TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway

ANTICANCER AND ANTIMICROBIAL PROPERTIES OF TAT-RasGAP317-326

Cancer ranks among the leading causes of death worldwide; this makes the development of novel and improved anticancer treatment a priority. Current oncological therapeutics aim to activate apoptosis to achieve disease control. However, cancer cells can adapt and become refractory to therapy by mutating and acquiring the ability to resist apoptotic stimuli. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. Our laboratory has developed a cell permeable peptide called TAT-RasGAP317-326, which bears several anticancer properties. First, the RasGAP-derived peptide has the ability to inhibit metastatic progression by increasing cell adhésion and decreasing cell migration. Second, it was shown to specifically sensitize tumor cells to anticancer treatments such as chemotherapy and radiotherapy both in vitro and in vivo. In the present study, we provide evidences that TAT-RasGAP317-326 peptide can specifically sensitize cancer cells to chemotherapy-induced apoptosis independently of several key apoptotic regulators such as p53 and Bcl-2 family members, including Bax and Bak. We also report that this peptide possesses the capacity to directly kill a subset of cancer cell lines. Using pharmacologie and genetic inhibitions, we demonstrate that TAT-RasGAP3i7-326-induced death differs from known regulated forms of death. To understand how TAT-RasGAP3i7-326-death is controlled, we performed a genome-wide CRISPR knockout screening to assess the requirement of each essential gene. We found that spécifié potassium channels such as KCNQ5 in Raji cells and KCNN4 and KCNK5 in SKW6.4 cells are necessary for the peptide to kill those cells. We reveal that inhibition of these potassium channels prevents proper cytosolic access of TAT-RasGAP3i7-326, and other TAT-constructs. This ability to block extracellular compound to enter cells was assessed on several viruses. Preliminary data suggest that potassium channel blockade hampers the infectivity of spécifié pathogenic agents. By sheer serendipity, we uncover the antimicrobial property of TAT-RasGAP317-326. In vitro data demonstrate that the RasGAP-derived peptide is able to efficiently kill several pathogenic bacteria. We could validate in vivo using E. coli peritonitis model that TAT-RasGAP317-326 is a promising antibiotic peptide. -- Le cancer est la deuxième cause de mortalité dans le monde, après les maladies cardiovasculaires. Pour combattre cette maladie, les traitements oncologiques traditionnels, tels que la radiothérapie et chimiothérapie, ont pour but d'induire la mort des cellules cancéreuses par apoptose. Toutefois, ces cellules peuvent s'adapter et devenir réfractaires aux thérapies via l'accumulation de mutation leur permettant de résister aux stimuli apoptotiques. Le développement de nouvelles thérapies plus efficaces est donc une priorité afin d'améliorer la survie des patients atteints de cancers. Notre laboratoire a développé un peptide appelé TAT-RasGAP317-326 possédant de multiples propriétés anti-cancers. Celui-ci à la capacité de réduire la formation de métastases en augmentant l'adhérence des cellules et en bloquant la migration cellulaire. De plus, il a été démontré que ce peptide à la faculté d'augmenter spécifiquement dans les cellules cancéreuses l'efficacité de la radiothérapie et chimiothérapie, à la fois in vitro et in vivo. Dans ce travail de thèse, nous avons montré que le peptide TAT-RasGAP317-326 est capable de sensibiliser les cellules cancéreuses à l'apoptose induite par des agents chimiothérapeutiques, et ce indépendamment de protéines clés telles que p53 et des membres de la famille Bcl-2, dont Bax et Bak. Nous avons aussi montré que ce peptide pouvait tuer directement certaines lignées cancéreuses. Par une approche d'inhibition, à la fois pharmacologique et génétique, nous avons démontré que la mort induite par TAT-RasGAP317-326 était différente des formes connues de mort régulées. Pour comprendre le fonctionnement de la mort induite par ce peptide, nous avons criblé le génome entier afin d'identifier les gènes essentiels nécessaires à sa toxicité. Nous avons mis en évidence l'implication de canaux à potassium, tels que KCNQ5 dans les cellules Raji ainsi que KCNN4 et KCNK5 dans les cellules SKW6.4. Nous avons établi que le blocage de ces canaux à potassium empêche au peptide TAT- RasGAP3i7.326 mais aussi à d'autres molécules comportant la séquence TAT d'être normalement internalisés jusqu'au cytosol. La capacité d'empêcher l'entrée de composés extracellulaires représente un grand intérêt dans d'autres domaines de la biologie, tels que la virologie. Des données préliminaires montrent que l'inhibition de canaux à potassium permet de diminuer l'infectivité de plusieurs agents viraux pathogènes. Par sérendipité, nous avons découvert que TAT-RasGAP317-326 possède des propriétés antimicrobiennes. Nos données in vitro montrent qu'il est capable de tuer certaines bactéries hautement pathogènes. De plus, son efficacité in vivo a pu être démontrée grâce au modèle de péritonite de la souris causée par E

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

International audienceFerredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP(+) reductase and ferredoxin-NAD(+) reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP(+) reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD(+) reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain

TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway