Régulations Naturelle et Artificielle

Gene therapy is a promising tool for the treatment of various cancers but is hindered by the physico-chemical properties of siRNA and needs a suitable vector for the delivery of siRNA to the target tissue. Bile acid-based block copolymers offers certain advantages for the loading and delivery of siRNA since they can efficiently complex siRNA and bile acids are biocompatible endogenous molecules. In this study, we demonstrate the use of lipids as co-surfactants for the preparation of mixed micelles to improve the siRNA delivery of cholic acid-based block copolymers. Poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) were polymerized on the surface of cholic acid to afford a star-shaped block copolymer with four arms (CA-PAGE-b-PEG)4. The allyl groups of PAGE were functionalized to bear primary or tertiary amines and folic acid was grafted onto the PEG chain end to increase cell uptake. (CA-PAGE-b-PEG)4 functionalized with either primary or tertiary amines show high siRNA complexation with close to 100% complexation at N/P ratio of 8. Uniform aggregates with diameters between 181 and 188 nm were obtained. DOPE, DSPE-PEG2k, and DSPE-PEG5k lipids were added as co-surfactants to help stabilize the nanoparticles in the cell culture media. Mixed micelles had high siRNA loading with close to 100% functionalization at N/P ratio of 16 and diameters ranging from 153 to 221 nm. The presence of lipids in the mixed micelles improved cell uptake with a concomitant siRNA transfection in HeLa and HeLa-GFP model cells, respectively.

This study introduces the synthesis of a new class of lipopolymers, composed of L-proline monomer units and a phospholipid initiator. These lipopolymers were incorporated into lipid giant unilamelar vesicles and exhibited thermal hysteresis. Phase separation and polymer multimerization at the lipid bilayer was observed through confocal microscopy and confirmed by FRAP experiments. Such temperature-triggered membrane reorganiza-tion was accompanied by the release of probes up to 10 kDa, as monitored by fluorescence. These findings demonstrate that specifically engineered proline-based protein-like polymer functionalized by a phospholipid can display controlled self aggregation in phospholipid bilayer and localized stimuli responsive permeability.

Ribosomes are macromolecular RNA-protein complexes that constitute the central machinery responsible for protein synthesis and quality control in the cell. Ribosomes also serve as a hub for multiple non-ribosomal proteins and RNAs that control protein synthesis. However, the purification of ribosomes and associated factors for functional and structural studies requires a large amount of starting biological material and a tedious workflow. Current methods are challenging as they combine ultracentrifugation, the use of sucrose cushions or gradients, expensive equipment, and multiple hours to days of work. Here, we present a rapid, facile, and cost-effective method to isolate ribosomes from in vivo or in vitro samples for functional and structural studies using single-step enrichment on magnetic beads – RAPPL (RNA Affinity Purification using Poly-Lysine). Using mass spectrometry and western blot analyses, we show that poly-lysine coated beads incubated with E. coli and HEK-293 cell lysates enrich specifically for ribosomes and ribosome-associated factors. We demonstrate the ability of RAPPL to isolate ribosomes and translation-associated factors from limited material quantities, as well as a wide variety of biological samples: cell lysates, cells, organs, and whole organisms. Using RAPPL, we characterized and visualized the different effects of various drugs and translation inhibitors on protein synthesis. Our method is compatible with traditional ribosome isolation. It can be used to purify specific complexes from fractions of sucrose gradients or in tandem affinity purifications for ribosome-associated factors. Ribosomes isolated using RAPPL are functionally active and can be used for rapid screening and in vitro characterization of ribosome antibiotic resistance. Lastly, we demonstrate the structural applications of RAPPL by purifying and solving the 2.7Å cryo-EM structure of ribosomes from the Cryptococcus neoformans, an encapsulated yeast causing cryptococcosis. Ribosomes and translational machinery purified with this method are suitable for subsequent functional or structural analyses and provide a solid foundation for researchers to carry out further applications – academic, clinical, or industrial – on ribosomes.

As antimicrobial resistance threatens our ability to treat common bacterial infections, new antibiotics with limited cross-resistance are urgently needed. In this regard, natural products that target the bacterial ribosome have the potential to be developed into potent drugs through structure-guided design, provided their mechanisms of action are well understood. Here we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X primarily inhibits peptide bond formation between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3' adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of tetracenomycin X on the bacterial ribosome and suggests a path forward for the development of novel aromatic polyketide antibiotics.

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules ("ligands") and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate "ready-to-screen" libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.

CD95 is a death receptor that can promote oncogenesis through molecular mechanisms that are not fully elucidated. Although the mature CD95 membrane receptor is considered to start with the arginine at position 17 after elimination of the signal peptide, this receptor can also be cleaved by MMP7 upstream of its leucine at position 37. This post-translational modification occurs in cancer cells but also in normal cells such as peripheral blood leukocytes. The non-cleaved CD95 amino-terminal region consists in a disordered domain and its in silico reconstitution suggests that it might contribute to receptor aggregation and thereby, regulate the downstream death signaling pathways. In agreement with this molecular modeling analysis, the comparison of CD95-deficient cells reconstituted with full-length or N-terminally truncated CD95 reveals that the loss of the amino-terminal region of CD95 impairs the initial steps of the apoptotic signal while favoring the induction of pro-survival signals, including the PI3K and MAPK pathways.

The combination between ion mobility mass spectrometry and molecular dynamics simulations is demonstrated for the first time to afford valuable information on structural changes undergone by dendriplexes containing ds-DNA and low-generation dendrimers when transferred from the solution to the gas phase. Dendriplex ions presenting 1:1 and 2:1 stoichiometries are identified using mass spectrometry experiments, and the collision cross sections (CCS) of the 1:1 ions are measured using drift time ion mobility experiments. Structural predictions using Molecular Dynamics (MD) simulations showed that gas-phase relevant structures, i.e., with a good match between the experimental and theoretical CCS, are generated when the global electrospray process is simulated, including the solvent molecule evaporation, rather than abruptly transferring the ions from the solution to the gas phase. The progressive migration of ammonium groups (either NH4+ from the buffer or protonated amines of the dendrimer) into the minor and major grooves of DNA all along the evaporation processes is shown to compact the DNA structure by electrostatic and hydrogen-bond interactions. The subsequent proton transfer from the ammonium (NH4+ or protonated amino groups) to the DNA phosphate groups allows creation of protonated phosphate/phosphate hydrogen bonds within the compact structures. MD simulations showed major structural differences between the dendriplexes in solution and in the gas phase, not only due to the loss of the solvent but also due to the proton transfers and the huge difference between the solution and gas-phase charge states.

Parkinson's disease (PD) is an age‐related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, associated with the accumulation of misfolded α‐synuclein and lysosomal impairment, two events deemed interconnected. Protein aggregation is linked to defects in degradation systems such as the autophagy‐lysosomal pathway, while lysosomal dysfunction is partly related to compromised acidification. We have recently proven that acidic nanoparticles (aNPs) can re‐acidify lysosomes and ameliorate neurotoxin‐mediated dopaminergic neurodegeneration in mice. However, no lysosome‐targeted approach has yet been tested in synucleinopathy models in vivo. Here, we show that aNPs increase α‐synuclein degradation through enhancing lysosomal activity in vitro. We further demonstrate in vivo that aNPs protect nigral dopaminergic neurons from cell death, ameliorate α‐synuclein pathology, and restore lysosomal function in mice injected with PD patient‐derived Lewy body extracts carrying toxic α‐synuclein aggregates. Our results support lysosomal re‐acidification as a disease‐modifying strategy for the treatment of PD and other age‐related proteinopathies.