Biology subjects

Burlingame, A.

Publications and source records attributed to Burlingame, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Optimization of Search and Acquisition Strategies for DSSO Crosslinking Mass Spectrometry using Protein Prospector and Touchstone.

Crosslinking mass spectrometry (XL-MS) enables residue-level characterization of protein-protein interactions (PPIs) in both purified assemblies and complex biological systems. Cleavable crosslinkers such as DSSO are widely used to facilitate analysis of complex samples, yet there remains limited consensus on how best to leverage their properties in both acquisition and data analysis workflows. In part, this reflects differences in scoring, classification, and false discovery rate (FDR) estimation across analysis pipelines. Therefore, an integrated XL-MS data analysis pipeline that enables confident identification of MS-cleavable cross-linked peptides through improved scoring and rigorous FDR assessment is essential for systematically comparing and benchmarking workflows across different sample preparations. To this end, we implemented a new capability within the Protein Prospector search engine to improve MS2-based identification of MS-cleavable cross-linked peptides. Next, we developed Touchstone, a framework for re-scoring, classification, FDR-controlled summarization, and visualization of Prospector XL-MS search results. Using this approach, we evaluate the contributions of DSSO-specific fragmentation features and compare multiple acquisition strategies across datasets ranging from purified macromolecular assemblies to proteome-scale samples. We find that the primary advantage of DSSO arises from enhanced backbone fragmentation of both linked peptides, rather than from diagnostic ion formation, and that incorporating diagnostic ion pairs into MS2-based scoring or filtering does not improve identification performance. Benchmarking against established datasets shows that Prospector-Touchstone performs comparably to leading methods for non-cleavable crosslinkers and provides improved performance for DSSO analysis. We further compare acquisition strategies and find that for purified protein assemblies, MS2.stHCD characterizes more residue-pairs than ETD-based, MS3-based, and hybrid methods. In contrast, for proteome-scale XL-MS, MS3-based acquisition yields similar numbers of inter-protein PPIs with lower error rates despite reduced sampling depth. Together, these results highlight complementary strengths of MS2- and MS3-based workflows and provide practical guidance for MS-cleavable XL-MS experiments with varying sample complexities.

bioinformatics↗

bioRxiv2026

Paralemmin 2 is a shared, alternatively-spliced regulator of axon initial segments and paranodal junctions in oligodendrocytes

Nervous system function depends on highly specialized axonal membrane domains. For example, axon initial segments (AIS), nodes of Ranvier, and paranodal junctions are essential for action potential initia-tion and saltatory conduction. These domains use shared molecular machinery converging on the master scaffolding protein AnkyrinG (AnkG). Using endogenous AnkG-TurboID proximity proteomics, we identify Paralemmin 2 (Palm2), a previously uncharacterized CNS protein, as a shared component of the AIS and nodes of Ranvier in neurons, and paranodes in oligodendrocytes. Palm2 localizes transiently to the AIS and nodes during development, but is found at paranodes throughout life, reflecting cell type-specific splicing of a lipid-modified domain both necessary and sufficient for AnkG interaction. Mice lacking oli-godendroglial Palm2 assemble normal paranodes during development, but aged mice lose paranode in-tegrity due to loss of paranodal AnkG and Neurofascin-155. Our findings identify Palm2 as a lipid raft-associated regulator of axonal and oligodendroglial membrane domains and reveal cell type-specific pro-tein variants as a recurring principle for specializing axonal and glial membrane architectures.

neuroscience↗

bioRxiv2026

Calcium-dependent synaptic proteomics reveals EGFR signaling at active synapses

Activity-dependent remodeling of the synaptic proteome is essential for circuit maturation, plasticity, and adaptation. However, these molecular changes are transient and spatially sparse in intact neural circuits, making them difficult to resolve under physiological conditions. To address this challenge, we developed synaptic Cal-ID, a synapse-targeted, calcium-dependent proximity labeling strategy that enables activity-resolved proteomic profiling under native physiological conditions. Using synaptic Cal-ID, we uncover an unexpected mechanism by which synaptic activity locally engages epidermal growth factor receptor (EGFR) signaling. We identify two novel synaptic proteins, Anks1a and Ubash3b, that are rapidly recruited following activity and cooperatively promote postsynaptic enrichment and signaling of EGFR. Disruption of this pathway impairs synaptic maturation, plasticity, and memory. Together, these findings reveal how neuronal activity establishes local signaling competence at active synapses by concentrating the molecular machinery required to engage a broadly acting growth factor pathway.

neuroscience↗

bioRxiv2026

Myelin basic protein is an RNA chaperone in microglial nuclear retro-transport

CNS oligodendrocytes generate myelin, an RNA-containing proteolipid substance that enhances axonal transmission. In multiple sclerosis (MS), myelin debris is phagocytosed by microglia (MG), and prior studies have detected myelin-derived mRNA in MG nuclei, suggesting a retrograde transport pathway. We report myelin basic protein (MBP) is a nucleic acid-binding and trafficking protein. We found that retro-transport of myelin RNA into the MG nucleus was phagocytosis and importin-dependent. Transcriptomic and proteomic analyses of MG nuclei revealed enrichment of myelin mRNAs and proteins, with MBP singularly detected in soluble and chromatin-associated fractions. MBP bound mRNA with high affinity (Kd {approx} 0.30 nM) and was sufficient to facilitate MG RNA nuclear import in vitro and in vivo. Functionally, MBP mediated the delivery of small interfering RNAs for targeted knockdown of toll-like receptor 4. These findings indicate MBP as an RNA-binding protein capable of MG nuclear import, providing insight into neuroinflammatory pathology of MS.

neuroscience↗

bioRxiv2026

COUNTERING AGE-ASSOCIATED ALTERATIONS IN OLIGODENDROCYTE-DERIVED EXTRACELLULAR MATRIX REJUVENATES COGNITION

Efforts to rejuvenate age-related cognitive decline have predominantly targeted neurons, often overlooking non-neuronal cell types in the aging brain. Here, we show that countering alterations in oligodendrocyte-derived extracellular matrix (ECM) in the aging hippocampus restores cognition. We identify broad age-associated transcriptional and proteomic changes in oligodendrocytes, including dysregulation of the matrisome, with marked upregulation of ECM components and associated regulators with age. Among these, we detect an increase in Hyaluronan and proteoglycan link protein 2 (HAPLN2), an oligodendrocyte-derived core matrisome protein that locates specifically at the nodes of Ranvier, in the hippocampus of aged mice and older humans. Hapln2 overexpression in oligodendrocytes of young mice recapitulated age-related memory impairments. Conversely, abrogating the age-related increase in Hapln2 induced synaptic plasticity-related hippocampal transcriptional signatures and improved memory in aged mice. Together, these data define oligodendrocyte-derived ECM remodeling as a hallmark of brain aging that can be targeted to rescue cognitive decline.

neuroscience↗

bioRxiv2025

Acetylation of Axonal G3BP1 through ELP3 Accelerates Axon Regeneration

Nerve injury triggers localized translation of axonal mRNAs to respond to injury and nerve regeneration. The core stress granule protein G3BP1 sequesters axonal mRNAs in granules before and after axotomy. G3BP1 granule disassembly can be regulated by post-translational modifications, including phosphorylation of S149 phosphorylation and acetylation of human K376 (mouse K374). Axonal G3BP1 undergoes phosphorylation after axotomy, but acetylation of G3BP1 in axons was unknown. Here we show that rodent G3BP1 undergoes K374 acetylation after axotomy is ELP3-dependent, which enhances axonal protein synthesis, accelerates nerve regeneration, and supports functional recovery. ELP3-depleted neurons exhibit reduced axon growth and increased axonal G3BP1 granules. The proximal axons degenerate rapidly despite maintaining soma connectivity, an effect prevented by expression of acetylmimetic G3BP1.Together, these findings identify G3BP1 acetylation via ELP3 as a critical regulator of both axonal regeneration and neuronal resilience, revealing a post-translational mechanism that links stress granule regulation to neuronal repair and protection.

neuroscience↗

bioRxiv2025

O-GlcNAc modifications regulate lamin A tail processing

Lamin A processing is highly regulated, and necessary for proper assembly of the nuclear lamina facilitating its role in nuclear structure and chromatin organization. Pre-lamin A is first farnesylated, and then a short C-terminal peptide is cleaved to produce mature lamin A. O-GlcNAc Transferase (OGT), a glucose sensitive post-translational modification enzyme, is a potential regulator for lamin A processing. To explore the role of OGT in lamin A biogenesis, we examined the effects of OGT levels and OGT inhibition. Variation in OGT dose or inhibition of its activity did not alter endogenous lamin A abundance or distribution. To more directly test the regulatory effects of O-GlcNAcylation on lamin A, we adapted a tail cleavage assay. Mutation of an OGT binding motif and O-GlcNAc modification sites reduced tail cleavage efficiency, suggesting that O-GlcNAcylation promotes lamin A processing. Our findings add to the understanding of the regulation of lamin A cleavage and identify a potential link between glucose metabolism and lamina biogenesis.

cell biology↗

bioRxiv2024

Functional screen for mediators of onco-mRNA translation specificity

Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). Using a functional genome-wide CRISPRi screen in PDAC cells, we identified activators of selective MYC translation through its 5 untranslated region (5UTR) and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. Among these RBPs, our top hit was RBM42, which is highly expressed in PDAC and predicts poor survival. Combining polysome sequencing and CLIP-seq analyses, we find that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, employing IP-mass spectrometry analysis, we find that RMB42 is a novel ribosome-associated protein (RAP). Using DMS-Seq and mutagenesis analysis, we show that RBM42 directly binds and remodels the MYC 5UTR RNA structure, facilitating the formation of the translation pre-initiation complex. Importantly, RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo. In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes.

cancer biology↗

bioRxiv2024

TAOK2 Drives Opposing Cilia Length Deficits in 16p11.2 Deletion and Duplication Carriers

Copy number variation (CNV) in the 16p11.2 (BP4-BP5) genomic locus is strongly associated with autism. Carriers of 16p11.2 deletion and duplication exhibit several common behavioral and social impairments, yet, show opposing brain structural changes and body mass index. To determine cellular mechanisms that might contribute to these opposing phenotypes, we performed quantitative tandem mass tag (TMT) proteomics on human dorsal forebrain neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSC) derived from 16p11.2 CNV carriers. Differentially phosphorylated proteins between unaffected individuals and 16p11.2 CNV carriers were significantly enriched for centrosomal and cilia proteins. Deletion patient-derived NPCs show increased primary cilium length compared to unaffected individuals, while stunted cilium growth was observed in 16p11.2 duplication NPCs. Through cellular shRNA and overexpression screens in human iPSC derived NPCs, we determined the contribution of genes within the 16p11.2 locus to cilium length. TAOK2, a serine threonine protein kinase, and PPP4C, a protein phosphatase, were found to regulate primary cilia length in a gene dosage-dependent manner. We found TAOK2 was localized at centrosomes and the base of the primary cilium, and NPCs differentiated from TAOK2 knockout iPSCs had longer cilia. In absence of TAOK2, there was increased pericentrin at the basal body, and aberrant accumulation of IFT88 at the ciliary distal tip. Further, pharmacological inhibition of TAO kinase activity led to increased ciliary length, indicating that TAOK2 negatively controls primary cilium length through its catalytic activity. These results implicate aberrant cilia length in the pathophysiology of 16p11.2 CNV, and establish the role of TAOK2 kinase as a regulator of primary cilium length.

neuroscience↗

bioRxiv2024

NEK1 modulates neurite outgrowth in motor neurons through coordinating retromer formation

Loss of function of a cell cycle-associated gene NEK1 causes amyotrophic lateral sclerosis (ALS), but how this leads to motor neuron degeneration is unknown. We studied the function of NEK1 in human stem cell-derived motor neurons and found that loss of NEK1 causes decreased neurite length accompanied by transcriptional alterations. We also found that NEK1 interacts with and modulates the formation of the retromer, and that impaired retromer function contributes to neurite outgrowth deficits. We identified SMC3, which interacts with NEK1 during the cell cycle, as a kinase substrate of NEK1 in motor neurons. Notably, loss of SMC3 not only recapitulates the decreased neurite outgrowth, but also affects retromer formation. We suggest that NEK1 interacts with multiple proteins in postmitotic neurons to coordinate retromer formation, and that loss of this leads to impaired neurite outgrowth.

neuroscience↗

bioRxiv2024

Assessing Heterogeneity in the N-Telopeptides of Type I Collagen by Mass Spectrometry

Collagen cross-links created by the lysyl oxidase and lysyl hydroxylase families of enzymes are a significant contributing factor to the biomechanical strength and rigidity of tissues, which in turn influence cell signaling and ultimately cell phenotype. In the clinic, the proteolytically liberated N-terminal cross-linked peptide of collagen I (NTX) is used as a biomarker of bone and connective tissue turnover, which is altered in several disease processes. Despite the clinical utility of these collagen breakdown products, the majority of the cross-linked peptide species have not been identified in proteomic datasets. Here we evaluate several parameters for the preparation and identification of these peptides from the collagen I-rich Achilles tendon. Our refined approach involving chemical digestion for protein solubilization coupled with mass spectrometry allows for the identification of the NTX cross-links in a range of modification states. Based on the specificity of the enzymatic cross-linking reaction we utilized follow-up variable modification searches to facilitate identification with a wider range of analytical workflows. We then applied a spectral library approach to identify differences in collagen cross-links in bovine pulmonary hypertension. The presented method offers unique opportunities to understand extracellular matrix remodeling events in development, aging, wound healing, and fibrotic disease that modulate collagen architecture through lysyl-hydroxylase and lysyl-oxidase enzymes.

biochemistry↗

bioRxiv2023

RAPIDASH: A tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues and stimulated macrophages

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translational control. However, a lack of technologies to enrich RAPs across many sample types has prevented systematic analysis of RAP number, dynamics, and functions. Here, we have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development that is linked to the translation of genes with long coding sequences. Finally, we characterized ribosome composition remodeling during immune activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs ranging from those with neuroregulatory functions to those activated by immune stimuli, thereby providing critical insights into how ribosomes are remodeled.

molecular biology↗

bioRxiv2023

Non-specific recognition of histone modifications by H3K9bhb antibody

Ketone bodies are short chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative source of energy for the brain, heart, and skeletal muscle. Beyond this classical metabolic role, {beta}-hydroxybutyrate (BHB), is gaining recognition as a pleiotropic signaling molecule. Lysine {beta}-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine {varepsilon}-amino groups. This novel protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments, including the mitochondria and nucleus. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against the {beta}-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s), which are increased by deacetylation inhibition and include likely acetylations. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

biochemistry↗

bioRxiv2023

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

neuroscience↗

bioRxiv2022

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

The spliceosome is a staggeringly complex machine comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and interrogated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A-complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B binding site but also to the G-patch (ATPase activator) domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

molecular biology↗

bioRxiv2022

In-cell chemical crosslinking identifies hotspots for p62-IκBα interaction that underscore a critical role of p62 in limiting NF-κB activation through IκBα-stabilization

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with I{kappa}B, an inhibitor of the transcriptional activator NF-{kappa}B. Such an intimate p62-I{kappa}B association we now document leads to a marked 18-fold proteolytic I{kappa}B-stabilization, enabling its nuclear entry and termination of the NF-{kappa}B-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-{kappa}B following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-I{kappa}B association. Accordingly, we have identified such I{kappa}B hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5th ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon I{kappa}B co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on I{kappa}B-protein-protein interactions. Interestingly, consistent with p62s capacity to proteolytically stabilize I{kappa}B, its presence greatly impaired I{kappa}Bs interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with I{kappa}B on an interface opposite to that of its NF-{kappa}B-interacting interface, p62 failed to significantly affect I{kappa}B-NF-{kappa}B interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling, leads us to speculate that it may be involved in "piggy-back" nuclear transport of I{kappa}B following its NF-{kappa}B-elicited transcriptional activation and de novo synthesis, required for the termination of the NF-{kappa}B-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252 harboring its positively charged patch, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-{kappa}B. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/512146v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@a2cce1org.highwire.dtl.DTLVardef@38da91org.highwire.dtl.DTLVardef@95847aorg.highwire.dtl.DTLVardef@2c829b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIp62 binds to and stabilizes I{kappa}B by preventing its proteolytic degradation C_LIO_LIIn-cell chemical crosslinking/LC-MS/MS identified the inter-crosslinked sites C_LIO_LIHotspots of p62-I{kappa}B association are defined C_LIO_LIAPEX proximity labeling revealed p62 impaired I{kappa}B-interaction with proteasome C_LIO_LIp62 chaperones newly synthesized I{kappa}B to terminate NF-{kappa}B activation. C_LI In BriefThe transcriptional activator NF-{kappa}B inhibitor, I{kappa}B is proteolytically unstable when uncomplexed. How newly synthesized I{kappa}B escapes degradation to terminate nuclear NF-{kappa}B-activation is unknown. Using in-cell chemical crosslinking and proximity labeling MS analyses, we uncovered a novel association of p62 with I{kappa}B via well-defined structural hotspots, which impairs its interaction with the 26S/20S proteasome, extending its life-span and enabling termination of NF-{kappa}B-activation. Mice carrying liver-specific genetic deletion of p62-I{kappa}B hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role.

cell biology↗

bioRxiv2022

Capturing the Hierarchically Assorted Modules of Protein Interaction in the Organized Nucleome

Nuclear proteins are major constituents and key regulators of the topological organization of nucleome. To elucidate the global connectivity of nucleomic proteins and to decipher the hierarchically organized modules of protein interaction that are involved in nucleomic organization and nuclear events, both formaldehyde and CBDPS crosslinkers were applied sequentially on the in vivo prefixed nuclei to perform a double chemical crosslinking with mass spectrometry (XL-MS) analysis. The integration of dimethyl-labelling with XL-MS generated a quantitative XL-MS workflow (qXL-MS) that consequently identified 5,340 cross-linked peptides (crosslinks) from nucleome. These crosslinks were construed into 1,297 nuclear protein-protein interactions (PPIs), from which discovered were 250 and 26 novel interactors of histones and nucleolar box C/D snoRNP complex, respectively. MONET-based modulomic analysis of their Arabidopsis orthoglous PPIs constructed 27 and 24 master nuclear protein interaction modules (NPIMs) that contain the condensate-forming protein(s) and the intrinsically disordered region (IDR)-containing proteins, respectively. These NPIMs successfully captured the previously reported nuclear protein complexes and nuclear bodies in nucleome. Surprisingly, modulomic analysis showed that these NPIMs were hierarchically assorted into four communities of NPIMs in nucleome including Genome Community and Nucleolus Community. The qXL-MS-based quantitative interactomics finally revealed 17 Hormone-specific module variants participating in a broad range of nuclear events. Thus, this integrated pipeline of qXL-MS and MONET modulomics, named as CHAMPION, is capable of capturing both nuclear protein complexes and nuclear bodies, constructing the topological architecture of protein interaction modules and module variants in nucleome and probably of mapping the protein compositions of condensates. HighlightsO_LIThe formaldehyde and CBDPS crosslinkers coupled qXL-MS discovered 5,340 crosslinked peptides. These crosslinks were construed into 1,297 nuclear protein-protein interactions (PPIs), protein components of which contained 250 and 26 novel interactors of histone octamer and nucleolar box C/D snoRNP complex, respectively, in the intricately organized nucleome. C_LIO_LIThe MONET-based modulomic analysis of these crosslinks captured 95 nuclear protein interaction modules (NPIMs), a portion of which contain both the condensate-forming and the intrinsically disordered region (IDR)-containing proteins. Especially, some NPIMs captured 6 previously reported nuclear protein complexes. C_LIO_LIA number of Hormone-specific module variants were identified by modulomics upon hormone treatment using the hormone significantly up-regulated crosslinks from qXL-MS. Several PPIs and NPIMs have been substantiated with alternative biological experiments. C_LIO_LIThis CHAMPION pipeline has partitioned these NPIMs into four hierarchically and topologically organized communities in nucleome. The molecular functions of those proteins partitioned into C1 and C2 community are specialized in genome organization and nucleolar functions, respectively. C_LI

bioinformatics↗

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↗