Biology subjects

Nelson, A.

Publications and source records attributed to Nelson, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Ketogenesis protects against MASLD-MASH progression through fat oxidation-independent mechanisms

AbstractThe progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) to metabolic-dysfunction-associated steatohepatitis (MASH) involves complex alterations in both liver-autonomous and systemic metabolism that influence the livers balance of fat accretion and disposal. Here, we quantify the relative contribution of hepatic oxidative pathways to liver injury in MASLD-MASH. Using NMR spectroscopy, UHPLC-MS, and GC-MS, we performed stable-isotope tracing and formal flux modeling to quantify hepatic oxidative fluxes in humans across the spectrum of MASLD-MASH, and in mouse models of impaired ketogenesis. We found in humans with MASH, that liver injury correlated positively with ketogenesis and total fat oxidation, but not with turnover of the tricarboxylic acid cycle. The use of loss-of-function mouse models demonstrated that disruption of mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting step of ketogenesis, impairs overall hepatic fat oxidation and induces a MASLD-MASH-like phenotype. Disruption of mitochondrial {beta}-hydroxybutyrate dehydrogenase (BDH1), the terminal step of ketogenesis, also impaired fat oxidation, but surprisingly did not exacerbate steatotic liver injury. Taken together, these findings suggest that quantifiable variations in overall hepatic fat oxidation may not be a primary determinant of MASLD-to-MASH progression, but rather, that maintenance of hepatic ketogenesis could serve a protective role through additional mechanisms that extend beyond quantified overall rates of fat oxidation.

biochemistry↗

bioRxiv2024

Dissecting the genetic regulation of lateral root development in tomato under salt stress

Roots are the primary site for sensing salt, making them critical to understanding how plants adapt to salinity. Unraveling the genetic and molecular mechanisms that maintain root growth under salt stress is essential for developing resilient crops. Tomatoes are vulnerable to salinity, as over 50% of arable land is projected to become saline by 2050. While salt stress effects on tomato seed germination, shoot growth, and fruit yield are well-documented, the genetic basis of root development under salinity remains underexplored. Previous studies focused on physiological responses of roots in a narrow range of cultivated-tomatoes, overlooking the genetic diversity for salt resilience in wild-relatives like Solanum pimpinellifolium. Here, we investigated salt-induced changes in root system architecture (RSA) across a natural diversity panel of 220 wild- and 25 cultivated-tomato varieties. We identified tolerant accessions with different RSA strategies; prioritizing lateral root elongation versus emergence. To identify genes involved in specific aspects of lateral root development, an F1 hybrid was generated that exhibited both parents combined characteristics. An F2-segregating population was then used to identify four distinct subpopulations for Bulk Segregant Analysis (BSA). Simultaneously, we conducted Genome-Wide-Association Studies (GWAS) on root architecture of wild-tomato accessions under salt stress. By integrating the results of BSA and GWAS, we identified 22 candidates involved in the maintenance of root architecture. We utilized RNA-Seq to examine transcriptome reprogramming of the 22 genes in tomato accessions with contrasting lateral root responses. This approach resulted in the identification of 2 genes, AP2-like-ethylene-responsive transcription factor TOE3 and L-ascorbate peroxidase. Using exogenous ethylene, we demonstrated that root architecture exhibits plasticity, which benefits the plant by reducing Na+ accumulation. We profiled H2O2 waves across root systems of tomatoes, emphasizing the role of ROS in maintaining root system architecture under salt stress. These findings provide novel genetic targets for enhancing salt resilience in tomatoes, opening avenues for future research and breeding programs.

plant biology↗

bioRxiv2024

Specification of human regional brain lineages using orthogonal gradients of WNT and SHH in organoids

The repertory of neurons generated by progenitor cells depends on their location along antero-posterior and dorso-ventral axes of the neural tube. To understand if recreating those axes was sufficient to specify human brain neuronal diversity, we designed a mesofluidic device termed Duo-MAPS to expose induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison of single cell transcriptomes with fetal human brain revealed that Duo-MAPS-patterned organoids generated the major neuronal lineages of the forebrain, midbrain, and hindbrain. Morphogens crosstalk translated into early patterns of gene expression programs predicting the generation of specific brain lineages. Human iPSC lines from six different genetic backgrounds showed substantial differences in response to morphogens, suggesting that interindividual genomic and epigenomic variations could impact brain lineages formation. Morphogen gradients promise to be a key approach to model the brain in its entirety.

neuroscience↗

bioRxiv2024

Structural basis of autoantibody binding in Neuromyelitis Optica

Neuromyelitis Optica (NMO) is an autoimmune disease of the central nervous system where pathogenic autoantibodies target the human astrocyte water channel aquaporin-4 causing neurological impairment. Autoantibody binding leads to complement dependent and complement independent cytotoxicity, ultimately resulting in astrocyte death, demyelination, and neuronal loss. Aquaporin-4 assembles in astrocyte plasma membranes as symmetric tetramers or as arrays of tetramers. We report molecular structures of aquaporin-4 alone and bound to Fab fragments from patient-derived NMO autoantibodies using cryogenic electron microscopy. Each antibody binds to epitopes comprised of three extracellular loops of aquaporin-4 with contributions from multiple molecules in the assembly. The structures distinguish between antibodies that bind to the tetrameric form of aquaporin-4, and those targeting higher order orthogonal arrays of tetramers that provide more diverse bridging epitopes. One-Sentence SummaryPathogenic autoantibodies in Neuromyelitis Optica distinguish multiply presented epitopes on arrays of human aquaporin-4.

immunology↗

bioRxiv2024

Neurochemical and Neurophysiological Effects of Intravenous Administration of N,N-dimethyltryptamine in Rats

N,N-dimethyltryptamine (DMT) is a serotonergic psychedelic that is being investigated clinically for the treatment of psychiatric disorders. Although the neurophysiological effects of DMT in humans are well-characterized, similar studies in animal models as well as data on the neurochemical effects of DMT are generally lacking, which are critical for mechanistic understanding. In the current study, we combined behavioral analysis, high-density (32-channel) electroencephalography, and ultra-high-performance liquid chromatography-tandem mass spectrometry to simultaneously quantify changes in behavior, cortical neural dynamics, and levels of 17 neurochemicals in medial prefrontal and somatosensory cortices before, during, and after intravenous administration of three different doses of DMT (0.75 mg/kg, 3.75 mg/kg, 7.5 mg/kg) in male and female adult rats. All three doses of DMT produced head twitch response with most twitches observed after the low dose. DMT caused dose-dependent increases in serotonin and dopamine levels in both cortical sites along with a reduction in EEG spectral power in theta (4-10 Hz) and low gamma (25-55 Hz), and increase in power in delta (1-4 Hz), medium gamma (65-115 ), and high gamma (125-155 Hz) bands. Functional connectivity decreased in the delta band and increased across the gamma bands. In addition, we provide the first measurements of endogenous DMT in these cortical sites at levels comparable to serotonin and dopamine, which together with a previous study in occipital cortex, suggests a physiological role for endogenous DMT. This study represents one of the most comprehensive characterizations of psychedelic drug action in rats and the first to be conducted with DMT. Significance StatementN,N-dimethyltryptamine (DMT) is a serotonergic psychedelic with potential as a tool for probing the neurobiology of consciousness and as a therapeutic agent for psychiatric disorders. However, the neurochemical and neurophysiological effects of DMT in rat, a preferred animal model for mechanistic studies, are unclear. We demonstrate that intravenous DMT caused a dose-dependent increase in serotonin and dopamine in medial prefrontal and somatosensory cortices, and simultaneously increased gamma functional connectivity. Similar effects have been shown for other serotonergic and atypical psychedelics, suggesting a shared mechanism of drug action. Additionally, we report DMT during normal wakefulness in two spatially and functionally distinct cortical sites -- prefrontal, somatosensory -- at levels comparable to those of serotonin and dopamine, supporting a physiological role for endogenous DMT.

neuroscience↗

bioRxiv2024

Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence

AO_SCPLOWBSTRACTC_SCPLOWBacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognize and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognizes cognate motifs has not been fully understood. We have characterised BREX from pathogenic Salmonella and generated the first X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that larger BREX complexes are required for methyltransferase activity and defence. Through rational engineering of PglX, we broadened both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is the sole specificity factor for BREX activity, providing motif recognition for both phage defence and host methylation.

microbiology↗

bioRxiv2024

Natural variation in salt-induced changes in root:shoot ratio reveals SR3G as a negative regulator of root suberization and salt resilience in Arabidopsis

Soil salinity is one of the major threats to agricultural productivity worldwide. Salt stress exposure alters root and shoot growth rates, thereby affecting overall plant performance. While past studies have extensively documented the effect of salt stress on root elongation and shoot development separately, here we take an innovative approach by examining the coordination of root and shoot growth under salt stress conditions. Utilizing a newly developed tool for quantifying the root:shoot ratio in agar-grown Arabidopsis seedlings, we found that salt stress results in a loss of coordination between root and shoot growth rates. We identify a specific gene cluster encoding domain-of-unknown-function 247 (DUF247), and characterize one of these genes as Salt Root:shoot Ratio Regulator Gene (SR3G). Further analysis elucidates the role of SR3G as a negative regulator of salt stress tolerance, revealing its function in regulating shoot growth, root suberization, and sodium accumulation. We further characterize that SR3G expression is modulated by WRKY75 transcription factor, known as a positive regulator of salt stress tolerance. Finally, we show that the salt stress sensitivity of wrky75 mutant is completely diminished when it is combined with sr3g mutation. Together, our results demonstrate that utilizing root:shoot ratio as an architectural feature leads to the discovery of new stress resilience gene. The studys innovative approach and findings not only contribute to our understanding of plant stress tolerance mechanisms but also open new avenues for genetic and agronomic strategies to enhance crop environmental resilience.

plant biology↗

bioRxiv2024

Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration

Parkinsons disease (PD) is characterized by the death of substantia nigra (SNc) dopamine (DA) neurons, but the pathophysiological mechanisms that precede and drive their death remain unknown. The activity of DA neurons is likely altered in PD, but we understand little about if or how chronic changes in activity may contribute to degeneration. To address this question, we developed a chemogenetic (DREADD) mouse model to chronically increase DA neuron activity, and confirmed this increase using ex vivo electrophysiology. Chronic hyperactivation of DA neurons resulted in prolonged increases in locomotor activity during the light cycle and decreases during the dark cycle, consistent with chronic changes in DA release and circadian disturbances. We also observed early, preferential degeneration of SNc projections, recapitulating the PD hallmarks of selective vulnerability of SNc axons and the comparative resilience of ventral tegmental area axons. This was followed by eventual loss of midbrain DA neurons. Continuous DREADD activation resulted in a sustained increase in baseline calcium levels, supporting a role for increased calcium in the neurodegeneration process. Finally, spatial transcriptomics from DREADD mice examining midbrain DA neurons and striatal targets, and cross-validation with human patient samples, provided insights into potential mechanisms of hyperactivity-induced toxicity and PD. Our results thus reveal the preferential vulnerability of SNc DA neurons to increased neural activity, and support a potential role for increased neural activity in driving degeneration in PD.

neuroscience↗

bioRxiv2024

Psilocybin induces dose-dependent changes in functional network organization in rat cortex

Psilocybin produces an altered state of consciousness in humans and is associated with complex spatiotemporal changes in brain networks. Given the emphasis on rodent models for mechanistic studies, there is a need for characterization of the effect of psilocybin on brain-wide network dynamics. Previous rodent studies of psychedelics, using electroencephalogram, have primarily been done with sparse electrode arrays that offered limited spatial resolution precluding network level analysis, and have been restricted to lower gamma frequencies. Therefore, in the study, we used electroencephalographic recordings from 27 sites (electrodes) across rat cortex (n=6 male, 6 female) to characterize the effect of psilocybin (0.1 mg/kg, 1 mg/kg, and 10 mg/kg delivered over an hour) on network organization as inferred through changes in node degree (index of network density) and connection strength (weighted phase-lag index). The removal of aperiodic component from the electroencephalogram localized the primary oscillatory changes to theta (4-10 Hz), medium gamma (70-110 Hz), and high gamma (110-150 Hz) bands, which were used for the network analysis. Additionally, we determined the concurrent changes in theta-gamma phase-amplitude coupling. We report that psilocybin, in a dose-dependent manner, 1) disrupted theta-gamma coupling [p<0.05], 2) increased frontal high gamma connectivity [p<0.05] and posterior theta connectivity [p[≤]0.049], and 3) increased frontal high gamma [p<0.05] and posterior theta [p[≤]0.046] network density. The medium gamma frontoparietal connectivity showed a nonlinear relationship with psilocybin dose. Our results suggest that high-frequency network organization, decoupled from local theta-phase, may be an important signature of psilocybin-induced non-ordinary state of consciousness.

neuroscience↗

bioRxiv2023

Topographical and cell type-specific connectivity of rostral and caudal forelimb corticospinal neuron populations

Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse group of neurons with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, no studies have mapped the diversity of spinal cell types targeted by CSNs, let alone compare CFA and RFA populations. Here we use anatomical and RNA-sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal cell types, with notable exceptions that suggest these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive surprisingly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior.

neuroscience↗

bioRxiv2023

Syntrichia ruralis: Emerging model moss genome reveals a conserved and previously unknown regulator of desiccation in flowering plants

Water scarcity poses a significant threat to ecosystems in the face of global climate change. Syntrichia ruralis, a dryland moss known for its desiccation tolerance, provides valuable insights into surviving water-limited conditions. In this study, the genome of S. ruralis was sequenced and assembled into 12 chromosomes encompassing 21,169 protein-coding genes. Additionally, 3,199 unplaced scaffolds were identified as non-nuclear and symbiont DNA. Transposable elements (TEs) constitute 51.24% of the genome. Notably, chromosome 12, the largest in size due to its high TE load, was identified as the putative sex chromosome. Comparative analysis with the closely related Syntrichia caninervis genome reveals significant large-scale synteny yet some rearrangements, as well as the occurrence of older duplication events that are shared by both. Desiccation and drought tolerance associated gene families, such as early light-inducible proteins (ELIPs) and late embryogenesis abundant (LEA) proteins, were characterized. In addition to a subset of LEA genes being species-specific, a comparative transcriptomic analysis revealed that some shared LEA genes respond differently to dehydration in these two species. Many ELIPs (9 out of 30) are the product of tandem duplication events. As expected, our analyses revealed the importance of the phytohormone abscisic acid (ABA) in the desiccation response of S. ruralis. A significant number of ABA responsive genes were found to be regulated by S. ruralis orthologs of ABA insensitive 3 (ABI3) and abscisic acid responsive element binding factor 2 (AREB2). Markedly, an uncharacterized, but deeply conserved MYB transcription factor, appears to act as a negative regulator of AREB2 in S. ruralis. Interestingly, we determined that the orthologous MYB TF is also involved in an ABA-dependent stress response in the model flowering plant A. thaliana. In sum, the new genomic resources from this emerging model moss offer new insights into the evolution of desiccation tolerance in land plants.

genomics↗

bioRxiv2023

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine. Significance statementReinforcement learning requires the phasic dopamine produced by burst firing but synaptic vesicle depletion limits the ability to convey information at high firing rates. We now find that AP-3 has two independent roles in dopamine release. First, AP-3 confers the axonal polarity of dopamine release by targeting vesicular monoamine transporter 2 (VMAT2) to the axon. Second, AP-3 acting locally at the nerve terminal produces synaptic vesicles that respond specifically to high frequency stimulation. Consistent with this, loss of AP-3 impairs reinforcement learning and this reflects the defect in release at high frequency, not the reduction in axonal dopamine.

neuroscience↗

bioRxiv2023

Development of a mobile, high-throughput, and low-cost image-based plant growth phenotyping system

Nondestructive plant phenotyping is fundamental for unraveling molecular processes underlying plant development and response to the environment. While the emergence of high-through phenotyping facilities can further our understanding of plant development and stress responses, their high costs significantly hinder scientific progress. To democratize high-throughput plant phenotyping, we developed sets of low-cost image- and weight-based devices to monitor plant growth and evapotranspiration. We paired these devices with a suite of computational pipelines for integrated and straightforward data analysis. We validated the suitability of our system for large screens by evaluating a cowpea diversity panel for responses to drought stress. The observed natural variation was subsequently used for Genome-Wide Association Study, where we identified nine genetic loci that putatively contribute to cowpea drought resilience during early vegetative development. We validated the homologs of the identified candidate genes in Arabidopsis using available mutant lines. These results demonstrate the varied applicability of this low-cost phenotyping system. In the future, we foresee these setups facilitating identification of genetic components of growth, plant architecture, and stress tolerance across a wide variety of species.

plant biology↗

bioRxiv2023

CyVerse: Cyberinfrastructure for Open Science

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 110,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in two countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

scientific communication and education↗

bioRxiv2023

The long and short of it: Benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing and hybrid approaches that combine long and short read technologies are now being widely implemented in bacterial genomics and metagenomics. However, the use of long-read sequencing to investigate viral communities is still in its infancy. While Nanopore and PacBio technologies have been applied to viral metagenomics, it is not known to what extent different technologies will impact the reconstruction of the viral community. Thus, we constructed a mock phage community of previously sequenced phage genomes and sequenced using Illumina, Nanopore, and PacBio sequencing technologies and tested a number of different assembly approaches. When using a single sequencing technology, Illumina assemblies were the best at recovering phage genomes. Nanopore- and PacBio-only assemblies performed poorly in comparison to Illumina in both genome recovery and error rates, which both varied with the assembler used. The best Nanopore assembly had errors that manifested as SNPs and INDELs at frequencies ~4x and 120x higher than found in Illumina only assemblies respectively. While the best PacBio assemblies had SNPs at frequencies ~3.5 x and 12x higher than found in Illumina only assemblies respectively. Despite high read coverage, long-read only assemblies failed to recover a complete genome for any of the 15 phage, down sampling of reads did increase the proportion of a genome that could be assembled into a single contig. Overall the best approach was assembly by a combination of Illumina and Nanopore reads, which reduced error rates to levels comparable with short read only assemblies. When using a single technology, Illumina only was the best approach. The differences in genome recovery and error rates between technology and assembler had downstream impacts on gene prediction, viral prediction, and subsequent estimates of diversity within a sample. These findings will provide a starting point for others in the choice of reads and assembly algorithms for the analysis of viromes. Data SummaryAll reads from virome sequencing were submitted to the ENA under study PRJEB56639. The assemblies are provided via FigShare (https://figshare.com/s/2d9b5121eb421d370455). Author NotesEight Supplementary Tables and nine Supplementary Figures are available with the online version of this article.

microbiology↗

bioRxiv2022

Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.

genetics↗

bioRxiv2022

Morphological Subprofile Analysis for Bioactivity Annotation of Small Molecules

Fast prediction of mode of action for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. A variety of target-based assays is available for addressing the modulation of druggable proteins. However, they cannot precisely predict how a compound would influence cellular processes due to polypharmacology. Furthermore, non-protein targets are often not considered. Morphological profiling, e.g., using the Cell Painting assay that monitors hundreds of morphological features upon compound perturbation and staining of cellular components, offers a fast, unbiased assessment of compound activity on various targets and cellular processes in one single experiment. However, due to incomplete bioactivity annotation and unknown activities of reference (landmark) compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the mode of action for both reference and unexplored compounds. We defined mode-of-action clusters for a group of reference compounds and extracted cluster subprofiles that contain only a subset of morphological features (i.e., subprofiles) to represent a consensus profile. Subprofile analysis allows for assignment of compounds to, currently, ten different targets or modes of action in one single assay and bypasses the need of exploring all biosimilar reference compounds for the generation of target hypothesis. This approach will enable rapid bioactivity annotation of compound collections, particularly of uncharacterized small molecules, and will be extended to further bioactivity clusters in future. The data is public accessible via https://github.com/mpimp-comas/2022_pahl_ziegler_subprofiles and the web app tool http://cpcse.pythonanywhere.com/.

cell biology↗

bioRxiv2022

Allosteric Regulation of a Synaptic Vesicle Glutamate Transporter

Concentration of neurotransmitter inside synaptic vesicles (SVs) underlies the quantal nature of synaptic transmission. In contrast to many transporters, SV uptake of the principal excitatory neurotransmitter glutamate is driven by membrane potential. To prevent nonquantal efflux of glutamate after SV exocytosis, the vesicular glutamate transporters (VGLUTs) are allosterically inhibited by the neutral pH of the synaptic cleft. We have now determined high-resolution structures of VGLUT2 with a cyclic analog of glutamate bound that defines the mechanism of substrate recognition, a positively charged cytoplasmic vestibule that electrostatically attracts the negatively charged substrate, and modification by palmitoylation that promotes retrieval of the transporter after exocytosis. The structure also incorporates an extensive, cytoplasmic network of electrostatic interactions that acts as a gate. Functional analysis shows how this cytoplasmic gate confers the allosteric requirement for lumenal H+ required to restrict VGLUT activity to SVs.

biochemistry↗