact its allosteric communication. We have characterized a series of novel inhibitors and tested their propensity to increase PARP1 DNA binding affinity. Our work highlights that bulky inhibitors that fill the active site will not necessarily promote PARP1 affinity for DNA lesions. Rather, it appears that inhibitors may trigger DNA trapping via their interaction with a neighboring region of the HD. Overall, our work deepens our understanding of PARP1 catalytic activation and allosteric communication. Properly understanding how PARP1 trapping occurs will help the design of specific drugs with the desired toxicity to kill cancer cells

Rest-activity disturbances and light exposure in myotonic dystrophy type 1 patients with apathy: An exploratory study using actigraphy

Background: Rest-activity rhythm disturbances have been reported in myotonic dystrophy type 1 (DM1) but no study has assessed its relationship with apathy and light exposure using actigraphy. Methods: Thirty-three DM1 patients wore an actigraph for two consecutive weeks. The Lille Apathy Rating Scale was used to assess apathy. Actigraphy was used to characterize sleep, activity, and light exposure and nonparametric analysis was performed to characterize intraday variability (IV), interday stability (IS), least active five-hour period (L5), most active ten-hour period (M10), and relative amplitude (RA). Results: Apathy was found in 42.4% of patients. Patients with apathy have a larger number of CTG repeat than those without (774 vs 381, p<0.01) Also, patients with apathy had higher IV values (0.98 vs 0.69, p<0.001) as well as lower IS (0.34 vs 0.47, p<0.05), RA (0.65 vs 0.83, p<0.05), and M10 (182.6 vs 493.8, p<0.001) values compared to patients without apathy. In addition, DM1 patients with apathy were exposed to less bright light (≥1000 lux) (41.4 vs 141.2 minutes p<0.01) than patients without. No difference was observed relatively to sleep quality and quantity. Conclusions: Daytime activity levels and bright light exposure were significantly lower in DM1 patients with apathy versus those without. The more fragmented, less stable, and lower amplitude rest-activity rhythm of DM1 patients with apathy, as revealed by non parametric analysis, can be partly ascribed to reduced daylight exposure. The use of bright light therapy as well as physical activity interventions should be explored to help prevent or reduce apathy levels in DM1

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field