Membrane Surface-Associated Helices Promote Lipid Interactions and Cellular Uptake of Human Calcitonin-Derived Cell Penetrating Peptides

Abstract

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.

Royal Jelly: An ancient remedy with remarkable antibacterial properties

“…After the formation of a binding between the peptide and the phospholipid head groups the peptide reaches the inner part of the cell without modified the membrane (a mechanism of transport through the lipid bilayer without the formation of a stable channel). Once inside, the peptide can interact with the targets (Pálffy et al, 2009;Xiao et al, 2015).Unfortunately, the type of aggregates that provide the insertion of the peptide inside the membrane is not well defined, so it is more difficult to predict molecular properties who can favour this mechanism (Herbig et al, 2005;Li et al, 2012). Besides the ability to interact with bacterial membranes, the AMPs could have other intracellular target (Ahn et al, 2006); in fact they can bind DNA, RNA and proteins and inhibit synthesis of different essential cell constituents as cell wall, DNA, RNA and proteins (Lan et al, 2010;Li et al, 2012).…”

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

“…In barrel-stave pore the peptide interaction with the target membrane is driven predominantly by hydrophobic interactions and the peptides should bind to phospholipid membranes irrespective of their charge [38], with a transmembrane domain that spans across the membrane [36]. These events are favoured in the absence of Trp residues in the peptide [40]. Pep-1 however does not fulfil any of such requirements for pore formation.…”

Administration de molécules actives dans les cellules

“…schémas, les modèles de Barrel-Stave et Carpet étant les plus connus [16,17]. Ces deux modèles ont été empruntés à l'étude des interactions des peptides antimicrobiens avec les bicouches lipidiques et la translocation des peptides est basée sur le concept de perturbation locale de la bicouche lipidique par des agrégats peptidiques.…”