Endocytosis Involved d-Oligopeptide of Tryptophan and Arginine Displays Ordered Nanostructures and Cancer Cell Stereoselective Toxicity by Autophagy.

Owing to their self-aggregation propensity and selective interaction with the anionic membranes, the peptides rich in tryptophan (Trp) and arginine (Arg) are considered for the development of novel anticancer therapeutics. However, the structural insights from the perspective of backbone chirality and spatial orientation of side chains into the selective toxicity of peptides are limited. Here, we investigated the selectivity and cellular uptake of HHC36, a Trp/Arg-rich nonapeptide, and its d-enantiomer (allDHHC36) and a retroinverso analogue in the lung A549 and breast MDA-MB-231 cancer cells. We realized that the d-peptides can specifically induce autophagy at nontoxic concentrations only in the A549 cells supported from the LC 3-II immunostaining expression in the vicinity of the nucleus and the ultrastructural analysis revealing the autophagosome formation. The autophagic flux was also remarkable in the cells exposed to d-peptides at a far lower concentration in synergism with doxorubicin (DOX). In marked contrast, nonselective cell death was observed only if a high amount of HHC36 was applied. HHC36 tended to irregular collagen-like fibrils relative to allDHHC36 that distinctly formed higher-order coiled nanostructures. Interestingly, the short d-peptide fragments were generated in a harsh oxidative condition. Compared with the direct membrane transduction of HHC36, the entry of d-peptides into the lung cancer cells was controlled by endocytosis through the contribution of heparan sulfate proteoglycans (HSPGs) and cholesterol (CHO). However, both l- and d-peptides feasibly crossed the membrane and localized inside the S-phase-arrested cell nucleus. This suggested the likelihood of peptide intercalation with DNA that might differently appear in selective and/or nonselective deaths. These results unraveled the d-handedness-selective toxicity of a self-assembling Trp/Arg-rich sequence that is dependent on the cell type from the aspects of the density of anionic charges and CHO in the outer leaflet of the plasma membrane, as well as the intracellular redox imbalance that may drive the formation of toxic peptide nanostructure fragments.

Turn-folded magainin lipopeptide analog induces cytoplasmic vacuoles in MDA-MB-231 cells through G2-phase arrest.

Selective induced non-canonical programmed deaths in the lipid raft type 1-enriched MDA-MB-231 is a promising treatment approach. Cationic amphiphilic peptides conjugated to relatively long fatty acyl chains that tend to self-aggregate are prone to upregulate necroptotic and paraptotic signaling. We investigated the toxic effects of an N-terminally palmitoylated magainin derivate (P1MK5E) in the MDA-MB-231 cells in relation to its structure at molecular level. The modeling showed that the palmitoylation reinforces a turn-like structural motif in the lipopeptide which is likely required for its activity. P1MK5E triggered intracellular generation of reactive oxygen species (ROS), G2-phase arrest, mitochondrial membrane potential (ΔΨmt) disturbance and presumable flopping of phosphatidylserine (PtdSer) to the cancer cell membrane outer surface in a comparable manner to doxorubicin (DOX) that induces apoptotic signaling. Despite forming extensive congregates of different sizes at the cell surface, P1MK5E had little impacts on the MDA-MB-231 membrane integrity. The cell death upon exposure to the lipopeptide was, however, caspase 3 independent and characterized by cytoplasmic vacuolation and no distinct nuclear fragmentation that is to be privileged in the treatment of apoptotic resistance pathways in triple-negative breast cancers (TNBCs).

Palmitoylation of Membrane-Penetrating Magainin Derivatives Reinforces Necroptosis in A549 Cells Dependent on Peptide Conformational Propensities.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike ß-protofibrils. At the close concentrations (∼7.5 µM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 µM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

Bacterial Aggregation Triggered by Fibril Forming Tryptophan-Rich Sequences: Effects of Peptide Side Chain and Membrane Phospholipids.

The influence of side chain residue and phospholipid characteristics of the cytoplasmic membrane upon the fibrillation and bacterial aggregation of arginine (Arg) and tryptophan (Trp) rich antimicrobial peptides (AMPs) has not been well described to date. Here, we utilized the structural advantages of HHC-10 and 4HarHHC-10 (Har, l-homoarginine) that are highly active Trp-rich AMPs and investigated their fibril formation and activity behavior against bacteria. The peptides revealed time-dependent self-assembly of polyproline II (PPII) α-helices, but by comparison, 4HarHHC-10 tended to form higher ordered fibrils due to relatively strong cation-π stacking of Trp with Har residue. Both peptides rapidly killed S. aureus and E. coli at their MICs and caused aggregation of bacteria at higher concentrations. This bacterial aggregation was accompanied by the formation of morphologically distinct electron-dense nanostructures, likely including but not limited to peptides alone. Both HHC-10-derived peptides caused blebs and buds in the E. coli membrane that are rich in POPE phospholipid that promotes negative curvature. However, the main population of S. aureus cells retained their cocci structure upon treatment with HHC peptides even at concentration higher than the MICs. In contrast, the cell aggregation was not induced by HHC fibrils that were most likely stabilized through intra-/intermolecular cation-π stacking. It is proposed that masking of these interactions might have resulted in diminished membrane association/insertion of the HHC nanostructures. The peptides caused aggregation of POPC/POPG (1/3) and POPE/POPG (3/1) liposomes. Nonetheless, disaggregation of the former vesicles was observed at ratios of lipid to peptide of greater than 6 and 24 for HHC-10 and 4HarHHC-10, respectively. Collectively, our results revealed dose-dependent bacterial aggregation mediated by Trp-rich AMPs that was profoundly influenced by the degree of peptide's self-association and the composition and intrinsic curvature of the cytoplasmic membrane lipids.