Biology subjects

Skidmore, J.

Publications and source records attributed to Skidmore, J..

9 recordsLinked to original sources

bioRxiv2026

Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration

Autophagy and mitophagy are essential cellular processes implicated in neurodegenerative diseases, yet few tractable targets have been robustly validated for therapeutic modulation of these pathways. In an industry-academia consortium we carried out an extensive literature review and expert curation, leading to selection of 29 genes previously reported to enhance autophagy upon genetic or pharmacological modulation. These genes were classified based on whether downregulation or overexpression induced autophagic activity. Using siRNA knockdown, small-molecule modulators, and transient transfection approaches, we systematically screened these targets in parallel across HeLa and HEK-293 cell lines using high- and low-content phenotypic imaging. Promising candidates were further evaluated in induced pluripotent stem cell (iPSC)-derived neurons and in Drosophila models. Three targets emerged as top candidates: HUWE1 downregulation consistently enhanced autophagy and aggregate clearance, while USP33 downregulation promoted mitophagy. We also identified USP20, a close homologue of USP33, as an inhibitor of mitophagy, a role not previously assigned to this protein. These effects on autophagy/mitophagy were corroborated in Drosophila, highlighting their potential functional relevance across species. This cross-platform, multi-laboratory study establishes a framework for identifying and validating regulators of autophagy and mitophagy and provides a route which enables a comprehensive reassessment of the current target literature in this field. The validation of HUWE1, USP20, and USP33 as candidate therapeutic targets offers new opportunities for intervention in neurodegenerative diseases.

neuroscience↗

bioRxiv2026

Resolving the Activation Mechanism of the Human 20S Proteasome

Proteasome activators (PAs) bind the -subunits of the human 20S proteasome (h20S), opening the "gates" and allowing entry of substrate proteins. Aging is associated with diminished proteasome activity, leading to interest in understanding this activation mechanism. Evolving models have been proposed regarding PAs C-terminal tails, yet the critical molecular contacts for gate-opening are unclear. Here, we show a conserved leucine in the 5th position (P5) of the C-terminus is essential for h20S gate opening. By engineering C-termini in a model activator, PA26E102A, we show mutations to P5 systematically modulate proteasome activity in vitro and in cells. Structures of PA26E102A:h20S complexes at 2.7-3.2 [A] resolution identify interactions between P5 and a conserved arginine in the h20S, leading to partial or full gate opening. These results clarify the essential contacts required for h20S gate opening, potentially enabling design of proteasome activation therapies.

biophysics↗

bioRxiv2025

Novel Predictive Spatial Biomarker in Non-Small Cell Lung Carcinoma: The Diversity of Niches Unlocking Treatment Sensitivity (DONUTS)

Probabilistic spatial modelling techniques developed on large-scale tumor-immune Atlases ([~]35M individually mapped cells; 50,000 high power fields) were used to characterize predictive features of treatment-responsive lung cancer. We identified CD8+FoxP3+ cell density as a robust pre-treatment biomarker for outcomes across disease stages and therapy types. In parallel, single-cell RNAseq studies of CD8+FoxP3+ T-cells revealed an activated, early effector phenotype, substantiating an anti-tumor role, and contrasting with CD4+FoxP3+ T-regulatory cells. A spatial biomarker was developed using an empirical probabilistic model to define the immediate cell neighbors or niche surrounding CD8+FoxP3+ cells and proximity to the tumor-stromal boundary. The resultant Diversity of Niches Unlocking Treatment Sensitivity (DONUTS) are more prevalent than the CD8+FoxP3+ cells themselves, mitigating sampling error in small biopsies. Further, the DONUTS only require four markers, are additive to PD-L1, and associate with tertiary lymphoid structure counts. Taken together, the DONUTS represent a next-generation predictive biomarker poised for clinical implementation. HIGHLIGHTSO_LILarge-scale tumor-immune Atlases drive robust computational biomarker development C_LIO_LICD8+FoxP3+ cells are anti-tumor T-cells and predict response to therapy C_LIO_LIThe niches or spatial donuts around CD8+FoxP3+ cells boost biomarker performance C_LIO_LICD8+FoxP3+ donuts are hallmarks of a larger immune organization that includes TLS C_LI

pathology↗

bioRxiv2025

A GCN1-independent activator of the kinase GCN2

Mutations of EIF2AK4, which encodes the eIF2 kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

cell biology↗

bioRxiv2024

Antibody-dependent Intracellular neutralisation by TRIM21 is potentiated by HOIP and linear ubiquitin chains

Antibody-dependent intracellular neutralisation (ADIN) promotes the rapid proteasomal degradation of viruses and other large substrates in the cytosol. It is dependent on detection of intracellular virus-bound antibodies by the Fc receptor and E3 ligase TRIM21, followed by disassembly of the virus by the unfoldase VCP/p97. It is not known how VCP is recruited to TRIM21. We performed a limited siRNA knock-down screen of known VCP adaptors to determine their involvement in ADIN. Knock-down of HOIP, the only ubiquitin E3 ligase capable of generating linear ubiquitin chains, resulted in impaired virus neutralisation. HOIPIN-8, a HOIP inhibitor, showed concentration-dependent reduction in virus neutralisation. We found that the activity of HOIP in ADIN is dependent on its ubiquitin binding domains and its PUB domain which recruits VCP. Knock-down of OTULIN, the only deubiquitinating enzyme that exclusively cleaves linear ubiquitin chains, potentiated neutralisation of the virus. Our results expand the role of HOIP and linear ubiquitin chains in proteostasis.

immunology↗

bioRxiv2024

LeGenD: determining N-glycoprofiles using an explainable AI-leveraged model with lectin profiling

Glycosylation affects many vital functions of organisms. Therefore, its surveillance is critical from basic science to biotechnology, including biopharmaceutical development and clinical diagnostics. However, conventional glycan structure analysis faces challenges with throughput and cost. Lectins offer an alternative approach for analyzing glycans, but they only provide glycan epitopes and not full glycan structure information. To overcome these limitations, we developed LeGenD, a lectin and AI-based approach to predict N-glycan structures and determine their relative abundance in purified proteins based on lectin-binding patterns. We trained the LeGenD model using 309 glycoprofiles from 10 recombinant proteins, produced in 30 glycoengineered CHO cell lines. Our approach accurately reconstructed experimentally-measured N-glycoprofiles of bovine Fetuin B and IgG from human sera. Explanatory AI analysis with SHapley Additive exPlanations (SHAP) helped identify the critical lectins for glycoprofile predictions. Our LeGenD approach thus presents an alternative approach for N-glycan analysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/587044v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11687e4org.highwire.dtl.DTLVardef@33b146org.highwire.dtl.DTLVardef@1bb6bcborg.highwire.dtl.DTLVardef@1a1e47e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Tau phosphorylated at serine 356 is associated with Alzheimer's disease pathology and can be lowered in mouse and human brain tissue using the NUAK inhibitor WZ4003

Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK) related enzyme NUAK1 been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimers disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD post-mortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype specific effects, we found that WZ4003 results in a culture-phase dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease.

neuroscience↗

bioRxiv2023

Selective inhibitors of the Aurora A-TPX2 protein-protein interaction exhibit in vivo efficacy as targeted anti-mitotic agents

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. Our biophysically, structurally and phenotypically validated lead compound, CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells, inhibition of Aurora A-TPX2 PPI synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

biochemistry↗

bioRxiv2020

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer biology↗