Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A 1 -binding sequence A. Galioot a, 1 , A.N. Godet a, 1, 2 , V. Maire a, 1, 3 , P.B. Falanga a , X. Cayla b , B. Baron c , P. England c , A. Garcia a, ⁎ a Laboratoire E3 Phosphatases, Unité Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, 25, rue du Dr Roux, 75015 Paris, France b Neurobiologie intégrative de la reproduction UMR 6175 INRA-CNRS-université Tours-Haras Nationaux Centre INRA de Tours, PRC F-37380 Nouzilly, France c Plateforme de Biophysique, Institut Pasteur, 25, rue du Dr Roux, 75015 Paris, France abstract article info Article history: Received 28 September 2012 Received in revised form 21 February 2013 Accepted 27 February 2013 Available online 14 March 2013 Keywords: PP2A E4orf4 DPT-peptides Cell-death Cancer Background: Induction of the death pathway resulting from the specific interaction of the PP2A 1 phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides. Methods: Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences. Results: We identified overlapping sequences, located within the N-terminal domain E4orf4 23-46 of canine ade- noviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A 1 holoenzyme. We characterized DPT-E4orf4 4 and TAT-E4orf4 4 , two bi-partite cell penetrating peptides containing the 12 PP2A 1 binding residues of the canine type 2 E4orf4 27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surpris- ingly DPT-E4orf4 4 , in contrast to inactive TAT-E4orf4 4 , adopted a well defined α-helical structure and co-precipitated PP2A 1 from HeLa cell extracts. DPT-E4orf4 4 also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4 4 or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4 4 also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts. Conclusions: We characterized the transducing properties of a new α-helical DPT-E4orf4 4 peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein. General significance: Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Protein phosphatase 2A (PP2A) is a large family of more than 95 highly conserved and ubiquitously expressed holoenzymes that comprises dimeric or heterotrimeric enzymes and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells [1]. The dimeric PP2A core enzyme represents approximately one-third of PP2A holoenzymes and consists of a catalytic C subunit (PP2Ac) and a structural A subunit. In mammals, two distinct genes encode closely related versions of both the PP2A A (Aα/PPP2R1A and Aβ/PPP2R1B) and C (Cα/PPP2CA and Cβ/PPP2CB) subunits giving rise to similar but functionally nonredundant α and β isoforms. The binding of a third variable B subunit to the core generates trimeric holoenzymes and regulates both their substrate specificity and their cellular localiza- tion. Interestingly, to generate the diversity of trimeric holoenzymes, 15 different human genes, that encode four unrelated families of B sub- units have been already identified today: B/B55/PPP2R2A, B′/B56/PR61/ PPP2R5, B″/PR72/PPP2R3, and Striatin/STRN [1]. The specific PP2A 1 ho- loenzyme is a trimeric protein composed of A, Bα and C subunits that represents a specific target for a variety of viral proteins originated from different viruses [2]. In addition, PP2A 1 plays a critical role in numerous cellular pro- cesses including cell growth, apoptosis and neurodegeneration, and thus represents an interesting therapeutic target [2–4]. In this regard, to specifically deregulate intra-cellular disease pathways controlled by PP2A holoenzymes, we previously published an approach, named Biochimica et Biophysica Acta 1830 (2013) 3578–3583 ⁎ Corresponding author. Tel.: +33 1 40 61 38 21; fax: +33 1 40 61 39 38. E-mail address: agarcia@pasteur.fr (A. Garcia). 1 Equal contribution. 2 Present address: Faculté de médecine Pierre et Marie Curie, Paris VI CHU Pitié- Salpêtrière porte 514, 105, Bd de l'Hôpital Paris, France. 3 Present address: Laboratoire de Signalisation Institut Curie, Département Transfert, Hopital Saint-Louis, Quadrilatère historique, Porte 131 Ave Claude Vellefaux 75475 Paris cedex 10, France. 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.02.024 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen