The cell-killing mechanism of TAT-RasGAP317-326 and the endosomal escape capacity of cell-penetrating peptides

Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver cargos, that normally would not cross the plasma membrane, into cells and, thus, they are very interesting in research and clinics. 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 leading to confusion and debate in which entry route CPPs take. We propose a methodological approach based in plasma membrane depolarization that blocks CPP direct translocation, but allows CPP uptake into endosomes in order to freely evaluate endosomal escape. 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. Therefore, cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs. In addition to that, we have characterized the cell death mechanism of an anticancer and antimicrobial CPP bearing peptide, called TAT-RasGAP317–326. Here we found that TAT-RasGAP317–326, after crossing the plasma membrane via direct translocation, binds lipids present at the inner-leaflet of the plasma membrane (PIP2 and PS) and from that position disrupts the cell membrane leading to a necrotic cell death. Moreover, we described that, 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. To sum up, TAT- RasGAP317-326 kills cancer cells in a manner that does not involve known genetically programmed cell death pathways. This is extremely useful for cancer therapy, since strategies that trigger non-genetically encoded forms of cell death would provide additional therapeutic options to fight cancer in a chemo- and radio- therapy relapse scenario. -- Les peptides pénétrant les cellules (CPP pour «cell-penetrating peptide» en anglais) sont de courts peptides (<30 acides aminés), généralement cationiques. Ils peuvent être couplés à des molécules ou substances, qui normalement ne traverseraient pas la membrane plasmique, et les transporter à l’intérieur de la cellule. Cette propriété rend les CPP très intéressants en recherche et en clinique. Les CPP accèdent au cytosol de la cellule soit par translocation directe à travers la membrane plasmique, soit par endocytose suivie d’un échappement endosomal («endosomal escape» en anglais). La translocation directe et l’échappement endosomal peuvent se produire simultanément, ce qui complique l’étude de l’accès cytosolique des CPP et alimente le débat sur les voies d'entrée empruntées par les CPP. Dans cette thèse, j’ai utilisé une approche méthodologique basée sur la dépolarisation de la membrane plasmique qui bloque la translocation directe des CPP, mais pas leur endocytose, afin d’étudier spécifiquement le processus de l’échappement endosomal. Mes résultats montrent que, malgré une bonne absorption endocytaire, les CPP que j’ai testé, inclus des CPP conçus pour sortir efficacement des endosomes, ne se sont pas retrouvés dans le cytosol une fois la translocation directe abrogée. Mes données révèlent donc que la présence de CPP dans le cytosol est le résultat de la translocation directe à travers la membrane plasmique. La dépolarisation cellulaire représente une manipulation simple pour tester rigoureusement la capacité d'échappement endosomal des CPP. J’ai également caractérisé la manière dont TAT-RasGAP317-326, un CPP ayant des propriétés anticancéreuses et antimicrobiennes, est capable d’induire la mort de cellules cancéreuses. Les données que j’ai obtenues montrent que TAT-RasGAP317- 326, après avoir traversé la membrane plasmique via une translocation directe, se lie aux lipides présents au niveau du feuillet interne de la membrane plasmique (PIP2 et PS). Cette liaison compromet l’intégrité de la membrane cellulaire, entrainant une mort cellulaire nécrotique. J’ai également montré que le mutant W317A de TAT-RasGAP317- 326, qui n’est pas capable de tuer les cellules cancéreuses, a des capacités réduites à se lier et à perméabiliser les membranes contenant PIP2 et PS. Ce mutant a également une capacité limitée à se déplacer à travers les membranes cellulaires, probablement en raison d'une plus grande propension à adopter un état α-hélicoïdal. En résumé, TAT-RasGAP317-326 tue les cellules cancéreuses d'une manière qui n'implique pas les voies de mort cellulaire génétiquement programmées connues. Ceci est extrêmement utile pour le traitement du cancer, car les stratégies qui déclenchent des formes de mort cellulaire non codées génétiquement fourniraient des options thérapeutiques supplémentaires pour lutter contre le cancer dans un scénario de rechute suivant des chimiothérapies ou la radiothérapie

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

ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

<div><p>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.</p></div

N- and C-terminal truncation mutants of ASPP2 can still promote Ras nanoclustering.

<p>(<b>A</b>) Schematic of full-length ASPP2, as well as ASPP2(1–360) and ASPP2(123–1128) truncation mutants. ASPP2 domains from left to right: Ubl, ubiquitin-like domain; α-helical domain; Pro, proline-rich domain; Ank, Ankyrin repeats; SH3, SRC homology 3 domain. (<b>B</b>) Confocal microscopic images of HEK cells cotransfected with mGFP-H-rasG12V (green) and full-length or truncated ASPP2 (red). (<b>C-E</b>) Nanoclustering-FRET analysis of HEK cells coexpressing mGFP- and mCherry-tagged (<b>C</b>) H-rasG12V, (<b>D</b>) K-rasG12V or (<b>E</b>) N-rasG12V. Cells were analysed after overexpression of Gal-1, full-length ASPP2 or its truncation mutants. (<b>C-E</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM, n = 3; ns, not significant; ****, p< 0.0001). (<b>F</b>) Western blot of anti-GFP immunoprecipitation samples probed with anti-ASPP2- (top) or anti-GFP- (bottom) antibodies. Samples were lysates prepared from mGFP-H-rasG12V transfected HEK cells that were cotransfected with full-length ASPP2 or its truncation mutants or an empty plasmid (control), as indicated. In, input; Ft, flow-through; W1, wash; E, elution. Red boxes indicate the immunoprecipitated ASPP2 fragments.</p

ASPP2 dominates over Gal-1 thus robustly inducing senescence and abrogating mammosphere formation.

<p>(<b>A</b>) SA-β-gal assay of MCF-7 cells transfected with plasmids encoding H-rasG12V, ASPP2, Gal-1 or the combination of the latter two, as indicated. Cells were stained 7 days after transfection. On the left, percentages of SA-β-gal positive cells are shown in the graph (mean ± SEM, n = 3). On the right, representative images from the assay. (<b>B</b>) Mammosphere formation assay with MCF-7, MDA-MB-231 or HS-578T breast cancer cell lines. Mammospheres were transfected with Gal-1, ASPP2, or both (1:1 ratio) and cells were then grown under non-adherent conditions for 9 days. On the right, representative images of mammospheres are shown as indicated. (<b>A, B</b>) Statistical significance of differences between controls and treated samples was examined using one-way ANOVA (mean ± SEM n≥3; ns, not significant; ****, p<0.0001).</p