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Contreversy<\/p>\n\n\n\n
Journal of Peptide Science <\/strong>(2025<\/em>)<\/p>\n\n\n\n Ali Hallaj, Francisca Tomas Ribeiro, Christian Widmann<\/p>\n\n\n\n In June 2024, at the CPP 2024 meeting in Montpelier (France), data from the group of Prof. Brock were presented suggesting that CPP direct translocation operated in distinct ways depending on which cell lines was tested. Specifically, it was reported that CPP direct translocation in HeLa cells was plasma membrane potential dependent, while in HEK cells, CPP direct translocation did not depend on the plasma membrane potential (Vm). The present paper revisited these points. Our data showed that CPP direct translocation required an adequate Vm in both HeLa and HEK cells as plasma membrane depolarization drastically reduced the cytosolic uptake of CPPs in these cell lines. The presented evidence indicates that hyperpolarization of the plasma membrane is the main driver of CPP direct translocation in cells and that there is no clear evidence for alternative modes of direct translocation. European Journal of Pharmaceutics and Biopharmaceutics <\/strong>(2023<\/em>)<\/p>\n\n\n\n Marc Serulla, Palapuravan Anees, Ali Hallaj, Evgeniya Trofimenko, Tara Kalia, Yamuna Krishnan, Christian Widmann<\/p>\n\n\n\n eLife <\/strong>(2021<\/em>)<\/p>\n\n\n\n Evgeniya Trofimenko, Gianvito Grasso, Mathieu Heulot, Nadja Chevalier, Marco Deriu, Gilles Dubuis, Yoan Arribat, Marc Serulla, S\u00e9bastien Michel, Gil Vantomme, Florine Ory, Linh Chi Dam, Julien Puyal, Francesca Amati, Anitha L\u00fcthi, Andrea Danani, and Christian Widmann<\/p>\n\n\n\n Cell Reports <\/strong>(2021<\/em>)<\/p>\n\n\n\n Evgeniya Trofimenko, Yuta Homma, Mitsunori Fukuda, and Christian Widmann<\/p>\n\n\n\n This work reports the characterization of an endocytic pathway that had escaped detection so far. In contrast to classical endocytic pathways that rely on the Rab5 and Rab7 proteins to move endocytosed material, such as transferrin, dextran, or EGF, to LAMP1-positive lysosomes, the newly described pathway move cationic substances (CPPs and polyamines for example) to LAMP1-positive compartments in a Rab14-dependent but Rab5\/7-independent manner. The initial process of vesicle formation in the Rab14 pathway also differs from classical endocytosis as it is not inhibited by dynamin inhibition, macropinocytosis inhibitors, or pan-PI3K inhibitors. Even though both classical endocytic pathways and the Rab14-dependent pathway end up in LAMP1-positive vesicles, the LAMP1-containing vesicles of classical pathways are acidic and correspond to lysosomes while the LAMP1-positive compartment in the Rab14 pathway are non- acidic (or less acidic) and presumably fulfil non-degradative functions.<\/p>\n\n\n iScience <\/strong>(2021<\/em>)<\/p>\n\n\n\n Maria Georgieva, Tytti Heinonen, Alessandra Vitale, Simone Hargraves, Senka Causevic, Trestan Pillonel, Leo Eberl, Christian Widmann and Nicolas Jacquier (last two authors share senior authorship)<\/p>\n\n\n\n This study investigated the parameters that influence the binding and activity of TAT-RasGAP317-326<\/sub> on bacteria. This study provides the basis for future investigation on the mode of action of TAT-RasGAP317-326<\/sub>, which may help developing antimicrobial treat- PNAS <\/strong>(2020<\/em>)<\/p>\n\n\n\n Marc Serulla, Gabriel Ichim, Filip Stojceski, Gianvito Grasso, Sergii Afonin, Mathieu Heulot, Tim Schober, Robyn Roth, C\u00e9dric Godefroy, Pierre-Emmanuel Milhiet, Kushal Das, Ana J Garc\u00eda-S\u00e1ez, Andrea Danani, Christian Widmann<\/p>\n\n\n\n TAT-RasGAP317-326<\/sub> is an anti-cancer cell-penetrating peptide that was designed in our laboratory in 2004. For many years, its ability to kill tumor cells had remained a mystery. We have finally elucidated its mode of action through the use of multiple experimental approaches, including cell imaging, cell biology, in vitro<\/em> vesicle biochemistry, and in silico<\/em> modeling. TAT-RasGAP317-326<\/sub> first penetrates cells by direct translocation through the plasma membrane and then targets inner-leaflet enriched phospholipids, such as phosphoserine and phosphatidylinositol-bisphosphate, to destabilize the cell’s membrane and induce its lysis.<\/p>\n\n\n\n Journal of Cell Science <\/strong>(2022<\/em>)<\/p>\n\n\n\n Adi Zheng, Gilles Dubuis, Maria Georgieva, Carla Susana Mendes Ferreira, Marc Serulla, Maria del Carmen Conde Rubio, Evgeniya Trofimenko, Thomas Mercier, Laurent Decosterd, and Christian Widmann<\/p>\n\n\n\n This paper concludes our research on HDLs, the “good cholesterol” that was supported by a Sinergia grant from the Swiss National Science Foundation. We show in this article that HDLs utilize their capacity of loading themselves with lipophilic compounds, akin to their ability to extract cellular cholesterol, to reduce the cell content of hydrophobic drugs. This can be beneficial if lipophilic xenobiotics are toxic but may be detrimental to the therapeutic benefit of lipophilic drugs such as glibenclamide.<\/p>\n\n\n Christian Widmann’s articles in journals with editorial boards<\/strong> (R: review; C: commentary or editorial; others: original articles).<\/p>\n\n\n\n <\/p>\n\n\n\n <\/p>\n\n\n\nNo Evidence for Plasma Membrane Potential-Independent Cell Penetrating Peptide Direct Translocation<\/a><\/h1>\n\n\n\n
Potential elements to explain the discrepency between our data and those of the Brock’s lab are presented in the article.<\/p>\n\n\n\nPlasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides<\/a><\/h1>\n\n\n\n
The capacity of cell-penetrating peptides (CPPs) to escape endosomes is a debated issue (check our movie<\/a> out on youtube). In the present paper, we monitored endosomal escape in conditions where cells were allowed to take up CPPs by endocytosis but not by direct translocation. In these conditions no endosomal escape could be detected even when we used CPPs supposedely designed for enhanced endosomal escape capacities. This indicates that CPPs are unable to escape endosomes in significant means. What was interpreted as endosomal escape in a number of earlier publications was thus most likely corresponding to CPP cytosolic uptake by direct translocation. CPP uptake by endocytosis occurs at lower CPP concentrations than those allowing direct translocation. In a number of conditions, endocytosis might thus be the predominant mode of entry for CPPs. Endocytosis of CPP is however a dead end for them. This is why we are currently trying to identify molecules able to favor CPP endosomal escape. These “escape promoting molecules” would increase the bioavailability and activity of CPPs and consequently their usefulness in biology and medicine.<\/p>\n\n\n\nGenetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore<\/a><\/strong><\/h1>\n\n\n\n
The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR\/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (\u2013150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (\u20135) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.<\/p>\n\n\n![\"\"](\"https:\/\/wp.unil.ch\/widmannlab\/files\/2021\/12\/00-002-1.png\")
The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7<\/a><\/strong><\/h1>\n\n\n\n
![\"\"](\"https:\/\/wp.unil.ch\/widmannlab\/files\/2021\/11\/Capture-1.png\")
Bacterial surface properties influence the activity of the TAT-RasGAP317-326<\/sub> antimicrobial peptide<\/a><\/strong><\/h1>\n\n\n\n
ments based on this peptide<\/p>\n\n\n\nTAT-RasGAP317-326<\/sub> kills cells by targeting inner-leaflet-enriched phospholipids<\/a><\/strong><\/h1>\n\n\n\n
![\"\"](\"https:\/\/wp.unil.ch\/widmannlab\/files\/2021\/02\/Capture-1024x591.png\")
HDLs extract lipophilic drugs from cells<\/a><\/strong><\/h1>\n\n\n\n
![\"\"](\"https:\/\/wp.unil.ch\/widmannlab\/files\/2022\/01\/Capture1.jpg\")
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