Laboratory of cell-permeable peptide biology.
Cell-permeable peptides (CPPs) allow the intracellular delivery of cargo molecules that are hooked to them. CPPs provide an efficient mode of transfer for bioactive molecules inside cells, especially in conditions when transcription or translation of these cargo-encoding sequences is undesirable or hard to achieve. CPP sequences are ubiquitous and are often detected in both anti-cancer and anti-microbial peptides. Despite their potential practical applications, the mechanisms of CPP entry inside cells are ill-defined and even controversial. No genes have been described that regulate the intracellular delivery of CPPs. The current focus of our laboratory is to characterize the mechanism of CPPs cell entry and CPP mode of action using as an example, the anti-cancer and anti-microbial CPP TAT-RasGAP317-326. Our research is relevant to the bioengineering, oncology and microbiology fields.
See the specific research sections for further details on our work.
CPP direct translocation into cells.
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.
How does the TAT-RasGAP317-326 anticancer CPP kills tumor cells?
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-RasGAP317-326, 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-RasGAP317-326. We are now finalizing the molecular characterization by which TAT-RasGAP317-326 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-RasGAP317-326 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-RasGAP317-326 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-RasGAP317-326 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-RasGAP317-326 and determine whether bacteria can develop resistance to this peptide and via what mechanism. Our data so far indicate that resistance to TAT-RasGAP317-326 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.
Christian Widmann, group leader
Christian Widmann (1963) received his MSc in biology in 1986. He obtained his PhD at the Biochemistry Institute of the University of Lausanne in 1991 under the supervision of prof. Giampietro Corradin. He then worked on the GLP1 receptor as a post-doctoral fellow in the laboratory of prof. Bernard Thorens until 1995. Dr. Widmann obtained a fellowship from the Swiss National Science Foundation in 1996 to move to the laboratory of prof. Gary L. Johnson at the National Jewish Medical and Research Center in Denver, Colorado, where he worked as a research associate on MAPK pathways until 1998. In 1999, he moved back to Europe and started his own research group at the University Hospital in Lausanne. In 2002, he got an assistant professor position at the Department of Cell Biology and Morphology at the University of Lausanne. Since 2006, Dr. Widmann is an associate professor at the Department of Physiology of the University of Lausanne. The first wave of his research was centered on the regulation of cellular stress and how a better understanding of these mechanisms at the molecular level could lead to the development of therapeutic compounds to treat various diseases such as cancer, microbial infections, and diabetes. The current focus of his labortory is on the mechanisms of entry of cell permeable peptides, which are promising but poorly understood cell biology and therapeutic tools .
Gilles Dubuis, laboratory technician
|Gilles Dubuis is the lab technician of the Widmann’s group. He joined the lab in 2003. He is involved in managing the laboratory and in many different projects on diabetes and cancer.|
Adi Zheng, postdoctoral fellow
Adi Zheng received her Bachelor’s degree in Bioengineering and PhD degree in Biochemistry and Molecular Biology from Xi’an Jiaotong University in China. She joined Dr. Christian Widmann’s group in January 2018 and now she is working on the topic of drug resistance in cancer.
Daniel Constantin, PhD student
|Daniel obtained his Bachelor degree in Biology and his Master degree in Medical Biology from the University of Bucharest, he then started working at the National Institute for Biological Sciences (INSB) in Bucharest where he worked with primary mesenchymal stem cells derived from bone marrow or adipose tissue and inducing differentiation into various lineages while growing the cells in collagen scaffolds enriched with various combinations of growth factors. From January 2014 to June 2015 he did a pre-doc stage in Dr. Moldovan’s laboratory at Penn State Hershey College of Medicine. There, he studied the impact of PCNA interacting proteins PARI and mono-ADP-ribosyltransferase (PARP14/ARTD8) on homologous recombination and genomic stability. In July 2015 he joined Dr. Widmann’s group where he studies the molecular functions of Src Homology 2 Domain Containing Adaptor Protein B (SHB).|
Evgeniya Trofimenko, PhD student
|Evgeniya obtained her Bachelor in Biology and Master in Molecular Life Science at the University of Lausanne. During the Master’s thesis she worked on adapting Next Generation Sequencing to measure the rates of trinucleotide repeat instability. She joined the Christian Widmann group in March 2016 as a PhD student and is currently working on characterisation of endosomal escape.|
Marc Serulla Llorens, PhD student
Marc Serulla obtained his Bachelor's Degree in Biomedical Sciences (2014) and his Master's Degree in Biomedical Research (2015) at the University of Lleida (Spain). He completed his undergraduate thesis at Abo Akademi (Finland), where he worked on the relationship between ASPP2 and Ras nanoclusters. He performed his master's thesis at the Arctic University of Norway (Norway) in a project dealing on neuroglial regulation of the hematopoietic stem cell niche in the transformation of acute myeloid leukemia. In January 2017, he joined Cristian Widmann’s group, where he focuses his work on the mechanisms of regulation and endosomal trafficking of the anticancer peptide TAT-RasGap317-326.
María del Carmen Conde Rubio, PhD student
|María del Carmen Conde Rubio obtained her Bachelor's Degree in Pharmacy in 2011 from the University Complutense of Madrid, she completed her Bachelor's Degree in Biotechnology from the University Francisco de Vitoria (Pozuelo de Alarcón, Spain ) in 2013 with a specialization in Clinical Biotechnology and her Master’s Degree in Biochemistry, Molecular Biology and Biomedicine from the University Complutense of Madrid in 2014. Currently, she is a PhD student and her research is focused on the characterization of cleaved proteins in stressed cells.|
Maria Georgieva, postdoctoral fellow
Maria Georgieva, PhD, joined the Widmann lab in August 2018. She received her PhD from Emory University in USA where she studied modulation of host immune responses by Mycobacterium tuberculosis. Prior to joining the Widmann Lab, Maria was a Research Fellow at Harvard University with Dr. Marc Lipsitch where she worked on immune escape by antibody antigens of Streptococcus pneumoniae. Now, in the Widmann Lab, Maria is working on antimicrobial peptides and elucidating their mechanism of action.
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