Laboratory Christian Widmann

We have identified potassium channels as key regulator of CPP translocation in cells. Using a CRISPR/Cas9-based screening, we discovered that various potassium channels positively modulate the direct cellular translocation of TAT-bound cargos by reducing the transmembrane potential of cells. Molecular dynamics simulations performed by our collaborators Gianvito Grasso and Andrea Danani at the Dalle Molle Institute for Artificial Intelligence in Ticino indicate that hyperpolarization favored the generation of 1.5 nm-wide water pores used by CPPs to enter cells. Hyperpolarization is also required for optimal cellular uptake of CPPs other than TAT, including poly-arginine peptides, model amphipatic peptide, and penetratin. Pharmacological manipulation to lower the transmembrane potential can boost the cellular uptake of CPPs in zebrafish and mouse models. This work identifies for the first time genes that regulate CPP translocation and provides the scientific ground to pharmacological strategies augmenting the susceptibility of cells to capture CPP-linked cargos in vitro and in vivo.

CPPs can enter cells through direct translocation as described in the previous section but they can also be taken up by cells via endocytosis. Our data indicate that CPP endocytosis differ markedly from most (all?) other forms of endocytosis by not relying on the Rab5 small GTP binding protein. The goal of the present proposal is to characterize this new form of endocytosis at the structural and mechanistic levels and assess whether cargos other than CPPs, pathogens in particular, use it for their entry into cells. A grant to the Swiss National Research Foundation has been submitted to support this research. This project is expected to provide important advances on several fronts. At the fundamental cell biology level, it will characterize a newly discovered endocytic pathway. At a preclinical level, it will give researchers working on the improvement of the bio-therapeutic delivering capacities of CPPs the mechanistic grounds to manipulate their entry into cells. Finally, this project will investigate whether pathogens use a Rab5-independent pathway to enter host cells, information that could identify novel targets for the development of anti-viral or anti-bacterial drugs.

Current anti-cancer treatments are often based on the combination of different drugs that induce tumor cell apoptosis. Consequently, mutations in one or a few key core components of apoptosis can be enough for tumor cells to become resistant to treatment. We have discovered in our laboratory that a cell-permeable peptide derived from the RasGAP protein, called TAT-RasGAP_317-326, can induce a non-programmed form of death in cancer cells. Indeed, the RasGAP-peptide is unhindered in its capacity to kill tumor cells when caspases, and hence apoptosis, are blocked. Other forms of death, such as necroptosis or autophagic cell death, can also be inhibited without compromising the tumor-specific killing activity of TAT-RasGAP_317-326. We are now finalizing the molecular characterization by which TAT-RasGAP_317-326 kills cells. Our data indicate that, after it enters cells by direct translocation, the peptide targets phospholipids that are enriched in the inner layer of the plasma membrane. Once bound to the inner layer, TAT-RasGAP_317-326 permeabilizes the cell membrane in a necrotic-like manner. It might be difficult for cancer cells to develop resistance to this type of non-programmed cell death and hence the type of lethal activity harbored by TAT-RasGAP_317-326 might represent a distinct avenue for the development of new anti-cancer therapies.

The emergence of antibiotic resistant bacterial strains calls for the development of new antibiotics and anti-microbial drugs. We have discovered that the anti-cancer TAT-RasGAP_317-326 CPP also possesses anti-microbial activity and efficiently kills a broad spectrum of bacteria species. In collaboration with Dr. Nicolas Jacquier from the Institute of Microbiology of the Lausanne University Hospital, we are currently trying to decipher the anti-microbial mode of action of TAT-RasGAP_317-326 and determine whether bacteria can develop resistance to this peptide and via what mechanism. Our data so far indicate that resistance to TAT-RasGAP_317-326 is mediated via the EnvZ/OmpR two-component system involved in osmoregulation in Gram-negative bacteria. These types of studies have the potential to facilitate the development of new strategies to fight microbial infections.