Biophysical and Biological Studies of End-Group-Modified Derivatives of Pep-1

Abstract

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.

Energy-independent translocation of cell-penetrating peptides occurs without formation of pores. A biophysical study with pep-1

“…In our previous studies, with model membranes, no evidence for pore formation was detected in the presence of pep-1 [7] and this result was confirmed by Weller et al [10]. However, this issue remains very controversial as electrophysiological studies using oocyte membrane [12] suggest that a transmembrane pore-like structure could be formed, mediated by pep-1 conformational changes.…”

“…These perturbations in the membrane occurred without evidence for pore formation. [7,10]. Fusion events without leakage were also observed for other CPPs such as R 7 W, TATP59W and TATLysP59W [31].…”

“…Fusion events without leakage were also observed for other CPPs such as R 7 W, TATP59W and TATLysP59W [31]. Moreover, membrane integrity in HeLa cells is only disrupted in the presence of high peptide concentration (concentration much higher than the one required for the translocation to occur) [10].…”

“…The peptide has an amphipathic nature, the hydrophobic domain, containing five Trp residues, inserts in the membrane with a shallow position [8,10], but not accessible to the aqueous environment [8]. The hydrophilic domain, with five basic residues, does not insert in the hydrophobic core of the membrane [9].…”

Thermodynamics, size, and dynamics of zwitterionic dodecylphosphocholine and anionic sodium dodecyl sulfate mixed micelles

“…DPC provides a zwitterionic surface on the micelles that adequately mimic biological membranes of the vertebrates, whereas the SDS micelles, with negatively charged headgroups, adequately imitate the bacterial membrane [15]. However, to mimic electrostatic properties of the vertebrate plasma, a membrane model characterized by a slight prevalence of the negative charge, such as that in the DPC/SDS mixed micelles, can be used as well [16][17][18]. The use of SDS to imitate membrane environment is not always rational because it has no structural analogues among the phospholipids that are components of biological membranes.…”