An Antimicrobial Peptide-Mimetic Methacrylate Random Copolymer Induces Domain Formation in a Model Bacterial Membrane.

To address the emerging issue of drug-resistant bacteria, membrane-active synthetic polymers have been designed and developed to mimic host-defense antimicrobial peptides (AMPs) as antibiotic alternatives. In this study, we investigated the domain formation induced by synthetic polymer mimics of AMPs using model membranes to elucidate the biophysical principles that govern their membrane-active mechanisms. To that end, lipid vesicles mimicking Escherichia coli (E. coli) membrane were prepared using an 8:2 (molar ratio) mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG). Our studies using differential scanning calorimetry (DSC) and fluorescence microscopy indicated that cationic amphiphilic methacrylate random copolymers induced the phase separation to form POPE- or POPG-rich domains. A rhodamine-labeled polymer also showed the binding to separated domains in the membrane. Based on these results, we propose the mechanism that the copolymers induce domain formation by clustering of anionic POPG lipids similar to natural AMPs. In addition, the time-course of polymer binding to the GUV membrane was sigmoidal, suggesting the positive feedback loop in the membrane binding. We also hypothesize that this cooperative binding of the polymer is driven by the domain formation. This study demonstrates the potential of the amphiphilic copolymers to modulate the lipid organization of cell membranes, which may provide a new strategy to design membrane-active antimicrobial agents.

Mechanistic Study of Membrane Disruption by Antimicrobial Methacrylate Random Copolymers by the Single Giant Vesicle Method.

Cationic amphiphilic polymers have been a platform to create new antimicrobial materials that act by disrupting bacterial cell membranes. While activity characterization and chemical optimization have been done in numerous studies, there remains a gap in our knowledge on the antimicrobial mechanisms of the polymers, which is needed to connect their chemical structures and biological activities. To that end, we used a single giant unilamellar vesicle (GUV) method to identify the membrane-disrupting mechanism of methacrylate random copolymers. The copolymers consist of random sequences of aminoethyl methacrylate and methyl (MMA) or butyl (BMA) methacrylate, with low molecular weights of 1600-2100 g·mol-1. GUVs consisting of an 8:2 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG) and those with only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared to mimic the bacterial (Escherichia coli) or mammalian membranes, respectively. The disruption of bacteria and mammalian cell membrane-mimetic lipid bilayers in GUVs reflected the antimicrobial and hemolytic activities of the copolymers, suggesting that the copolymers act by disrupting cell membranes. The copolymer with BMA formed pores in the lipid bilayer, while that with MMA caused GUVs to burst. Therefore, we propose that the mechanism is inherent to the chemical identity or properties of hydrophobic groups. The copolymer with MMA showed characteristic sigmoid curves of the time course of GUV burst. We propose a new kinetic model with a positive feedback loop in the insertion of the polymer chains in the lipid bilayer. The novel finding of alkyl-dependent membrane-disrupting mechanisms will provide a new insight into the role of hydrophobic groups in the optimization strategy for antimicrobial activity and selectivity.

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions.

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-ß, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

Disruption of the mouse Slc39a14 gene encoding zinc transporter ZIP14 is associated with decreased bone mass, likely caused by enhanced bone resorption.

Osteoclasts are bone-resorbing cells that play an essential role in maintaining bone homeostasis. Zinc (Zn) has been reported to inhibit osteoclast-mediated bone resorption, but the mechanism of this action has not been clarified. Zn homeostasis is tightly controlled by the coordinated actions of many Zn transporters. The Zn transporter ZIP14/Slc39a14 is involved in various physiological functions; hence, Zip14-knockout (KO) mice exhibit multiple phenotypes. In this study, we thoroughly investigated the bone phenotypes of Zip14-KO mice, demonstrating that the KO mice exhibited osteopenia in both trabecular and cortical bones. In Zip14-KO mice, bone resorption was increased, whereas the bone formation rate was unchanged. Zip14 mRNA was expressed in normal osteoclasts both in vivo and in vitro, but receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis was not impaired in bone marrow-derived macrophages prepared from Zip14-KO mice. These results suggest that ZIP14 regulates bone homeostasis by inhibiting bore resorption and that in Zip14-KO mice, bone resorption is increased due to the elimination of this inhibitory regulation. Further studies are necessary to conclude whether the enhancement of bone resorption in Zip14-KO mice is due to a cell-autonomous or a non-cell-autonomous osteoclast defect.

Lipid-membrane-incorporated hydrophobic photochromic molecules prepared by the exchange method using cyclodextrins.

It was found that the exchange method for the preparation of lipid-membrane-incorporated guest molecules was applicable not only to fullerenes but also to other hydrophobic molecules such as azobenzene and stilbene. The advantages of this method are that the long-term stability of the lipid-membrane-incorporated azobenzene solution and the maximum ratio of [stilbene]/[lipid] were higher than those prepared by the classical method, which we call the 'premixing method'. Photoisomerisations of these photochromic guest molecules in the lipid membranes maintained the morphology of liposomes.

Modulation of raft domains in a lipid bilayer by boundary-active curcumin.

The function and mode of action of curcumin in modulating the formation of lipid raft domains were investigated by microscopic observation using model membranes. Curcumin induces fusion of lipid raft domains at extremely low concentrations through the alteration of the boundary between the ordered and disordered phases.

Dynamic behaviour of giant unilamellar vesicles induced by the uptake of [70]fullerene.

To obtain direct evidence for the fullerene-exchange reactions from the γ-cyclodextrin cavities to the lipid membranes following the addition of a C70·Î³-cyclodextrin complex, we monitored the dynamic behaviours of the giant unilamellar vesicles. A number of C70 aggregates generated in the lipid membranes moved about vigorously.