RESUMEN
Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral ß3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.
Asunto(s)
Antibacterianos , Membrana Celular , Simulación de Dinámica Molecular , Antibacterianos/farmacología , Antibacterianos/química , Membrana Celular/efectos de los fármacos , Lipopolisacáridos/farmacología , Pruebas de Sensibilidad Microbiana , Péptidos/química , Péptidos/farmacología , Microscopía Electrónica , Bacterias Gramnegativas/efectos de los fármacos , Escherichia coli/efectos de los fármacosRESUMEN
The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the 1HN-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, 1Hα-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407-Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1-140 nt) highlight that the Thr367-Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein-RNA interaction as a "fuzzy" complex.
Asunto(s)
ARN Largo no Codificante , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Protein unfolding and denaturation are main issues in biochemical and pharmaceutical research. Using a global parameter, the translational diffusion coefficient D, folded, unfolded, and intrinsically disordered proteins of a given molar mass M can be distinguished based on their distinct hydrodynamic properties. For broader applications, we provide generalized, PFG-NMR-based empirical D-M relations validated at different temperatures and ready to use with the corresponding corrections in different media. We demonstrate that these relations enable a more accurate molecular mass determination and show fewer potential errors than those of the common methods based on small-molecular diffusion standards. We monitor unfolding of three model proteins using 8 M urea and dimethyl sulfoxide (DMSO)-water mixtures as denaturing agents, highlighting the effect of disulfide bonds. Denaturation in 8 M urea is pH-dependent; in addition, for proteins with highly stable disulfide bonds, a reducing agent (TCEP) is required to achieve complete unfolding. Regarding the effect of local parameters, we show that at low DMSO concentrationsâcommon conditions in pharmaceutical binding studiesâthe PFG-NMR-derived global parameters are not significantly affected. Still, the atomic environments can change, and the bound solvent molecule can inhibit the binding of a partner molecule. Using proteins with natural isotopic abundance, this effect can be proven by fast 1H-15N 2D correlation spectra. Our results enable fast and easy estimation of protein molecular mass and the degree of folding in various media; moreover, the effect of the cosolvent on the atomic-level structure can be traced without the need of isotope labeling.
Asunto(s)
Dimetilsulfóxido , Proteínas , Difusión , Dimetilsulfóxido/química , Disulfuros , Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Termodinámica , UreaRESUMEN
Inspired by the well-established clinical evidence about the interplay between apoptotic TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) mechanism and reactive oxygen species (ROS)-mediated oxidative stress, a set of novel ONC201 hybrids containing the impiridone core and one or two differently positioned ferrocenylalkyl groups were synthesised in our present work. These two types of residues have been implicated in the aforementioned mechanisms associated with cytotoxic activity. A straightforward, primary amine-based synthetic approach was used allowing the introduction of a variety of N-substituents into the two opposite regions of the heterocyclic skeleton. Reference model compounds with benzyl and halogenated benzyl groups were also synthesised and tested. The in vitro assays of the novel impiridones on five malignant cell lines disclosed characteristic structure-activity relationship (SAR) featuring significant substituent-dependent activity and cell-selectivity. A possible contribution of ROS-mechanism to the cytotoxicity of the novel metallocenes was suggested by density functional theory (DFT)studies on simplified models. Accordingly, unlike the mono-ferrocenylalkyl-substituted products, the compounds containing two ferrocenylalkyl substituents in the opposite regions of the impiridone core display a much more pronounced long-term cytotoxic effect against A-2058 cell line than do the organic impiridones including ONC201 and ONC212. Furthermore, the prepared bis-metallocene derivatives also present substantial activity against COLO-205- and EBC-1 cell lines.