Use of Penetrating Peptides Interacting with PP1/PP2A Proteins As a General Approach for a Drug Phosphatase Technology

Abstract

Protein phosphatase types 1 (PP1) and 2A (PP2A) represent two major families of serine/threonine protein phosphatases that have been implicated in the regulation of many cellular processes, including cell growth and apoptosis in mammalian cells. PP1 and PP2A proteins are composed of oligomeric complexes comprising a catalytic structure (PP1c or PP2AC) containing the enzymatic activity and at least one more interacting subunit. The binding of different subunits to a catalytic structure generates a broad variety of holoenzymes. We showed here that casein kinase 2␣ (Ck2␣) and simian virus 40 small t antigen share a putative common ␤-strand structure required for PP2A1 trimeric holoenzyme binding. We have also characterized DPT-sh1, a short basic peptide from Ck2␣ that interacted only in vitro with the PP2A-A subunit and behaves as a nontoxic penetrating shuttle in several cultivated human cell lines and chick embryos. In addition, DPT-sh1 specifically accumulated in human red cells infected with Plasmodium falciparum malaria parasites. We therefore designed bipartite peptides containing DPT-sh1 and PP1-or PP2A-interacting sequences. We found that DPT-5, a DPT-sh1-derived peptide containing a short sequence identified in CD28 antigen, interacts with PP2A-B␣, and DPT-7, another DPT-sh1-derived peptide containing a short sequence identified in Bad as a PP1 catalytic consensus docking motif, induce apoptosis in cultivated cell lines. These results clearly indicate that the rational design of PP1/PP2A interacting peptides is a pertinent strategy to deregulate intracellular survival pathways.In eukaryotic cells, biological activity of nearly 30% of proteins is modulated by phosphorylation. Reversible protein phosphorylation regulates multiple cellular processes, including metabolism, signal transduction pathways, cell cycle progression, oncogenic transformation, cell differentiation, and apoptosis (Garcia et al., , 2003. Protein phosphorylation regulates the activities of protein kinases and protein phosphatases themselves (Hunter, 2000). Type 1 (PP1) and type 2A (PP2A) Ser/Thr protein phosphatases are major regulators of cell dephosphorylation. Binding of PP1 catalytic subunit (PP1c) to specific regulatory subunits generates a large family of PP1 holoenzymes (Cohen, 2002). For PP2AThis work was supported in part by the Institut-Pasteur (PTR-136 and DVPI 27147-27188) and by the Association pour la Recherche sur le Cancer grant 4437 (to A.G.) and grant 4812 (to S.A.S.). F.D. was supported by a fellowship grant from Institut-Pasteur (DVPI). J.G. is a recipient of a Ministere de la Recherche et Technologie fellowship from the French Government. V.J.Y. was supported by a Marie Curie IntraEuropean fellowship within the 6th European Community Framework Programme (contract MEIF-2003-501887

La famille des protéine phosphatases PP2A

“…Ce résultat illustre bien un des prérequis de ces approches : les agents pharmacologiques doivent avoir une très haute affinité pour les sous-unités de PP2A qui sont ciblées, de façon à pouvoir contrecarrer efficacement la liaison à PP2A d'oncogènes cellulaires ou viraux. Dans ce contexte, nous avons proposé une approche thérapeu-tique appelée drug phosphatase technology, ou DPT, basée sur l'inhibition de processus pathologiques par transfert intracellulaire de peptides pénétrants mimant des sites d'interaction avec des protéines de la famille des protéine phosphatases PP1/PP2A (Figure 4) [28]. En particulier, nous avons caractérisé DPT-5, un peptide pénétrant contenant, à l'instar d'E4orF4, une séquence d'interaction (dans ce cas issue de l'antigène CD28) avec la sous-unité B de PP2A 1 , qui induit l'apoptose des cellules humaines [28].…”

“…Dans ce contexte, nous avons proposé une approche thérapeu-tique appelée drug phosphatase technology, ou DPT, basée sur l'inhibition de processus pathologiques par transfert intracellulaire de peptides pénétrants mimant des sites d'interaction avec des protéines de la famille des protéine phosphatases PP1/PP2A (Figure 4) [28]. En particulier, nous avons caractérisé DPT-5, un peptide pénétrant contenant, à l'instar d'E4orF4, une séquence d'interaction (dans ce cas issue de l'antigène CD28) avec la sous-unité B de PP2A 1 , qui induit l'apoptose des cellules humaines [28]. Plus récemment, nous avons montré qu'un peptide DPT contenant un site de liaison de la protéine E4orf4 de chien qui interagit avec la sous-unité structurale B de PP2A 1 provoque l'apoptose des cellules tumorales humaines sans affecter les cellules saines (Galioot et al, en préparation).…”

“…Ce peptide appelé DPTsh1 est une navette non toxique qui permet d'introduire dans les cellules en culture des séquences apoptotiques impliquées dans l'interaction avec PP1/ PP2A. En particulier, une séquence d'interaction de la protéine apoptotique Bad avec PP1c, ou de l'antigène CD28 avec PP2A 1 , provoquent l'apoptose des cellules humaines [28]. Plus récemment, nous avons caractérisé DPT-AIF 1 , une nouvelle séquence pénétrante, issue du domaine carboxy-terminal de la protéine apoptotique AIF (apoptosis inducing factor) qui se lie à la protéine PP1c pour induire l'apoptose.…”

Targeting Protein Serine/Threonine Phosphatases for Drug Development

“…1, A-C). A recent study exploited cell-permeable peptides that mimic the structure of "phosphatase-interacting motifs" in specific subcellular targets of PP1 and PP2A; these peptides bind the phosphatase in the same manner as the substrate (Guergnon et al, 2006). The determination of phosphatase binding sites within a PP1 interacting protein (Bad) and a PP2A interacting protein (CD28) allowed for the construction of short corresponding peptides fused with a basic stretch of amino acids to confer cell permeability.…”

Cell-Penetrating Peptide TP10 Shows Broad-Spectrum Activity against both Plasmodium falciparum and Trypanosoma brucei brucei

“…While the main route of TP10 uptake is believed to be via the endocytic pathway (25), it has recently been suggested that perturbation of the lipid bilayer is required to maximize TP10 uptake (35). However, uptake does not necessarily translate into toxicity, as two newly designed CPPs are reported to accumulate in P. falciparum-infected red blood cells (RBCs) without killing the parasite but never enter noninfected RBCs (10). This may be related to the changes in erythrocyte membrane composition due to the formation of new permeability pathways (19).…”