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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 (Nüst, 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-recognitionContactSoftwareEngineering@md.catapult.org.uk, ian.dunlop@md.catapult.org.uk

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27–Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27–Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

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.

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.

Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans-Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex – a key regulator of retrograde CI-MPR sorting – revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting., Summary: Retromer, a master controller of endosomal cargo sorting, is deregulated in neurodegenerative disease. Here, we develop and apply a retromer knocksideways methodology to quantify endosomal cargo sorting upon acute perturbation.

Biomarkers predicting fingolimod (FTY) treatment response in relapsing-remitting multiple sclerosis (RRMS) are lacking. Here, we performed extensive functional immunophenotyping using multiparametric flow cytometry to examine peripheral immune changes under FTY treatment and explore biomarkers of FTY treatment response. From among 135 RRMS patients who initiated FTY in a 2-year multicentre observational study, 36 were classified as ‘Active’ or ‘Stable’ based on clinical and/or radiological activity on-treatment. Flow cytometric analysis of immune cell subsets was performed on pre- and on-treatment peripheral blood mononuclear cells (PBMC) samples. Decreased absolute counts of B cells and most T-cell subsets were seen on-treatment. Senescent CD8 + T cells, CD56 + T cells, CD56dim natural killer cells, monocytes and dendritic cells were not reduced in number and hence relatively increased in frequency on-treatment. An unbiased multiparametric and traditional manual analysis of T-cell subsets suggested a higher pre-treatment frequency of CD4 + central memory T cells (TCM) in patients who were subsequently Active versus Stable on-treatment. Lower pre-treatment terminally differentiated effector memory (TEMRA) cell frequencies were also seen in the subsequently Active cohort. Together, our data highlight differential effects of FTY on peripheral immune cell subsets and suggest that pre-treatment T-cell subset frequencies may have value in predicting FTY treatment response.

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. Retromer’s 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.

Cannabinoid type one 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 in cultured rat hippocampal neurons. 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-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 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., eLife digest The brain contains around 100 billion neurons that are in constant communication with one another. Each consists of a cell body, plus two components specialized for exchanging information. These are the axon, which delivers information, and the dendrites, which receive it. This exchange takes place at contact points between neurons called synapses. To send a message, a neuron releases chemicals called neurotransmitters from its axon terminals into the synapse. The neurotransmitters cross the synapse and bind to receptor proteins on the dendrites of another neuron. In doing so, they pass on the message. Cannabinoid type 1 receptors (CB1Rs) help control the flow of information at synapses. They do this by binding neurotransmitters called endocannabinoids, which are unusual among neurotransmitters. Rather than sending messages from axons to dendrites, endocannabinoids send them in the opposite direction. Thus, it is dendrites that release endocannabinoids, which then bind to CB1Rs in axon terminals. This backwards, or 'retrograde', signalling dampens the release of other neurotransmitters. This slows down brain activity, and gives rise to the 'mellow' sensation that recreational cannabis users often describe. Like most other proteins, CB1Rs are built inside the cell body. So, how do these receptors end up in the axon terminals where they are needed? Are they initially sent to both axons and dendrites, with the CB1Rs that travel to dendrites being rerouted back to axons? Or do the receptors travel directly to the axon itself? Fletcher-Jones et al. tracked newly made CB1Rs in rat neurons growing in a dish. The results revealed that the receptors go directly to the axon, before moving on to the axon terminals. A specific region of the CB1R protein is crucial for sending the receptors to the axon, and for ensuring that they do not get diverted to the dendrite surface. This region stabilizes CB1Rs at the axon surface, and helps to make the receptors available to bind endocannabinoids. CB1Rs also respond to medical marijuana, a topic that continues to generate interest as well as controversy. Activating CB1Rs could help treat a wide range of diseases, such as chronic pain, epilepsy and multiple sclerosis. Future studies should build on our understanding of CB1Rs to explore and optimize new therapeutic approaches.

Kainate receptors (KARs) are glutamate-gated ion channels that play fundamental roles in regulating neuronal excitability and network function in the brain. After being cloned in the 1990s, important progress has been made in understanding the mechanisms controlling the molecular and cellular properties of KARs, and the nature and extent of their regulation of wider neuronal activity. However, there have been significant recent advances towards understanding KAR trafficking through the secretory pathway, their precise synaptic positioning, and their roles in synaptic plasticity and disease. Here we provide an overview highlighting these new findings about the mechanisms controlling KARs and how KARs, in turn, regulate other proteins and pathways to influence synaptic function.

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, CB1RΔH9 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.

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 (also known as GRIK2), and the properties of these receptors are determined in part by ADAR2 (also known as ADARB1)-mediated mRNA editing of GluK2, which changes a genomically encoded glutamine residue into an arginine residue (Q/R editing). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here, we show that induction of KAR upscaling, a phenomenon in which surface expression of receptors is increased in response to a chronic decrease in synaptic activity, results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently, this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPA receptor (AMPAR) subunit GluA2 (also known as GRIA2) also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling. This article has an associated First Person interview with the first author of the paper.

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.

The GTPase dynamin-related protein 1 (Drp1) is essential for physiological and pathophysiological mitochondrial fission. DeSUMOylation of Drp1 by the enzyme SENP3 promotes cell death during reperfusion after ischaemia by enhancing Drp1 partitioning to the mitochondrial outer membrane (MOM), which causes cytochrome c release and apoptosis. However, how deSUMOylation recruits Drp1 to the MOM is unknown. Here we show that deSUMOylation selectively promotes Drp1 binding to the MOM resident adaptor protein mitochondrial fission factor (Mff). Consistent with this, preventing Drp1 SUMOylation by mutating the SUMO acceptor sites enhances binding to Mff. Conversely, increasing Drp1 SUMOylation by knocking down SENP3 reduces both Drp1 binding to Mff and stress-induced cytochrome c release. Directly tethering Drp1 to the MOM bypasses the need for Mff to evoke cytochrome c release, and occludes the effect of SENP3 overexpression. Thus, Drp1 deSUMOylation promotes cell death by enhancing Mff-mediated mitochondrial recruitment. These data provide a mechanistic explanation for how the SUMOylation status of Drp1 acts as a key switch in cell death/survival decisions following extreme cell stress.