Biology subjects

Scott, D. J.

Publications and source records attributed to Scott, D. J..

8 recordsLinked to original sources

bioRxiv2025

ASAP-ID: Proximity labelling with small tags

Biotinylation-based proximity labelling methods are valuable for discovering protein-protein interactions within cellular systems. However, one limitation of these approaches is that most require fusing the target protein with the enzyme that biotinylates nearby proteins (i.e., TurboID or APEX2), which risks sterically disrupting the proteins native function. Here, we present a method designed to reduce the steric impact of these fusions and offer greater flexibility in labelling modalities. The method, Antibody and Small-tag Assembly on Proteins for Interaction Detection (ASAP-ID), involves a bipartite system. Target proteins are fused to a small peptide antigen that recruits TurboID or APEX2 fused to an antibody directed to the antigen. Using two different antigen/antibody systems (SunTag and MoonTag), we show that ASAP-ID can specifically label human Lamin A in cells. The method works when the target protein and nanobody are co-expressed together in cis (ASAP-IDIC). We also demonstrate that the approach works when the antibody fusion is added in trans to fixed cells post-expression (ASAP-IDIT). ASAP-IDIT identified more than 448 known and previously undescribed interactors of lamin. We further used ASAP-IDIT to study how ALS-mutant profilin 1 affected its interactome. The method identified proteins involved in protein quality control that correlated with aggregation propensity. Moreover, the different mutants showed variation in the cellular location where aggregates formed. ASAP-IDIT revealed preferences for mitochondrial proteins for the two profilin mutants that tend to aggregate in the cytoplasm, C71G and M114T, and nuclear proteins for a mutant more prone to nuclear aggregation. These findings position ASAP-ID as a powerful addition to the proximity labelling toolkit, capable of probing subtle differences in interactomes in a less invasive manner.

biochemistry↗

bioRxiv2023

Defining the mechanism of galectin-3-mediated TGF-β1 activation and its role in lung fibrosis.

Integrin-mediated activation of the pro-fibrotic mediator transforming growth factor-{beta}1 (TGF-{beta}1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF individuals, although its mechanism of action is not precisely defined. We hypothesised that galectin-3 potentiates TGF-{beta}1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-{beta}1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L--lysophosphatidic acid sodium salt (LPA)-induced integrin-mediated TGF-{beta}1 activation. Surface plasmon resonance (SPR) analysis confirmed that galectin-3 binds to the v integrins, v{beta}1, v{beta}S and v{beta}6 and also to the TGF{beta}RII subunit in a glycosylation-dependent manner. This galectin-3 binding is heterogeneous and not a 1:1 binding stoichiometry. These binding interactions were blocked by small molecule inhibitors of galectin-3 which target the carbohydrate recognition domain. Binding of galectin-3 to the {beta}1 integrin was validated in vitro by co-immunoprecipitation (Co-IP) in HLFs. In addition, proximity ligation assay (PLA) data indicates that galectin-3 and the {beta}1 integrin colocalize closely (40 nm) on the cell surface of HLFs, that colocalization is increased by TGF-{beta}1 treatment and galectin-3 inhibitors prevented this colocalization. In the absence of TGF-{beta}1 stimulation, such colocalization was detectable only in HLFs isolated from IPF patients suggesting that the proteins are inherently more closely associated in the disease state. Taken together, this data suggests that galectin-3 promotes TGF-{beta}1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-{beta}1 signaling cascade. Declaration of InterestsRGJ reports grants or contracts from AstraZeneca, Biogen, Galecto Biotech, GlaxoSmithKline, Nordic Biosciences, RedX, Plaint, consulting fees from Bristol Myers Squibb, Chiesi, Daewoong Veracyre, Resolution Therapeutics and Pliant, honoraria from Boehringer Ingelheim, Chiesi, Roche, PatientMPower, AstraZeneca, advisory roles with Boehringer Ingelheim, Galapagos and Vicore, non-financial support from NuMedii, and is a Trustee for Action for Pulmonary Fibrosis. AEJ reports grant funding from Galecto Biotech. BG reports CASE student project partnership with Galecto Biotech, honoraria from GlaxoSmithKline and Vertex and grants and fellowships from UKRI MRC and BBSRC, Alpha-1 Foundation, BLF/A+LUK, Wellcome Trust. RJS, ACM, FRZ, JFC are Galecto employees with shares/options in the company. NRP is a Roche employee. GH, RML, PS, SBC, DJS declare no competing interests.

molecular biology↗

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗

bioRxiv2022

Ligands tune the local and global motions of neurotensin receptor 1 (NTS1): a DFT-guided solution NMR analysis

Unlike many signaling proteins that function as binary switches between on and off states, G protein-coupled receptors (GPCRs) exhibit basal activity that can be increased or decreased by numerous ligands. A given receptor can recognize multiple ligands, allosteric modulators, and transducers to create a complex free energy landscape. Many of the lowest energy states have been captured by static structural techniques while detailing the wells widths, metastable states, and the transition between them, is still in its infancy. Nuclear magnetic resonance (NMR) spectroscopy can monitor the structure and dynamics of GPCR ensembles across fifteen orders-of-magnitude, but technical challenges have limited its application to super-microsecond timescales. Focusing on a prototypical peptide-binding GPCR, the neurotensin receptor 1 (NTS1), we employed NMR and density functional theory (DFT) to probe global sub-nanosecond motions. The near random coil chemical shifts of the apo receptor produced a poor correlation with theoretical predictions that may indicate a high degree of conformational averaging in solution, a crystallization artifact, or both. Whereas orthosteric agonists and antagonists both rigidified the receptor, but to varying degrees, which suggests conformational entropy differentially contributes to their respective pharmacology. The strong correlations of observed and theoretical chemical shifts lend confidence to interpreting spectra in terms of local structure, methyl dihedral angle geometry, and pico-second timescale transitions. Together, our results suggest a role for sub-nanosecond dynamics and conformational entropy in GPCR ligand discrimination.

biophysics↗

bioRxiv2021

Structural insights into the modes of relaxin-binding and tethered-agonist activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signalling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.

biophysics↗

bioRxiv2019

α1A-adrenoceptor inverse agonists and agonists modulate receptor signalling through a conformational selection mechanism

G-Protein Coupled Receptors (GPCRs) transmit signals across the cell membrane via an allosteric network from the ligand-binding site to the G-protein binding site via a series of conserved microswitches. Crystal structures of GPCRs provide snapshots of inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here we analyse the correlation between ligand binding and receptor conformation of the 1A-adrenoceptor, known for stimulating smooth muscle contraction in response to binding noradrenaline. NMR of 13C{varepsilon}H3-methionine labelled 1A-adrenoreceptor mutants, each exhibiting differing signalling capacities, revealed how different classes of ligands modulate receptor conformational equilibria. 13C{varepsilon}H3-methionine residues near the microswitches revealed distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling between the microswitches controls receptor conformation and underlies the mechanism of ligand modulation of GPCR signalling in cells.

biochemistry↗

bioRxiv2019

Structure-guided enhancement of selectivity of chemical probe inhibitors targeting bacterial seryl-tRNA synthetase

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

biochemistry↗

bioRxiv2017

Reconstructing Promoter Activity From Lux Bioluminescent Reporters

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for slaughter. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a modelling approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

bioinformatics↗