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

Abstract

30Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs 31 provide efficient methodology to transfer bioactive molecules in cells, in particular in 32 conditions when transcription or translation of cargo-encoding sequences is not 33 desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined 34 and controversial. This work identifies potassium channels as key regulators of cationic 35 CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that 36 KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by 37 reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to 38 megapolarization values (about -150 mV) leading to the formation of ~2 nm-wide water 39 pores used by CPPs to access the cell's cytoplasm. Pharmacological manipulation to 40 lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse 41 models. Besides identifying the first genes that regulate CPP translocation, this work 42 characterizes key mechanistic steps used by CPPs to cross cellular membrane. This 43 opens the ground for pharmacological strategies augmenting the susceptibility of cells 44 to capture CPP-linked cargos in vitro and in vivo. 45 46Cell-penetrating peptides (CPPs) are non-toxic molecules of 5-30 amino acids that can 47 translocate into living cells. CPPs can be hooked to a variety of cargos (siRNAs, DNA, 48 polypeptides, liposomes, nanoparticles, etc.) to allow their transport into cells for 49 therapeutic or experimental purposes (1-10). The origin of CPPs is diverse. For 50 example, TAT48-57 is a 10 amino-acid fragment derived from the trans-activator of 51 transcription (TAT) HIV-1 protein (11, 12), penetratin is a 16 amino-acid peptide 52 derived from the Antennapedia Drosophila melanogaster protein (13), and MAP (model 53 amphipatic peptide) is a synthetic alanine/leucine/lysine-rich peptide (14). The vast 54 majority of CPPs are cationic (1, 3, 6, 7). 55How CPPs enter cells is debated and not fully characterized at the molecular level 56 (reviewed in (1-8)). Due to this knowledge gap, it is difficult to ameliorate CPP cellular 57 entry and this slows down development of CPP-based interventions. Two main modes 58 of CPP entry have been described: endocytosis and direct translocation(1-8). 59Endocytosed CPPs gain cytosolic access by escaping endosomes. Direct 60 translocation has been proposed to occur through transient pore formation or 61 membrane destabilization. Endocytosis and direct translocation are not mutually 62 exclusive. Several entry routes can be followed simultaneously by a given CPP in a 63 given cell line (9, 10). 64Here, we used CRISPR/Cas9-screenings to identify proteins required for the cellular 65 uptake of CPPs. This approach identified three potassium channels as mandatory for 66 the direct translocation of CPPs into various cell types. Further, we highlighted the 67 requirement of an appropriate membrane potential to generate a 2 nm-wide water 68 pores through which...

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

“…However, there is no consensus and clarity regarding the precise nature of the endosomal pathway used by CPPs and its underlying mechanisms. CPPs additionally enter cells through direct translocation via water pores that are formed as a consequence of membrane megapolarization induced by the CPP themselves and the activity of potassium channels 32 . Direct translocation can be inhibited through plasma membrane depolarization or invalidation of specific potassium channels (e.g.…”

“…Direct translocation can be inhibited through plasma membrane depolarization or invalidation of specific potassium channels (e.g. KCNN4 in HeLa cells), without affecting endocytosis of CPPs 32 , transferrin 33 or vesicular stomatitis virus (VSV) 33 . Here, we took advantage of KCNN4 knockout HeLa cells to study specifically endocytosis in the absence of possible confounding effects mediated by CPP direct translocation.…”

“…To investigate the endocytic pathway employed by CPPs, we phenotypically characterized CPP containing vesicles (Figure S1A-B) for the presence of early (Rab5 and EEA1) and late (Rab7 and Lamp1) endosomal markers. We selected five most commonly used CPPs in research and in clinic (TAT, R9, Penetratin, MAP and Transportan) as well as TAT-RasGAP317-326, a prototypical TAT-cargo complex 32, 34–44 . Pulse-chase experiments (Figure 1A-B and S1B) demonstrated colocalization of transferrin, EGF and dextran with EEA1, Rab5A and Rab5B at early time points, and Rab7 and Lamp1 at later time points.…”

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

“…TAT-RasGAP 317-326 and compounds with similar anticancer activities can potentially complement existing anticancer therapies based on the use of genotoxins or radiation that induce apoptosis. membrane in a membrane-potential-dependent manner (13). Direct translocation of TAT-RasGAP 317-326 and other cationic cell-penetrating peptides can be prevented by membranedepolarizing agents such as gramicidin or extracellular highpotassium concentrations (13,14).…”

“…membrane in a membrane-potential-dependent manner (13). Direct translocation of TAT-RasGAP 317-326 and other cationic cell-penetrating peptides can be prevented by membranedepolarizing agents such as gramicidin or extracellular highpotassium concentrations (13,14). Depolarizing cells with 100 mM extracellular KCl does not affect, or only minimally affects, at higher peptide concentrations, the initial cell-surface binding of TAT-RasGAP 317-326 (SI Appendix, Fig.…”