Biology subjects

Healy, M.

Publications and source records attributed to Healy, M..

7 recordsLinked to original sources

bioRxiv2026

The GTPase activating protein Gyp6 binds Retromer and inactivates Rab7/Ypt7 to coordinate the formation of endosomal carriers

The Retromer coat is conserved in all eukaryotes and is crucial for the correct intracellular sorting of many transmembrane receptors and lysosomal hydrolases. Retromer is an effector of the late endosomal small GTPase RAB7 and is also implicated in its inactivation required for proper endosomal maturation. Here, we explore the role of controlled GTP hydrolysis by the RAB7 ortholog Ypt7 in the formation of Retromer-coated membrane carriers in yeast. Proximity labelling and genetic ablation identify the GTPase Activating Protein (GAP) Gyp6 as a critical regulator of Ypt7 activity in the context or Retromer. Structural studies show that Retromer recruits Gyp6 through its Vps29 subunit, which recognises a specific PL motif and a secondary binding site in the C-terminal domain of Gyp6. This interaction does not occur with other yeast GAPs. Ablation of the Gyp6-Retromer interface or the catalytic activity of Gyp6 leads to the accumulation of tubular structures on endo-lysosomal compartments and to increased association of Ypt7 with Retromer and its cargo Vps10. These results support a model in which Gyp6 controls the switch from Ypt7-dependent Retromer coat assembly and cargo collection to the departure of the carrier through membrane fission and uncoating.

cell biology↗

bioRxiv2025

Extrachromosomal circular DNA promotes inflammation and hepatocellular carcinoma development

Two decades after the initial report on increased micronuclei in human chronic liver disease (CLD) and hepatocellular carcinoma (HCC), their role in HCC development is still poorly understood. Here we show that micronuclei in hepatocytes trigger hepatic immune response and promote HCC development via an increased level of extrachromosomal circular DNA (eccDNA). Livers from a CLD model (Mcl1{Delta}hep mice) show increased micronuclei in parallel to eccDNA. Circular sequencing confirms higher eccDNA levels in micronuclei compared to primary nuclei. We developed nuclei-segregated DNA fiber (NuSeF) assay to show that micronuclei are more susceptible to replication stress, showing increased replication fork slowing. By comparing different murine liver disease models, high eccDNA is correlated with increased tumor incidence. EccDNA is a strong immunostimulant and promotes a crosstalk between hepatocytes and immune cells through the cGAS-STING pathway. Deletion of Sting1 in Mcl1{Delta}hep mice reduces immune cell chemotaxis, as well as tumor incidence. Our findings suggest that eccDNA from micronuclei mediates inflammation-driven liver carcinogenesis in CLD.

cancer biology↗

bioRxiv2023

Mapping Structural and Dynamic Divergence Across the MBOAT family

Membrane Bound O-acyltransferases (MBOATs) are membrane embedded enzymes which catalyse acyl chain transfer to a diverse group of substrates including lipids, small-molecules and proteins. Recent MBOAT structures reveal a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain, hindering effective therapeutic targeting. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify specific lipid binding sites surrounding MBOAT reactant gates into the surrounding membrane. We use bioinformatics to reveal a conserved role for re-entrant loop-2 in stabilisation of the MBOAT fold and identify a key hydrogen bond involved in DGAT1 dimerisation. Finally, we predict differences in MBOAT core solvation and water gating properties across the family. These data are pertinent to the design of MBOAT specific inhibitors that encompass dynamic information within cellular mimetic environments.

biochemistry↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2020

An fMRI study of initiation and inhibition of manual responses in people who stutter

Developmental stuttering is a speech motor disorder characterised by difficulties initiating speech and frequent interruptions to the speech flow. Previous work suggests that people who stutter (PWS) have an overactive response suppression mechanism. Imaging studies of speech production in PWS consistently reveal greater activity of the right inferior frontal cortex, an area robustly implicated in inhibitory control of both manual and spoken responses. Here, we used a manual response version of the stop-signal task during fMRI to investigate neural differences related to response initiation and inhibition in PWS. Behaviourally, PWS were slower to respond to go stimuli than people who are typically fluent (PWTF), but there was no difference in stop-signal reaction time. Our fMRI results were consistent with these behavioural results. The fMRI analysis revealed the expected networks associated with manual response initiation and inhibition in both groups. However, all contrasts between the two groups were characterised by overactivity in PWS relative to PWTF. This overactivity was significantly different for the initiation of responses (i.e. the go trials) but not for response inhibition (i.e. the stop trials). One explanation of these results is that PWS are consistently in a heightened inhibition state, i.e. areas of the inhibition network are more active, generally. This interpretation is consistent with predictions from the global response suppression hypothesis.

neuroscience↗

bioRxiv2019

Single Cell Spatial Chromatin Analysis of Fixed Immunocytochemically Identified Neuronal Cells

Assays examining the open-chromatin landscape in single cells require isolation of the nucleus, resulting in the loss of spatial/microenvironment information. Here we describe CHEX-seq (CHromatin EXposed) for identifying single-stranded open-chromatin DNA regions in paraformaldehyde-fixed single cells. CHEX-seq uses light-activated DNA probes that binds to single-stranded DNA in open chromatin. In situ laser activation of the annealed probes 3-Lightning Terminator in selected cells permits the probe to act as a primer for in situ enzymatic copying of single-stranded DNA that is then sequenced. CHEX-seq is benchmarked with human K562 cells and its utility is demonstrated in dispersed primary mouse and human brain cells, and immunostained cells in mouse brain sections. Further, CHEX-seq queries the openness of mitochondrial DNA in single cells. Evaluation of an individual cells chromatin landscape in its tissue context enables \"spatial chromatin analysis\".\n\nOne Sentence SummaryA new method, CHEX-seq (CHromatin eXposed), identifies the open-chromatin landscape in single fixed cells thereby allowing spatial chromatin analysis of selected cells in complex cellular environments.

neuroscience↗

bioRxiv2019

Genetic and functional evidence relates a missense variant in B4GALT1 to lower LDL-C and fibrinogen

Increased LDL-cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease (CVD). We identified novel associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.5 mg/dl lower LDL-C (p=1.6E-15), and 26 mg/dl lower plasma fibrinogen (p= 9.8E-05). N-linked glycan profiling found p.Asn352Ser to be associated (p-values from 1.4E-06 to 1.0E-17) with decreased glycosylation of glycoproteins including: fibrinogen, ApoB100, immunoglobulin G (IgG), and transferrin. In vitro assays found that the mutant (352Ser) protein had 50% lower galactosyltransferase activity compared to wild type (352Asn) protein. Knockdown of b4galt1 in zebrafish embryos resulted in significantly lower LDL-C compared to control, which was fully rescued by co-expression of 352Asn human B4GALT1 mRNA but only partially rescued by co-expression of 352Ser human B4GALT1 mRNA. Our findings establish B4GALT1 as a novel gene associated with lower LDL-C and fibrinogen and suggest that targeted modulation of protein glycosylation may represent a therapeutic approach to decrease CVD risk.

genetics↗