Biology subjects

Barsukov, I.

Publications and source records attributed to Barsukov, I..

8 recordsLinked to original sources

bioRxiv2026

An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

SHANK proteins form core postsynaptic density (PSD) scaffolds that organise synaptic signalling complexes through multiple protein-protein interaction domains, including PDZ domains that typically recognise C-terminal peptide motifs. Here, we identify an internal PDZ recognition mechanism that links the synaptic protein Densin-180 to SHANK and promotes SHANK scaffold assembly. We map SHANK binding to an internal PDZ-binding motif in Densin-180 (residues 843-863) and show by NMR spectroscopy and fluorescence polarisation that this motif binds SHANK1-3 PDZ domains with high affinity and specificity, competing with canonical C-terminal ligands. The crystal structure of the Densin-180-SHANK complex reveals that Phe858 inserts into the hydrophobic pocket of the PDZ domain despite the absence of a terminal carboxylate. In neurons, this interaction mediates Densin-180 recruitment to dendritic spines and drives activity-dependent reorganisation of postsynaptic SHANK3 assemblies, whereas mutation of Phe858 disrupts both processes. Disrupting the Densin-180-SHANK interaction using a Densin-180-derived peptide impairs activity-dependent structural plasticity of spines, accompanied by defects in PSD organisation and actin cytoskeleton remodelling. These findings define an internal mode of PDZ recognition and reveal how Densin-180 couples neuronal activity to the remodelling of postsynaptic SHANK scaffolds.

neuroscience↗

bioRxiv2024

Dock-and-lock binding of SxIP ligands is required for stable and selective EB1 interactions.

End Binding protein 1 (EB1) is a key component of the signalling networks located at the plus ends of microtubules. It incorporates an N-terminal microtubule binding CH domain and the C-terminal EBH domain that interacts with the SxIP-containing sequences of other microtubule plus end tracking proteins (+TIPs). By using a series of SxIP containing peptides derived from the microtubule-actin cross-linking factor, MACF, we show that the SxIP motif itself binds to EBH with low affinity, and the full interaction requires contribution of the post-SxIP residues. Based on the solution structure and dynamics of the EBH/MACF complex we proposed a two-step dock-and-lock model for the EBH interaction with targets, where the SxIP motif initially binds to a partially formed EBH pocket, which subsequently induces folding of the unstructured C-terminus and transition to the stable complex. We dissect contributions from different interactions into the binding and design MACF mutations of the post-SxIP region that enhance the affinity by two orders of magnitude, leading to a nanomolar interaction. We verify the enhanced recruitment of the mutated peptide to the dynamic plus ends of MTs in a live cell experiment. Our model explains EB1s interaction with the SxIP-containing ligands and can be used to design of small molecule inhibitors that can block SxIP interaction with EB1.

biochemistry↗

bioRxiv2023

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

cell biology↗

bioRxiv2023

The Shank/ProSAP N-terminal (SPN) domain of Shank3 regulates targeting to postsynaptic sites and postsynaptic signalling.

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, CaMKII and cytoskeletal proteins. Here we analyse the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive CaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on CaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analysed in primary cultured neurons; here we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of dendritic spines. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signalling, and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.

neuroscience↗

bioRxiv2021

Tensin3 interaction with talin drives formationof fibronectin-associated fibrillar adhesions

The formation of healthy tissue involves continuous remodelling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here we examine how tensin3 contributes to formation of fibrillar adhesions (FBs) and fibronectin fibrillo-genesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for formation of 5{beta}1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.

cell biology↗

bioRxiv2021

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The relevance of missense mutations for these pathologies is unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (i) a loss of binding to {delta}-catenin (e.g. in the p.L270M variant), and (ii) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the -subunit of the calcium-/calmodulin dependent kinase II (CaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

neuroscience↗

bioRxiv2019

Force-independent interactions of talin and vinculin govern integrin-mediated mechanotransduction

Talin, vinculin and paxillin are core components of the dynamic link between integrins and actomyosin. Here we study the mechanisms that mediate their activation and association using a mitochondrial-targeting assay, structure-based mutants, and advanced microscopy. As expected, full-length vinculin and talin are auto-inhibited and do not interact with each other in this state. Contrary to previous models that propose a critical role for forces driving talin-vinculin association, our data show that force-independent relief of auto-inhibition is sufficient to mediate their tight interaction. Interestingly, paxillin can bind to both talin and vinculin when either is inactive. Further experiments demonstrate that adhesions containing paxillin and vinculin can form without talin following integrin activation. However, these are largely deficient in exerting traction forces to the matrix. Our observations lead to a model whereby paxillin contributes to talin and vinculin recruitment into nascent adhesions. Activation of the talin-vinculin axis subsequently leads to the engagement with the traction force-machinery and focal adhesion maturation.

cell biology↗

bioRxiv2018

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to the 2-O position of -L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.\n\nSUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

biochemistry↗