Biology subjects

Volkov, O.

Publications and source records attributed to Volkov, O..

4 recordsLinked to original sources

bioRxiv2023

Application of polystyrene sulfonate (PSS) for inhibiting toxicity of ALS/FTD-linked dipeptide repeats

The GGGGCC (G4C2) expansion in the noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat region is translated into five different dipeptide repeat proteins (DPRs), of which the arginine-rich DPRs (R-DPRs) poly- GR (GRn) and poly-PR (PRn) are highly neurotoxic. In this study, we characterized the protective effect against R- DPR toxicity of polystyrene sulfonate (PSS), an FDA-approved drug applied in hyperkalemia, in biochemical, cellular and iPSC-derived motor neuron (MN) models. We found that PSS, in a length-dependent manner, interacts very tightly with R-DPRs, and releases their bound RNA in R-DPR - RNA mixtures. PSS significantly influences the liquid- liquid phase separation (LLPS) of R-DPRs elicited by RNA and reduces their ensuing cell toxicity in Neuro-2a cells. PSS is cell penetrable, and it can effectively rescue the inhibitory effect R-DPRs on axonal mitochondrial transport in iPSC-derived MNs. Shorter (n < 340) variants of PSS are not toxic either to cells or to mice upon intracerebroventricular injection up to 1 mM concentration. Our results suggest that its polymeric nature endows PSS with an advantageous effect in C9-ALS providing a potential therapeutic tool against this debilitating neurodegenerative disease.

molecular biology↗

bioRxiv2023

Functional GPCR expression in eukaryotic LEXSY system

G protein-coupled receptors (GPCRs) represent an important class of drug targets, and their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Here we tested the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We expressed the human A2A adenosine receptor (A2AAR) in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression.

biophysics↗

bioRxiv2023

Identification of small molecule antivirals against HTLV-1 by targeting the hDLG1-Tax-1 protein-protein interaction

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analysed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.

biochemistry↗

bioRxiv2020

Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Forster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptors constitutive activity. For the agonist-bound A2AAR, we detected faster (390{+/-}80 s) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

biophysics↗