Biology subjects

Evans, A. J.

Publications and source records attributed to Evans, A. J..

8 recordsLinked to original sources

bioRxiv2024

Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation.

Endosomal retrieval and recycling of integral cargo proteins is essential for cell, tissue and organism-level development and homeostasis and is orchestrated through a specialised retrieval sub-domain on the endosomal vacuole. However, although sub-domain dysfunction is associated with human disease our appreciation of the molecular details and functional components of the retrieval sub-domain(s) remains poorly described. Here, using comparative proximity proteomics of critical retrieval sub-domain components Retromer and Retriever, their cargo adaptors, and a component of the opposing ESCRT-degradative sub-domain, we provide a data-rich resource that identifies new molecular details of retrieval sub-domain composition and organization, including an unrecognised complexity in the interface of Retromer with RAB GTPases. Combining X-ray crystallography and in silico predictions with extensive biochemical and cellular analysis, we dissect the direct association of Retromer with RAB10 regulators DENND4A, DENND4C, TBC1D1, and TBC1D4, and the RAB35 regulator TBC1D13. Overall, we conclude that the Retromer retrieval sub-domain constitutes a major hub for the regulated switching of selected RAB GTPases and propose that this constitutes a major component of the role of Retromer in neuroprotection.

cell biology↗

bioRxiv2023

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression.

Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis. Significance StatementPost-translational modifications (PTMs) are biochemical switches that control substrate protein properties and interactions. In consequence, PTMs are critical regulators of essentially all cellular pathways and are vital for eukaryotic cell survival. In the brain, among other roles, PTMs influence neuronal growth, differentiation, synaptic activity and plasticity. Kainate receptors (KARs) play core roles in all these processes and previous work has shown that the GluK2 subunit of KARs is subject to multiple PTMs. Here, using GluK2 as an exemplar protein, we delineate the sequence, coordination, and consequences of the PTMs palmitoylation, phosphorylation and SUMOylation on KAR surface expression. Our data show how the complex interplay between PTMs dynamically regulates synaptic proteins and neuronal function.

neuroscience↗

bioRxiv2022

Bio-Aurac an open-source browser plugin to better navigate literature content.

SummaryBio-Aurac is an open-source web browser plugin designed to support the research community in drug discovery and life sciences. The tool has been designed to help researchers, scientists, and curators to better explore, navigate and understand content from literature accessing valuable integrated information from third party resources. It identifies, highlights, and provides additionally knowledge for names of biochemical entities like genes and proteins. Availability and ImplementationBio-Aurac has been implemented using a microservice architecture which is open-source and abstracted from non-technical users by utilizing Docker containers (Nust, 2020). It can be run with minimal prerequisites on both Chrome and Firefox browsers. The code for installing and running the web browser plugin can be found here: https://github.com/mdcatapult/bio-aurac (Along with detailed installation instructions). A comprehensive collection of all the code involved in building this open-source project can be found: https://github.com/mdcatapult/aurac-web-plugin, https://github.com/mdcatapult/PDF-Converter, https://github.com/mdcatapult/entity-recognition ContactSoftwareEngineering@md.catapult.org.uk, ian.dunlop@md.catapult.org.uk

biochemistry↗

bioRxiv2021

Mechanistic basis for SNX27-Retromer coupling to ESCPE-1 in promoting endosomal cargo recycling.

Sorting nexin-27 (SNX27)-Retromer is an endosomal sorting complex that orchestrates endosome-to-plasma membrane recycling of hundreds of internalized receptors, channels and transporters, enzymes and adhesion molecules. While SNX27-Retromer is essential for development, subtle functional defects are observed in human disease, most notably neurodegenerative and neurological disorders. Achieving a thorough mechanistic dissection of SNX27-Retromer is central to understanding endosomal sorting in health and disease. Here we combine biochemical, structural and cellular analyses to establish the mechanistic basis through which SNX27-Retromer couples to the membrane tubulating ESCPE-1 complex (Endosomal SNX-BAR sorting complex for promoting exit 1). We show that a conserved surface in the FERM (4.1/ezrin/radixin/moesin) domain of SNX27 directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the disordered amino-termini of the SNX1 and SNX2 subunits of ESCPE-1. This interaction hands-over SNX27-Retromer captured integral membrane proteins into ESCPE-1 tubular profiles to promote their cell surface recycling. Through phylogenetic analysis, we reveal that SNX27:Retromer:ESCPE-1 assembly evolved in a stepwise manner during the early evolution of metazoans, which reflects the increasing complexity of endosomal sorting from the ancestral opisthokont to modern animals.

cell biology↗

bioRxiv2020

Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2

The endosome-associated cargo adaptor sorting nexin-27 (SNX27) is linked to various neuropathologies through sorting of integral proteins to the synaptic surface, most notably AMPA receptors. To provide a broader view of SNX27-associated pathologies we have performed unbiased proteomics to identify new neuronal SNX27-dependent cargoes, and identified proteins linked to excitotoxicity (SLC1A3, SLC4A7, SLC6A11), epilepsy, intellectual disabilities and working memory deficits (KCNT2, ADAM22, KIDINS220, LRFN2). Focusing on the synaptic adhesion molecule leucine-rich repeat and fibronectin type-III domain-containing protein 2 (LRFN2), we establish that SNX27 binds to LRFN2 and is responsible for regulating its endosomal sorting. LRFN2 associates with AMPA receptors and knockdown of LRFN2 phenocopies SNX27 depletion in decreasing surface expression of AMPA receptors, reducing synaptic activity and attenuating hippocampal long-term potentiation. Our evidence suggests that, in contrast to previous reports, SNX27 does not directly bind to AMPA receptors, and instead controls AMPA receptor-mediated synaptic transmission and plasticity indirectly through the endosomal sorting of LRFN2. Overall, our study provides new molecular insight into the perturbed function of SNX27 and LRFN2 in a range of neurological conditions.

molecular biology↗

bioRxiv2019

Acute perturbation of retromer and ESCPE-1 leads to functionally distinct and temporally resolved defects in endosomal cargo sorting.

The human retromer is a stable heterotrimer of VPS35, VPS29 and VPS26 whose principal role is to orchestrate the endosomal retrieval of hundreds of internalised cargo and promote their recycling to the cell surface; a prototypical cargo being the glucose transporter GLUT1. Retromers role in a distinct endosomal retrieval pathway, the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network (TGN), remains controversial. Here we have developed and applied knocksideways to acutely inactivate retromer and by visualising the sorting of endogenous GLUT1 and CI-MPR provide insight into the temporal dynamics of endosomal cargo sorting in HeLa and H4 human neuroglioma cells. While retromer knocksideways led to the development of time-resolved defects in cell surface sorting of GLUT1 we failed to observe defects in the sorting of the CI-MPR. In contrast knocksideways of ESCPE-1, a key regulator of retrograde CI-MPR sorting, resulted in a time-resolved defect in CI-MPR sorting. Together these data provide independent evidence consistent with a comparatively limited role for retromer in ESCPE-1 dependent CI-MPR retrograde sorting in HeLa and H4 human neuroglioma cells.

cell biology↗

bioRxiv2018

The C-terminal Helix 9 motif regulates cannabinoid receptor type 1 trafficking and surface expression

Cannabinoid type 1 receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminal and involves the Helix 9 (H9) domain. Removal of the H9 domain increases dendrite secretory pathway delivery and decreases in surface stability. Furthermore, CB1R{Delta}H9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

neuroscience↗

bioRxiv2018

ADAR2 mediated Q/R editing of GluK2 regulates homeostatic plasticity of kainate receptors

Kainate receptors (KARs) are heteromeric glutamate-gated ion channels that regulate neuronal excitability and network function in the brain. Most KARs contain the subunit GluK2 and the precise properties of these GluK2-containing KARs are determined by additional factors including ADAR2-mediated mRNA editing of a single codon that changes a genomically encoded glutamine (Q) to arginine (R) in the pore-lining region of GluK2. ADAR2-dependent Q/R editing of GluK2 is dynamically regulated during homeostatic plasticity (scaling) elicited by suppression of synaptic activity with TTX. Here we show that TTX decreases levels of ADAR2 by enhancing its proteasomal degradation. This selectively reduces the numbers of GluK2 subunits that are edited and increases the surface expression of GluK2-containing KARs. Furthermore, we show that partial ADAR2 knockdown phenocopies and occludes TTX-induced scaling of KARs. These data indicate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing provides a mechanism for KAR homeostatic plasticity.

neuroscience↗