Membrane interaction and cellular internalization of penetratin peptides

Abstract

Penetratin is a 16-residue peptide ] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the a-helical structure of the peptides. The R53A/K57A and R52A/ K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.Keywords: penetratin; Antennapedia homeodomain; lipid vesicles; membrane; cellular internalization.Homeoproteins are a class of well-conserved transcription factors, involved in multiple morphological processes [1,2]. The 60-residue DNA-binding homeodomain of the Drosophila Antennapedia homeoprotein was shown to internalize into cells via a receptor-independent mechanism [3][4][5]. This homeodomain consists of three helices and a loop separating helix 2 and 3 [2]. Mutagenesis experiments demonstrated that the third helix drives the homeodomain internalization [6]. The short 16-residue penetratin peptide, corresponding to residues RQIKIWFQNRRMK WKK(43-58) of helix 3, is able to translocate through the plasma membrane to the cytosol and nucleus of living cells, both at 37°C and 4°C.Translocation of the penetratin peptide occurs even when it is coupled to hydrophilic molecules (e.g. phosphopeptides, oligonucleotides, peptidic nucleic acids, drugs, etc.) [7][8][9][10][11][12][13][14][15][16]. The penetratin peptide is therefore considered as a member of the so-called ÔTrojanÕ peptides or cell-penetrating peptides (CPP) that are water-soluble peptides with a low lytic activity that can be used as vectors for cellular internalization of hydrophilic biomolecules and drugs [17,18]. De...

Penetratin and derivatives acting as antifungal agents

“…It has been previously reported that peptideelipid association occurs through formation of salt bridges between the positively charged residues K4, R11 and K15 and the lipid phosphate groups [33]. In addition, tryptophan fluorescence studies previously showed the importance of peptide with positively charged residues for the initial binding to negatively charged vesicles, since double R/K / A mutations involving the residues K4/R10/ R11/K13/K15 significantly decreased the binding affinity [38]. The MEPs of 1 suggest that the above-mentioned residues (R1, K4, R11 and K15) could be responsible for the initial binding.…”

Liposome destabilization induced by synthetic lipopeptides corresponding to envelope and non-structural domains of GBV-C/HGV virus. Conformational requirements for leakage

“…To simulate protein-mediated fusion, many studies on peptide-induced membrane fusion have been conducted on model membranes such as liposomes and have employed synthetic peptides corresponding to the putative fusion sequences of viral proteins [9][10][11]. On the other hand and due to the fact that membraneassociated peptides often show a remarkable structural behaviour, the conformational study of these sequences is important to get information about their activities [12][13][14][15].…”

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

“…Other models, suggesting translocation of the CPPs across the lipid bilayer through stable or transiently formed pores or inverted micelles, have also been hypothesized (11). The basic nature due to the high content of arginine or lysine, the amphipathic character, and/or the presence of a hydrophobic core sequences have been suggested as important structural features of CPPs (12)(13)(14). Little is known about the cellular functions of CPPs; they have generally been studied as vectors for intracellular delivery of cargo molecules with pharmacologic potential (11,15).…”

“…Short basic peptides referred to as cell-penetrating peptides (CPPs) 1 are able to translocate across the plasma membrane into cells (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Several prototypical CPPs include the human immunodeficiency virus transactivator of transcription (Tat) protein and the Antennapedia homeodomain or represent oligoarginine polymers or chimeric peptides (1)(2)(3)(4)(5)(6).…”