Biology subjects

McDonald, N. Q.

Publications and source records attributed to McDonald, N. Q..

11 recordsLinked to original sources

bioRxiv2026

Discovery of a sulfotyrosine-motif in the human TrkB extracellular domain required for agonist activation

The brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signalling axis is a key effector of synaptic plasticity and neuroprotection. While TrkB activation is a major objective towards preventing dysfunction of the nervous system, it cannot be reached with exogenous BDNF administration given the unfavourable physiochemical properties of BDNF. In addition, BDNF also activates a tumour necrosis factor pathway by binding to the neurotrophin receptor p75. The TrkB agonist ZEB85 provides an alternative route to the selective activation of TrkB. We report here the structural basis for the interaction between human TrkB, and both ZEB85 and BDNF, and reveal that a sulfated tyrosine modification is indispensable for ZEB85 activation of TrkB signalling. Using structure-guided BDNF- and ZEB85-binding deficient TrkB mutants, we assessed their ability to sequester ligands from full-length TrkB in cultured human neurons. We found that the BDNF binding site extends into the extracellular juxtamembrane domain of TrkB but does not require the sulfotyrosine at residue 400 to activate TrkB. Together with biophysical analysis and AlphaFold modelling these results also explain how BDNF can displace ZEB85 from TrkB through an overlapping epitope. Our findings reveal unique features of TrkB, not present in the related neurotrophin receptors TrkA and TrkC, and suggest new directions to explore the role of sulfotyrosine in TrkB signalling and identify new TrkB-specific protein ligands. One Sentence SummaryInvestigation of the mechanism of action of TrkB agonist ZEB85 extends molecular understanding of TrkB activation.

biochemistry↗

bioRxiv2026

A long-chain heparan sulfate capture mechanism directs paracrine GDNF-GFRα1 signalling through RET

The receptor tyrosine kinase RET is activated by GDNF and GFR1 together as a bipartite ligand, driving receptor activation and signalling in developmental and neuroprotective contexts. Evidence from developmental and cell models has suggested that heparan sulfate (HS) functions as a fourth component in RET signalling by binding to GDNF, but the molecular details remain unclear. Here, we present the cryo-EM structure of the heterohexameric RET:GDNF:GFR1 complex with a fully resolved heparin ligand, revealing an unexpected extended HS binding site spanning all three proteins. The architecture of the complex and binding mode of the HS chain in this complex enables the formation of a higher order 4:4:4 assembly bound to a single 30-saccharide HS chain which bridges two intimately bound complexes. This multi-protein interface selectively binds the highly sulfated domains of HS over other GAG classes, and is essential for RET activation in trans with soluble GFR1, but not in cis when GFR1 is membrane-bound. Our data suggest that HS shapes the dynamics of RET signalling at every stage, from ligand diffusion to signalling complex formation. Thus, GDNF-GFRa1 paracrine signalling reveals a surprising dependence for long-chain GAG function in which the glycan engages in both receptor complex assembly and clustering.

biochemistry↗

bioRxiv2025

PKN2 regulates cell-junctions to limit colitis and colon tumour formation

Intestinal barrier failure is a defining feature of inflammatory bowel disease and a key driver of inflammation-associated colorectal cancer. However, the epithelial mechanisms that preserve barrier stability during inflammatory stress remain incompletely defined. Here, we identify the Rho-regulated kinase PKN2 as a non-redundant safeguard of tight junction integrity and a candidate tumour suppressor in colitis-associated colorectal cancer. Conditional PKN2 deletion in mice sensitised the colon to inflammatory injury and significantly increased adenoma formation in an inflammatory cancer model, with tumour burden tightly correlating with colitis severity. Mechanistically, PKN2 localises to tight junctions and is required to stabilise barrier integrity during injury in mouse and organoid models. PKN2 loss induces transcriptional programs conserved in human inflammatory bowel disease, where reduced PKN2 expression associates with increased disease severity and altered therapeutic response. Notably, PKN2 is encoded within genomic loci previously linked to intestinal inflammation susceptibility and tumour suppression in both humans and mice. Our findings identify PKN2 as a functional effector underlying these phenotypes and demonstrate that even heterozygous loss is sufficient to confer heightened sensitivity to inflammatory injury and tumour initiation, positioning PKN2 as a central regulator of tight junction stability that shapes disease severity, treatment response and cancer risk.

cell biology↗

bioRxiv2025

The penetrant chordoid glioma PRKCA mutation is an oncogenic gain-of-function kinase inactivation eliciting early onset chondrosarcoma in mice.

The penetrant PRKCA D463H mutation, a biomarker and potential driver in chordoid glioma, was found to provoke the development of chondrosarcomas in heterozygous knock-in mice. This mutation entirely abrogates kinase activity, but strikingly no oncogenic phenotype is observed for the related inactivating mutation D463N indicating that the lack of activity is not the driver. In cells, the D463H mutant closely mirrored PKC WT behaviours and retained ATP binding, contrary to the related D463N mutant. Mechanistically, the PKC D463H mutant protein was found to display quantitative alterations to the PKC interactome, enhancing association with epigenetic regulators. This aligned with transcriptomic changes which resembled an augmented PKC expression program, with enhanced BRD4, Myc and TGF{beta} signatures. D463H dependent reduced sensitivity to the BET inhibitors JQ1 and AZD5153 indicates the functional importance of these pathways. The data show that D463H is a dominant gain-of-function oncogenic mutant, operating through a non-catalytic allosteric mechanism. One Sentence SummaryA PKC catalytic inactivating mutation confers gain-of-function properties - a paradigm shift in kinase actions.

cancer biology↗

bioRxiv2024

Capture, mutual inhibition and release mechanism for aPKC-Par6 and its multi-site polarity substrate Lgl

The mutually antagonistic kinase-substrate relationship between the apical aPKC-Par6 heterodimer and the basolateral substrate Lgl is key to the establishment and maintenance of cell polarity across metazoa. Although aPKC-Par6 can phosphorylate Lgl at three serine sites to exclude it from the apical domain, paradoxically, aPKC-Par6 and Lgl can also form a stable kinase-substrate complex whose function remains unclear and with conflicting roles proposed for Par6. We report the structure of human aPKC{iota}-Par6 bound to full-length Llgl1, captured through an aPKC{iota} docking site and a Par6PDZ contact. This soluble tripartite complex traps a phospho-S663 Llgl1 intermediate bridging between aPKC and Par6, impeding phosphorylation progression. Thus, aPKC{iota} is effectively inhibited by Llgl1pS663 whilst Llgl1 is captured by aPKC{iota}-Par6. Mutational disruption of Lgl-aPKC interaction impedes complex assembly and Lgl phosphorylation, whereas disrupting the Lgl-Par6PDZ contact promotes complex dissociation and completion of Lgl phosphorylation cycle. We incorporate these findings into a Par6PDZ-regulated substrate capture-and-release model that we demonstrate requires binding by Cdc42-GTP and the apical partner Crumbs to drive complex disassembly. Our results provide an explanation for the opposing roles of Par6 underpinning the spatial control of aPKC-Par6 activity by Lgl relevant to polarised membrane contexts across multi-cellular organisms.

biochemistry↗

bioRxiv2023

Optimized dimerization of the PAR-2 RING domain drives cooperative and selective membrane recruitment for robust feedback-driven cell polarization

The behavior of cell polarity networks is defined by the quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues that arise during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by dimerisation of the polarity protein PAR-2 via ubiquitin-independent function of its N-terminal RING domain. Specifically, we combine theory and experiment to show that dimer affinity is optimized to achieve dynamic, selective, and cooperative recruitment of PAR-2 to the plasma membrane during polarization. Reducing dimerization results in loss of positive feedback and compromises robustness of symmetry-breaking, while enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to membrane displacement by the polarity kinase, aPKC/PKC-3. Thus, our data reveal how a dynamically oligomeric RING domain results in a cell polarity network that is both robust and responsive and highlight how tuning of oligomerization kinetics can serve as a general strategy for optimizing dynamic and cooperative intracellular targeting.

cell biology↗

bioRxiv2023

Control of atypical PKC{iota} membrane dissociation by tyrosine phosphorylation within a PB1-C1 interdomain interface

Atypical PKCs are cell polarity kinases that operate at the plasma membrane where they function within multiple molecular complexes to contribute to the establishment and maintenance of polarity. In contrast to the classical and novel PKCs, atypical PKCs do not respond to diacylglycerol cues to bind the membrane compartment. Until recently it was not clear how aPKCs are recruited; whether aPKCs can directly interact with membranes or whether they are dependent on other protein interactors to do so. Two recent studies identified the pseudo-substrate region and the C1 domain as direct membrane interaction modules, however their relative importance and coupling are unknown. We combined molecular modelling and functional assays to show that the regulatory module of aPKC{iota}, comprising the PB1 pseudo-substrate and C1 domains, forms a cooperative and spatially continuous invariant membrane interaction platform. Furthermore, we show the coordinated orientation of membrane-binding elements within the regulatory module requires a key PB1-C1 interfacial {beta}-strand (BSL). We show this element contains a highly conserved Tyr residue that can be phosphorylated and that negatively regulates the integrity of the regulatory module, leading to membrane release. We thus expose a novel regulatory mechanism of aPKC{iota} membrane binding and release during cell polarization.

biochemistry↗

bioRxiv2022

Bimodal regulation of axonal transport by the GDNF-RET signalling axis in healthy and diseased motor neurons

Deficits in axonal transport are one of the earliest pathological outcomes in several models of amyotrophic lateral sclerosis (ALS), including SOD1G93A mice. Evidence suggests that rescuing these deficits prevents disease progression, stops denervation, and extends survival. Kinase inhibitors have been previously identified as transport enhancers, and are being investigated as potential therapies for ALS. For example, inhibitors of p38 mitogen-activated protein kinase and insulin growth factor receptor 1 have been shown to rescue axonal transport deficits in vivo in symptomatic SOD1G93A mice. In this work, we investigated the impact of RET, the tyrosine kinase receptor for glial cell-line-derived neurotrophic factor (GDNF), as a modifier of axonal transport. We identified fundamental interplay between RET signalling and axonal transport in both wild type and SOD1G93A motor neurons in vitro. We demonstrated that blockade of RET signalling using pharmacological inhibitors and genetic knockdown enhances signalling endosome transport in wild type motor neurons and uncovered a divergence in the response of primary motor neurons to GDNF compared with cell lines. Finally, we demonstrated that inhibition of the GDNF-RET signalling axis rescues in vivo transport deficits in early symptomatic SOD1G93A mice, promoting RET as a potential therapeutic target in the treatment of ALS.

neuroscience↗

bioRxiv2021

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria

Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each others functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.

neuroscience↗

bioRxiv2020

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFR co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFR1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFR co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFR1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands. HighlightsO_LICrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interface C_LIO_LIComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loop C_LIO_LIStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimer C_LIO_LIDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms C_LI

biophysics↗

bioRxiv2019

Cryo-EM Structures of the XPF-ERCC1 Endonuclease Reveal an Auto-Inhibited Conformation and the Basis for Activation

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. We report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks ERCC1 DNA-binding elements and restricts access to the XPF catalytic site. Binding of a model DNA junction separates the XPF helical and ERCC1 (HhH)2 domains, promoting activation. Using these structural data, we propose a model for a 5-NER incision complex involving XPF-ERCC1-XPA and a DNA junction substrate. Structure-function data suggest xeroderma pigmentosum patient mutations often compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.

molecular biology↗