Alain Chavanieu

Background and Purpose: Gamma‐glutamyl dipeptides are essentially produced after glutathione (GSH) degradation. While several γ‐glutamyl dipeptides have been detected in different organs and body fluids, their biological activity remains elusive. This is the case for γ‐Glutamyl‐Glycine (γ‐Glu‐Gly) which has been found in different brain areas. γ‐Glutamyl‐glycine may bind to ionotropic glutamate receptors. Here, we have investigated whether γ‐Glu‐Gly could exhibit modulatory actions on excitatory synaptic transmission and plasticity by performing its functional characterization on glutamate, GABA and glycine receptors. Experimental Approach: Electrophysiological and calcium imaging experiments were performed on cells heterologously expressing either Glutamate, GABA or Glycine receptors and on cultured hippocampal neurons and acute hippocampal slices. The γ‐Glu‐Gly production by cultured cells and hippocampal slices was measured by LC‐MS analysis, following incubation with modulators of the glutathione metabolic cycle. Key Results: γ‐Glu‐Gly exerted partial agonist effects on both NMDA GluN1 and mGlu5 receptors, but without any binding to the glutamate binding domain on both AMPA and NMDA GluN2A receptors. γ‐Glu‐Gly was devoid of any effect on GABA, glycine and GluN3A receptors. γ‐Glu‐Gly was able to trigger a long‐term increase in synaptic transmission in the CA1 area of mouse hippocampus. Conclusion and implications: γ‐Glu‐Gly partially retains the excitatory actions of glutamate on specific receptor sub‐types and may trigger plastic events in the hippocampus. Its accumulation under pathological conditions associated with oxidative stress and a high GSH consumption could thus interfere with endogenous synaptic transmission and disrupt natural plasticity, which is a hallmark of neurodegenerative diseases.

Targeting specific protein–protein interactions, particularly those involving DNA methyltransferases (DNMTs), offers a promising strategy to resensitise glioblastoma multiforme cells to temozolomide.

Background/Objectives: Antibody-dependent cellular cytotoxicity relies on the interaction between the Fc region of immunoglobulin G1 (IgG1) and the CD16a receptor. While removal of core fucosylation on Fc and introduction of the DFTE mutation set (S239D, H268F, S324T, I332E) are known to enhance CD16a binding, the detailed contributions of these engineered sites in solution remain incompletely defined. Methods: Here, we employed 1 µs molecular dynamics simulations to map, at atomic resolution, the interaction networks stabilizing pre-formed Fc-CD16a complexes, including afucosylated Fc-wild-type, DFTE-engineered, Fc-fucosylated, and asymmetrically engineered Fc variants. Results: Our results show that only S239D, present on both Fc chains, and H268F on chain A consistently contribute to stabilizing the CD16a interface, while I332E does not form persistent interactions. Glycan–protein contacts are primarily intrachain, with transient interchain glycan–glycan interactions not contributing significantly to complex stability. Fucosylation on Fc significantly reduces binding stability by disrupting peripheral interactions and critical glycan-mediated contacts. Notably, the asymmetric Fc variant, in which the two heavy chains carry distinct sets of substitutions, retains high-affinity binding despite lacking S239D and carrying core fucose, through a novel hydrophobic cluster and reinforced peripheral electrostatic interactions. Conclusions: Altogether, these findings provide a quantitative framework for how targeted mutations and fucose modifications remodel Fc-CD16a interactions, offering insights for the rational design of next-generation therapeutic antibodies.

The recently characterized honeybee Ca_V4 channel is a high-voltage–activated Ca²⁺ channel ortholog to the DSC1 channel identified in Drosophila. While sequence similarities to Na_V channels are obvious, permeation properties and current kinetics are more closely aligned with those of Ca_V channels. Ca_V4 exhibits a distinctive cation-dependent inactivation pattern, a hallmark of Ca²⁺ channel behavior, and nonetheless displays sensitivity to a Na⁺ channel–specific regulator, veratrine. Calcium channel facilitation is a phenomenon whereby the probability of calcium channel opening increases with successive depolarization pulses, resulting in an enhanced Ca²⁺ influx during repetitive or sustained electrical activity. In this study, we have identified an additional specific property of Ca_V4 in the form of an atypical voltage-dependent facilitation of the Ca²⁺ or Ba²⁺ currents by strong pre-depolarizations or prepulses (pPs). This physiologically relevant phenomenon, known as pP-induced facilitation (PiF), is subject to positive regulation by the amplitude of the pP but to negative regulation by its duration. It produces a hyperpolarizing shift of the I–V curve without any change in the reversal potential and macroscopic or single channel conductance. PiF is thus more pronounced for small depolarizations and almost absent when channels reach their maximal open probability. A mutation that affects the inactivation of the Ca_V4 channel prevents the occurrence of PiF. This previously undocumented form of facilitation appears exclusive to Ca_V4 channels. A strong pP may lock Ca_V4 channels in a pre-open state, rendering them more susceptible to activation and thereby shifting the activation curve toward more negative potentials. This, in turn, would accelerate channel opening and increase current amplitude. Lastly, we show that the inactivation particle of Ca_V4 (MFLT sequence, equivalent to the IFMT motif in human Na_V, or MFMT in Apis Na_V channel), in addition to its role in the initiation of the voltage-dependent inactivation, also modulates PiF.

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.

DNA methyltransferase (DNMT) inhibitors are emerging as a promising class of agents for personalized and targeted cancer therapy, particularly in malignancies with limited therapeutic options such as glioblastoma (GB). In GB, the MGMT/DGKI methylation profile serves as a biomarker for stratifying patients by treatment response. Specifically, the MGMTmethylated/DGKIunmethylated profile is associated with favorable outcomes, whereas the MGMTmethylated/DGKImethylated profile correlates with poor outcome. These findings suggest that selective demethylation of DGKI without altering MGMT or inducing widespread genomic hypomethylation, which may promote tumorigenesis, could represent a novel and more effective therapeutic strategy. Current DNMT inhibitors lack specificity for glioblastoma relevant methylation profiles, thereby limiting their therapeutic efficacy. To address this challenge, AlphaFold-based protein–protein interaction predictions were integrated with sequential chromatin immunoprecipitation assays and established DNMT1 interactome data. This integrative approach led to the identification of a DNMT1/ELK1 complex as a critical regulator of DGKI methylation. A peptide mimicking the DNMT1/ELK1 interface, designated EXDDNMT1/ELK1, was designed and shown to induce selective DGKI demethylation without altering MGMT or inducing global DNA hypomethylation. Notably, EXDDNMT1/ELK1 did note promote cellular proliferation or invasion, and successfully restored sensitivity to standard glioblastoma therapy in both cellular and in vivo models. These findings also support the use of MGMT and DGKI methylation levels in cell-free DNA as potential biomarkers to identify patients likely to benefit from EXDDNMT1/ELK1 treatment. This study identifies a clinically actionable biomarker (MGMTMethylated/DGKIMethylated), detectable in both solid and liquid biopsies, enabling patient stratification. Furthermore, it establishes EXDDNMT1/ELK1 as a highly selective epigenetic therapeutic agent to treat GB patients.

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (Na_V), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca²⁺). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca²⁺, while excluding sodium ions (Na⁺), thus defining a new family of Ca²⁺ channels, known as Ca_V4. In this study, we characterize Ca_V4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like Na_V and Ca_V1 channels, respectively. Ca_V4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as Na_V but permeates Ca²⁺ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium-dependent modulation of voltage-gated Ca²⁺ and Na⁺ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of Ca_V4 and defines this channel as a novel class of voltage-gated Ca²⁺ channels.

Cav2.1 channels are expressed throughout the brain and are the predominant Ca²⁺ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1−e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

The intensive use of insecticides, combined with other factors such as habitat loss, might explain worldwide decrease of insect populations documented in the past twenty years. However, due to the involvement of pest species in crop destruction and in vector‐borne diseases, insecticides will probably continue to be required still for decades. The most commercially successful insecticides are neurotoxicants acting on ion channels of the central nervous system affecting thereby cellular excitability and synaptic transmission and causing insect paralysis and fatality. In this article, we provide an overview of the insecticides acting on voltage‐gated sodium channels, GABA‐gated chloride channels and nicotinic acetylcholine receptors. We summarize the current knowledge on those ion channels from the honeybee Apis mellifera and discuss the possible mode of action of neurotoxic insecticides.

Protein‐polymer bioconjugates have shown great promise in biomedical and life science applications including drug delivery and diagnosis. The current bioconjugation strategies suffer from lack of efficiency and versatility. In this article, poly(styrene‐ alt ‐maleic anhydride) copolymers were first prepared by RAFT polymerization and characterized by different analytical techniques. Then, the poly(styrene‐ alt ‐maleic anhydride) precursors were functionalized with primary amine such as azidopropylamine and amino poly(ethylene glycol). The reaction of amino compounds with maleic anhydride was found to be a highly efficient, a versatile, and a facile chemical ligation reaction for the synthesis of macromolecules with quantitative yield under mild conditions. The main benefit is the incorporation of a wide range of functionality by easily changing the primary amine compound. For the amphiphilic graft copolymers based on poly(ethylene glycol), aggregation behavior in water was investigated. In a second part, azido‐functionalized polystyrene copolymers were used to prepare a new protein‐polymer bioconjugate by copper‐free click chemistry reaction.