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 (TG), a sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) inhibitor, by extracting the drug from cells. Drug efflux could also be triggered to some extent by low-density lipoproteins and serum. HDLs did not reverse the non-lethal mild ER stress response induced by low TG concentrations or by SERCA knockdown, but HDLs inhibited the toxic SERCA-independent effects mediated by high TG concentrations. HDLs could extract other lipophilic compounds, but not hydrophilic substances. 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. This can be beneficial if lipophilic xenobiotics are toxic but may be detrimental to the therapeutic benefit of lipophilic drugs such as glibenclamide.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

Plasma membrane depolarization reveals endosomal escape incapacity of cell-penetrating peptides.

Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver diverse cargos into cells. CPPs access the cytosol either by direct translocation through the plasma membrane or via endocytosis followed by endosomal escape. Both direct translocation and endosomal escape can occur simultaneously, making it non-trivial to specifically study endosomal escape alone. Here we depolarize the plasma membrane and showed that it inhibits the direct translocation of several CPPs but does not affect their uptake into endosomes. Despite good endocytic uptake many CPPs previously considered to access the cytosol via endosomal escape, failed to access the cytosol once direct translocation was abrogated. Even CPPs designed for enhanced endosomal escape actually showed negligible endosomal escape into the cytosol. Our data reveal that cytosolic localization of CPPs occurs mainly by direct translocation across the plasma membrane. Cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs.

Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. 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 dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (-5) 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.

Before a drug can have its desired effect, it must reach its target tissue or organ, and enter its cells. This is not easy because cells are surrounded by the plasma membrane, a fat-based barrier that separates the cell from its external environment. The plasma membrane contains proteins that act as channels, shuttling specific molecules in and out of the cell, and it also holds charge, with its inside surface being more negatively charged than its outside surface. Cell-penetrating peptides are short sequences of amino acids (the building blocks that form proteins) that carry positive charges. These positive charges allow them to cross the membrane easily, but it is not well understood how. To find out how cell-penetrating peptides cross the membrane, Trofimenko et al. attached them to dyes of different sizes. This revealed that the cell-penetrating peptides enter the cell through temporary holes called water pores, which measure about two nanometres across. The water pores form when the membrane becomes 'megapolarized', this is, when the difference in charge between the inside and the outside of the membrane becomes greater than normal. This can happen when the negative charge on the inside surface or the positive charge on the outer surface of the membrane increase. Megapolarization depends on potassium channels, which transport positive potassium ions outside the cell, making the outside of the membrane positive. When cell-penetrating peptides arrive at the outer surface of the cell near potassium channels, they make it even more positive. This increases the charge difference between the inside and the outside of the cell, allowing water pores to form. Once the peptides pass through the pores, the charge difference between the inside and the outside of the cell membrane dissipates, and the pores collapse. Drug developers are experimenting with attaching cell-penetrating peptides to drugs to help them get inside their target cells. Currently there are several experimental medications of this kind in clinical trials. Understanding how these peptides gain entry, and what size of molecule they could carry with them, provides solid ground for further drug development.

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold.

Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2's interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2's ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events.