Biology subjects

Oxley, D.

Publications and source records attributed to Oxley, D..

11 recordsLinked to original sources

bioRxiv2026

PTPRF is a stress responsive cytoskeletal checkpoint that coordinates metabolic adaptation in hepatocytes and β cells

Cytoskeletal remodeling is essential for adaptation to nutrient availability, yet how cells coordinate actin dynamics with glucose homeostasis in metabolic organs remains unclear. Here, we identify a pathway linking metabolic stress to actin reorganization in hepatocytes and pancreatic {beta} cells. This mechanism involves transcriptional repression of the receptor protein tyrosine phosphatase PTPRF by spliced XBP1, a key unfolded protein response factor. In hepatocytes, PTPRF loss under dietary stress enhances insulin signaling, increases mitochondrial respiration and reduces steatosis. Proteomic analyses show that PTPRF interacts with regulators of actin polymerization and cell junctions, and its deletion promotes actin filament organization, shifting metabolism toward oxidative pathways. In {beta} cells, PTPRF deficiency similarly enhances actin polymerization and augments glucose-stimulated insulin secretion in obesity. Collectively, these findings place PTPRF as a nutrient-responsive regulator of cytoskeletal remodeling that coordinates hepatic metabolism and {beta}-cell function, highlighting its potential as a therapeutic target for improving systemic glucose control.

physiology↗

bioRxiv2026

Phosphoproteomics identifies the DYRK1B protein kinase as a regulator of processing bodies

Dual-specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) modulates the cell cycle, cell fate during development, and is deregulated in cancer and metabolic syndrome. However, only a few DYRK1B substrates have been defined, so we undertook a phosphoproteomics screen in cells that exhibit inducible DYRK1B expression. Motif analysis revealed enrichment for proline-directed serine or threonine phosphorylation sites (pSer/pThr-Pro), consistent with the consensus motif of class I DYRKs. Gene ontology analysis revealed enrichment of proteins involved in mRNA binding, mRNA processing and ribonucleoprotein complexes. Several processing body (PB) components, including DCP1A, PATL1(PAT1B), EDC3 and 4E-T, were identified as DYRK1B-inducible phosphoproteins. DYRK1B also co-immunoprecipitated with DCP1A, PAT1B, EDC3, EDC4, DDX6 and XRN1. Super-resolution microscopy demonstrated that DYRK1B co-localised with DCP1A, DCP1B and DDX6 in PBs. Activation of DYRK1B increased PB abundance, whereas inhibition, depletion or knockout of DYRK1B reduced phosphorylation of DCP1A and 4E-T and decreased PB number. Re-expression of wild type, but not kinase-dead, DYRK1B restored PB numbers in knockout cells. These findings reveal novel DYRK1B targets and establish DYRK1B as a regulator of processing body abundance. HighlightsO_LIDYRK1B induces phosphorylation of a cluster of RNA binding and processing body associated proteins. C_LIO_LIDYRK1B localises to PBs and associates with multiple PB components. C_LIO_LIDYRK1B controls P-body abundance in a kinase-dependent manner. C_LI

cell biology↗

bioRxiv2026

Loss of PTPRK in hepatocytes reduces steatosis and carcinogen-induced tumour development in obesity

Protein tyrosine phosphatases are crucial regulators of metabolism with specific roles in different tissues. To investigate hepatocyte-specific function of protein tyrosine phosphatase receptor type K (PTPRK), we generated mice carrying floxed Ptprk alleles and crossed them with Alb-Cre mice (Ptprk{Delta}Hep mice). Under chow feeding, Ptprk{Delta}Hepmice were largely comparable to littermate controls. In contrast, Ptprk{Delta}Hepmice fed a high-fat, high-fructose, high-cholesterol diet exhibited reduced steatosis, lower hepatic PPAR{gamma}, and blunted hepatocyte hypertrophy, accompanied by improved systemic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps. We identified PTPRK-interacting proteins enriched for metabolic functions associated with glycolysis and lipid biosynthesis using pull downs from primary hepatocyte lysates. In line with these findings, Ptprk{Delta}Hep mice developed fewer tumours than controls in an obesity and carcinogen-induced hepatocellular carcinoma (HCC) model. Our data show that under nutrient excess PTPRK is functionally engaged in hepatocytes to support PPAR{gamma}-linked steatotic growth, insulin resistance, and tumour initiation, highlighting PTPRK as a potential therapeutic target in MASLD-associated HCC.

pathology↗

bioRxiv2025

An Nlrp5-null mutation leads to attenuated de novo methylation in oocytes, accompanied by a significant reduction in DNMT3L

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5- null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

developmental biology↗

bioRxiv2024

Protein degradation shapes developmental tempo in mouse and human neural progenitors

The pace of embryonic development differs markedly across mammalian species, yet the molecular mechanisms underlying these tempo differences remain largely unknown. Here, we systematically compared protein dynamics in mouse and human neural progenitors (NPs) and examined how protein stability influences developmental timing. We find that mouse NPs exhibit faster protein production and degradation than human NPs. Human NPs display broadly increased protein half-lives, independent of cellular compartment or protein function, and this difference persists in post-mitotic neurons. Consistent with this, proteasomal activity is lower in human embryonic spinal cord and stem cell-derived neural progenitors than mouse, correlating with reduced expression of proteasome-associated proteins. Functionally, accelerating the degradation of the key transcriptional repressor IRX3 in mouse NPs speeds the activation of its target gene, providing causal evidence that protein turnover modulates developmental tempo. These results reveal that species-specific regulation of protein degradation shapes the timing of neural development and suggest that evolutionary tuning of proteasomal activity contributes to differences in embryonic developmental pace.

developmental biology↗

bioRxiv2024

Erosion of X-Chromosome Inactivation in female hiPSCs is heterogeneous and persists during differentiation

During culture, female human pluripotent stem cells (hPSCs), including human induced PSCs (hiPSCs) exhibit a propensity for erosion of X-chromosome inactivation (XCI). This phenomenon is characterized by the loss of XIST RNA expression and reactivation of a subset of X-linked genes from the inactive X chromosome (Xi). XCI erosion, despite its common occurrence, is often overlooked by the stem cell community, hindering a complete understanding of its impact on both fundamental and translational applications of hiPSCs. Investigating erosion dynamics in female hiPSCs, our study reveals that XCI erosion is a frequent yet heterogeneous phenomenon, resulting in the reactivation of several X-linked genes. The likelihood of a gene to erode increases for those located on the short arm of the X chromosome and within H3K27me3-enriched domains. Paradoxically, genes that typically escape XCI are hypersensitive to loss of XIST RNA and XCI erosion. This implies that XIST RNA normally restrains expression levels of these genes on the Xi. Importantly, increased X-linked gene expression upon erosion does not globally impact (hydroxy)methylation levels in hiPSCs or at imprinted regions. By exploring diverse differentiation paradigms, such as trilineage commitment and cardiac differentiation, our study reveals the persistence of abnormal XCI patterns throughout differentiation. This finding has significant implications for fundamental research, translational applications, and clinical use of stem cells. We underscore the importance of raising awareness within the stem cell community regarding XCI erosion and advocate for its inclusion in comprehensive hiPSC quality control.

molecular biology↗

bioRxiv2023

A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV (PADI4) regulation

Peptidylarginine deiminase IV (PADI4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. Genetic or pharmacological PADI4 inhibition is therapeutically efficacious in mice, but no clinically relevant inhibitors currently exist. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to lack of appropriate systems and tools. Here, we developed and applied a set of potent and selective PADI4 modulators. Using the mRNA- display-based RaPID system, we screened >1012 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, an inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalised for the isolation and study of cellular PADI4; and PADI4_11, a first-in-class activator. Using a newly developed method for the quantification of cellular PADI4 activity, we show that PADI4_3 and PADI4_11 are effective in cells. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work offers new understanding of PADI4 regulation and a toolkit for the study and modulation of PADI4 across physiological and pathophysiological contexts.

biochemistry↗

bioRxiv2023

Stability of Genomic Imprinting and X-Chromosome Inactivation in the Aging Brain

Epigenetic drift is a hallmark of aging that contributes to the irreversible decline in organismal fitness ultimately leading to aging-related diseases. Epigenetic modifications regulate the cellular memory of the epigenetic processes of genomic imprinting and X-chromosome inactivation to ensure monoallelic expression of imprinted and X-linked genes. Whether epigenetic drift affects maintenance of genomic imprinting and X-chromosome inactivation has not been comprehensively studied. Here, we investigate the allele-specific transcriptional and epigenetic signatures of the aging brain, by comparing juvenile and old hybrid mice obtained from C57BL/6J (BL6) & CAST/EiJ (CAST) reciprocal crosses, with an emphasis on the hippocampus (HCP). We confirm that the aged HCP shows an increase of DNA hydroxymethylation, a sign of epigenetic drift, and a typical aging transcriptional signature. Genomic imprinting was found to be largely unaffected with stable parent-of-origin-specific DNA methylation in HCP, but also other brain regions such as the cerebellum (CB), nucleus accumbens, hypothalamus and prefrontal cortex. Consistently, transcriptomics analysis confirmed unaltered imprinting expression in the aged HCP. An exception are three novel non-coding transcripts (B230209E15Rik, Ube2nl and A330076H08Rik) at the Prader-Willi syndrome/Angelman syndrome (PWS/AS) imprinted locus which lose strict monoallelic expression during aging. Like imprinting, X-chromosome inactivation was remarkably stable with no signs of aging-driven skewing or relaxation of monoallelic expression of X-linked genes. Our study provides a valuable resource for evaluating monoallelic expression in the aging brain and reveals that, despite epigenetic drift during aging, genomic imprinting and X-chromosome inactivation remain predominantly stable throughout the process of physiological aging in the mouse brain.

molecular biology↗

bioRxiv2023

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2-phosphatase, PTEN. Despite huge investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signalling and constrained by pathway-feedback. In the absence of PTEN, the network is dramatically remodelled. A poorly understood, YXXM and PIP3/PI(3,4)P2-binding PH domain-containing, adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT-phosphorylation and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K and SRC-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1-mRNA and activating-Y419-phosphorylation of hSRC correlated with PI3K-pathway activity in human prostate cancers. We propose that in PTEN-null cells, receptor-independent, SRC-dependent tyrosine-phosphorylation of PLEKHS1 creates positive-feedback that escapes homeostasis, drives PIP3- signalling and supports tumour progression.

cell biology↗

bioRxiv2021

Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads

Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture proteins, with subsequently washes allowing contaminant removal. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in protein recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief centrifugation--either with or without low-cost inert glass beads--as the means of aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1-5000 {micro}g preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling (n = 9,076) also demonstrated improved recovery by SP4 and a significant enrichment of membrane and low-solubility proteins vs. SP3. The effectiveness of SP4 was verified in three other labs, each confirming equivalent or improved proteome characterisation over SP3. This work suggests that protein precipitation is the primary mechanism of SP3, and reliance on magnetic beads presents protein losses, especially at higher concentrations and amongst hydrophobic proteins. SP4 represents an efficient and effective alternative to SP3, provides the option to omit beads entirely, and offers virtually unlimited scalability of input and volume--all whilst retaining the speed and universality of SP3. BRIEFSolvent precipitation SP3 (SP4) captures aggregated protein for proteomics sample clean-up by omitting magnetic beads, instead employing brief centrifugation--with or without low-cost inert glass beads. SP4 offers improvements to protein yields, higher reproducibility, and greater recovery of membrane proteins, with verifications from three labs. Protein precipitation appears to be the primary mechanism of SP3, with reliance on magnetic beads presenting protein losses, especially at higher concentrations. SP4 presents an effective alternative to SP3 with improved scalability and equal speed and universality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/461247v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ba615corg.highwire.dtl.DTLVardef@14df25dorg.highwire.dtl.DTLVardef@62f0c1org.highwire.dtl.DTLVardef@1ac436a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Autophagy is a fundamental catabolic process essential for development, homeostasis and proper immune function 1. During autophagy, a cascade of ATG proteins target intracellular cargoes for lysosomal degradation and recycling 2. This pathway utilises a unique post-translational modification, the conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at autophagosomes, which modulates cargo selection and maturation. ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal compartments, which plays a key role in phagocytosis and other processes 3. It has been widely assumed that SMAC involves the same lipidation of ATG8 to PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes alternative lipidation to phosphatidylserine (PS) during non-canonical autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, by pharmacological agents 4,5, or during non-canonical autophagy processes such as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8, induces the covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-PE adducts are differentially delipidated by isoforms of the ATG4 family, indicating significant molecular distinctions and mechanisms between these two species. Together, these results provide an important new insight into autophagy signalling, revealing an alternative form of the hallmark ATG8-lipidation event, so widely used to define and assay autophagy. Furthermore, ATG8-PS lipidation provides a specific molecular signature for non-canonical autophagy, uncovering a novel means of detecting and monitoring this emerging pathway.

cell biology↗