Biology subjects

Widmann, C.

Publications and source records attributed to Widmann, C..

8 recordsLinked to original sources

bioRxiv2026

The cell cycle regulates cell-penetrating peptide cytosolic uptake via differential expression of KCNH1 potassium channel

Membrane potential fluctuations, driven by ion channel activity, are important regulators of processes such as action potential propagation in neurons and muscle cell contraction. These variations also occur in non-excitable cells, influencing various cellular processes, including, but not limited to, direct translocation of cell-penetrating peptides (CPPs) into the cytosol. CPPs are cationic peptide vectors that facilitate the delivery of cargos into cells. The efficiency of CPP direct translocation varies significantly within the same cell population, a phenomenon that is not understood mechanistically. In this study, we used an unbiased transcriptomic approach to identify genes involved in efficient CPP cytosolic uptake. This revealed a close correlation between the expression of cell cycle-related genes and the ability of cells to take up CPPs by direct translocation. Monitoring phases of the cell cycle with the PIP-FUCCI system showed that CPP direct translocation was most efficient in the G2/M and S phases and least efficient during the G1 phase. This was correlated with the cells plasma membrane potential: cells in G2/M and S phases being more polarized than cells in the G1 phase. The expression of the KCNH1 voltage-gated potassium channel was found to be highest in the phases of the cell cycle most permissive for CPP direct translocation. Pharmacological inhibition of KCNH potassium channels efficiently blocked CPP direct translocation in these cell cycle phases. These data indicate that KCNH1 controls the cell membrane potential in a cell cycle-dependent manner and consequently the ability of cells to take up CPPs by direct translocation. Our findings highlight the importance of the cell cycle in regulating CPP uptake. Variations in the plasma membrane potential along the cell cycle should be considered in the development of CPP-based therapeutics and their applications.

cell biology↗

bioRxiv2022

A database of accurate electrophoretic migration patterns for human proteins in cell lines.

Native molecular weight (MW) is one of the defining features of proteins. Denaturing gel electrophoresis (SDS-PAGE) is a very popular technique for separating proteins and determining their MW. Coupled with antibody-based detection, SDS-PAGE is widely applied for protein identification and quantitation. Yet, electrophoresis is poorly reproducible and the MWs obtained are often inaccurate. This hampers antibody validation and negatively impacts the reliability of western blot data, resulting worldwide in a considerable waste of reagents and labour. To alleviate these problems there is a need to establish a database of reference MWs measured by SDS-PAGE. Using mass spectrometry as an orthogonal detection method, we acquired electrophoretic migration patterns for approximately 10000 human proteins in five commonly used cell lines. We applied a robust internal calibration of migration to determine accurate and reproducible molecular weights. This in turn allows merging replicates to increase accuracy, but also enables comparing different cell lines. Mining of the data obtained highlights structural factors that affect migration of distinct classes of proteins. We also show that the information produced recapitulates known post-translational modifications and differential splicing and can be used to formulate hypotheses on new or poorly known processing events. The full information is freely accessible as a web resource through a user friendly graphical interface (https://pumba.dcsr.unil.ch/). We anticipate that this database will be useful to investigators worldwide for troubleshooting western blot experiments, but could also contribute to the characterization of human proteoforms.

biochemistry↗

bioRxiv2021

Identification of a new, Rab14-dependent, endo-lysosomal pathway

Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

cell biology↗

bioRxiv2020

HDLs extract lipophilic drugs from cells

High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The underlying mechanisms are however still poorly understood. Here we present evidence that HDLs efficiently protect cells against thapsigargin (a SERCA inhibitor) by extracting the drug from cells. Drug efflux could also be triggered to some extent by low-density lipoproteins (LDLs) and serum, which contains lipoproteins. HDLs did not reverse the non-lethal mild endoplasmic reticulum (ER) stress response induced by low thapsigargin concentrations or by SERCA knock-down but HDLs inhibited the toxic SERCA-independent effects mediated by high thapsigargin concentrations. HDLs were also found to extract other lipophilic compounds, such as the anti-diabetic drug glibenclamide. In contrast, hydrophilic substances (doxorubicin hydrochloride, rhodamine 123) were not extracted from cells by HDLs. This work shows 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. Silencing of the P-glycoprotein/ABCB1 transporter reduced the capacity of cells to load thapsigargin on HDLs. This work suggests that HDL-mediated cell efflux of toxic lipophilic xenobiotic is beneficial but also that HDL-mediated efflux can be detrimental to the therapeutic benefit of lipophilic drugs such as glibenclamide. Lipoprotein-mediated drug efflux should therefore be considered when evaluating drug efficacy.

cell biology↗

bioRxiv2020

Characterization of the antimicrobial activity of the cell-penetrating peptide TAT-RasGAP317-326

Antibiotic resistance is an increasing threat for public health, underscoring the need for new antibacterial agents. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics. TAT-RasGAP317-326 is a recently described AMP effective against a broad range of bacteria, but little is known about the conditions that may influence its activity. Using RNA-sequencing and screening of mutant libraries, we show that Escherichia coli and Pseudomonas aeruginosa respond to TAT-RasGAP317-326 by regulating metabolic and stress response pathways, possibly implicating two-component systems. Our results also indicate that bacterial surface properties, in particular integrity of the lipopolysaccharide layer, influence peptide binding and entry. Finally, we found differences between bacterial species with respect to their rate of resistance emergence against this peptide. Our findings provide the basis for future investigation on the mode of action of this peptide and its potential clinical use as an antibacterial agent.

microbiology↗

bioRxiv2020

The antimicrobial peptide TAT-RasGAP317-326 inhibits the formation and the expansion of bacterial biofilms in vitro

Biofilms are structured aggregates of bacteria embedded in a self-produced matrix. Pathogenic bacteria can form biofilms on surfaces and in tissues leading to nosocomial and chronic infections. While antibiotics are largely inefficient in limiting biofilm formation and expansion, antimicrobial peptides (AMPs) are emerging as alternative anti-biofilm treatments. In this study, we explore the effect of the newly described AMP TAT-RasGAP317-326 on Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus biofilms. We observe that TAT-RasGAP317-326 inhibits the formation of biofilms at concentrations equivalent or two times superior to the minimal inhibitory concentration (MIC) of the corresponding planktonic bacteria. Moreover, TAT-RasGAP317-326 limits the expansion of A. baumannii and P. aeruginosa established biofilms at concentrations 2-4 times superior to the MIC. These results further confirm the potential of AMPs against biofilms, expand the antimicrobial potential of TAT-RasGAP317-326 and support further development of this peptide as an alternative antimicrobial treatment.

microbiology↗

bioRxiv2020

ASH2L drives proliferation and sensitivity to bleomycin and other genotoxins in Hodgkin's lymphoma and testicular cancer cells

It is of clinical importance to identify biomarkers predicting the efficacy of DNA damaging drugs (genotoxins) so that non-responders are not unduly exposed to the deleterious effects of otherwise inefficient drugs. Using a whole genome CRISPR/Cas9 gene knockout approach we have identified that low levels of ASH2L cause resistance to genotoxins. ASH2L is a core component of the H3K4 methyl transferase complex. We show that ASH2L absence decreases cell proliferation and favors DNA repair upon genotoxin exposure. The cell models we have used are derived from cancers currently treated either partially (Hodgkins lymphoma), or entirely (testicular cancer) with genotoxins. For such cancers, ASH2L levels could be used as a biomarker to predict the response to genotoxins. Our data also indicate that patients with low ASH2L expressing tumors do not develop resistance to ATR inhibitors. In these patients, such inhibitors may represent an alternative treatment to DNA damaging drugs.

cancer biology↗

bioRxiv2020

Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. 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 (-150 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 sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs 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 membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

cell biology↗