Cholesterols Work as a Molecular Regulator of the Antimicrobial Peptide-Membrane Interactions

Abstract

The existing cholesterols (Chols) in animal cell membranes play key roles in many fundamental cellular processes, which also promise the possibility to modulate the bioactivity of various membrane-active biomacromolecules. Here, combining dynamic giant unilamellar vesicle leakage experiments and molecular dynamics simulations, the inhibitory effect of Chols on the membrane poration activity of melittin (Mel), a typical natural antimicrobial peptide, is demonstrated. Molecular details of the Mel-Chol interactions in membrane show that, for a Chol-contained lipid membrane, Mel exposure would perturb the symmetric bilayer structure of the membrane and specifically influence the location and orientation distributions of Chol molecules to an asymmetric state between the two leaflets; moreover, the Mel-Chol interactions are significantly influenced by the membrane environment such as unsaturation degree of the lipid components. Such inhibitory effect is normally ascribed to an accumulation of Chol molecules around the membrane-bound peptide chains and formation of Chol-Mel complexes in the membrane, which hinder the further insertion of peptides into the membrane. This work clarifies the molecular interactions between membrane-active peptides and Chol-contained membranes, and suggest the possibility to develop targeted drugs due to the membrane component specificity between bacterial and animal cells.

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design

“…The above discussed studies considered only peptides statically located at a membrane; the main mechanism through which antimicrobial peptides kill cells is, however, the formation of transmembrane pores. Simulating pore formation is computationally expensive; thus in a few older studies, to avoid long simulations, antimicrobial peptides were initially placed in the lipid bilayer core [818][819][820] or simulations were performed using the coarse-grained MARTINI model [821][822][823][824][825]. A few studies set out to elucidate the entire process of pore formation; however, the protocol of the MD simulations was altered e.g., by running simulations at an artificially high temperature or through a biased selection of the initial configuration.…”

Membrane permeability and antimicrobial peptides: Much more than just making a hole

“…[58] Also the transmembrane entry process of the probe into GUVs can be evaluated (Figure 4b). [59] For instance, using these methodologies, the process of pore formation induced by a well-known AMP, magainin 2, could be revealed, obtaining the rate constants of several elementary steps, such as peptide/protein-induced pore formation in lipid membranes and the membrane permeation of fluorescent probes through the pores. [57] Moreover, it is also possible to introduce technical modifications, such as the addition of another fluorescent agent to the vesicles to evaluate whether the permeabilization is temporary or not, or through the use of confocal microscopy, to define whether membrane permeabilization occurs through a pore formation mechanism or total membrane disruption.…”

“…This inhibitory effect of Cho on the membrane permeabilization efficiency of melittin would be beneficial for the specific activity of melittin on bacteria (without Chol in its composition) rather than mammalian cell membranes (with Cho). [59] The experimental permeability measurements of solutes through lipid membranes have contributed not only to the description of the interaction of AMPs but also represent a measure of the structural alteration they can cause. However, there are certain limitations in the experimental approaches, as in the case of the description of the molecular mechanism of passive transport.…”

“…[ 58 ] Also the transmembrane entry process of the probe into GUVs can be evaluated (Figure 4b). [ 59 ]…”

Amphiphilic Gold Nanoparticles: A Biomimetic Tool to Gain Mechanistic Insights into Peptide-Lipid Interactions

“…When looking at the combination of cholesterol and zwitterionic PC phospholipids in biomimetic systems, numerous examples reveal a dramatic reduction in the membrane-binding properties of Arg-containing amphipathic AMPs [ 127 ]. This is the case, for instance, of honeybee melittin (one of the most studied antimicrobial peptides) [ 128 ], temporin L derived from the frog Rana temporaria [ 129 ] and Arg-rich protegrin-1 isolated from porcine leukocytes [ 130 , 131 ]. Remarkably, in homogeneous PC bilayers with a high cholesterol content, the spontaneous membrane incorporation of melittin has been reported to be strongly delayed compared to phase-separated lipid systems with the same amount of cholesterol [ 132 ].…”