Biology subjects

Burlingame, A. L.

Publications and source records attributed to Burlingame, A. L..

At least 19 recordsLinked to original sources

bioRxiv2025

Identification and Characterization of PLUTO-201, a Novel Long Non-Coding RNA Associated with Poor Outcomes in Prostate Cancer

Background: Despite extensive investigation, the factors promoting aggressive prostate cancer are poorly understood. In particular, despite a few prominent examples, the role of long non-coding RNAs (lncRNAs) is largely unknown. Methods: We performed a comprehensive analysis of whole-genome transcriptome data to identify differential expression across 1,567 patients with prostate cancer, then correlated differential expression with risk of metastatic recurrence. We characterized the lncRNA most associated with metastasis in vitro and in vivo to investigate the mechanism by which it promotes aggressive prostate cancer. Results: We have identified a novel lncRNA, Prostate Cancer Associated hnRNPK Interacting Transcript (PCAHIT), which is strongly associated with metastasis and poor overall survival in men with prostate cancer. We find that overexpression/knockdown of PCAHIT in pre-clinical models of prostate cancer modulates proliferation rates and markers of an aggressive phenotype through regulation of steroid biosynthesis and expression of the MHC class I complex, driving increased growth in androgen-depleted conditions and decreased susceptibility to T cell-mediated cytotoxicity. We further find that the heterogeneous nuclear ribonucleoprotein hnRNPK directly binds PCAHIT and is indispensable for its activity. Conclusions: Overall, our findings indicate that PCAHIT is a driver of aggressive prostate cancer phenotypes and poor clinical outcomes through suppression of the immune response and increased androgen-independent cancer growth. PCAHIT is a potential biomarker of aggressive disease.

cancer biology↗

bioRxiv2025

Synaptic vesicles that store monoamines and glutamate differ in protein composition.

Neuromodulators such as the monoamines are known to differ from classical neurotransmitters like glutamate in the time scale of signaling due to activation of slower G protein-coupled receptors. Recent work has suggested that the mode of release also differs between classical and modulatory transmitters. Although many components of neurotransmitter release machinery have been identified, we still understand little about the mechanisms responsible for differences in release. In this study, we address the differences between release of dopamine and glutamate by comparing the composition of synaptic vesicles (SVs) that contain the vesicular monoamine transporter 2 (VMAT2) versus vesicular glutamate transporter 2 (VGLUT2). Previous work has shown that these SV populations differ in frequency dependence, recycling kinetics and biogenesis. Taking advantage of a CRISPR-generated knock-in mouse with a cytoplasmic hemagglutinin (HA) tag at the N-terminus of VMAT2 to immunoisolate monoamine SVs, we find differences in the abundance and isoform expression of many SV protein families. Validation in primary neurons and in brain tissue confirms these differences in SV protein abundance between dopamine and glutamate release sites. Functional analysis reveals that the loss of differentially expressed SCAMP5 selectively impairs the recycling of VGLUT2 SVs, sparing VMAT2 vesicles in the same neuronal population. These findings provide new insights into the molecular diversity of SVs and the mechanisms that regulate the release of dopamine and glutamate, with implications for the physiological role of these transmitters and behavior.

neuroscience↗

bioRxiv2025

An intrinsically disordered region of histone demethylase KDM5A activates catalysis through interactions with the nucleosomal acidic patch and DNA

Lysine demethylase 5A (KDM5A) plays a key role in the regulation of chromatin accessibility by catalyzing the removal of trimethyl marks on histone H3K4 (H3K4me3). KDM5A is also an oncogenic driver, with overexpression of KDM5A observed in various cancers, including breast, lung, and ovarian cancer. Past studies have characterized the functions of KDM5A domains, including KDM5A interactions with the histone H3 tail, but have yet to identify the broader mechanisms that drive KDM5A binding to the nucleosome. Through investigation of binding and catalysis on nucleosome substrates, we uncovered multivalent interactions of KDM5A with the H2A/H2B acidic patch and DNA that play crucial roles in the regulation of catalytic activity. We also identified an intrinsically disordered region (IDR) containing bifunctional arginine-rich motifs capable of binding to both the histone H2A/H2B acidic patch and nucleosomal DNA that is necessary for catalysis on nucleosome substrates. Our findings both elucidate previously unknown mechanisms that regulate KDM5A catalytic activity and reveal the ability of an IDR to engage in multiple interactions with chromatin. ARTICLE HIGHLIGHTSO_LIThe intrinsically disordered region of KDM5A binds the acidic patch and DNA. C_LIO_LIInteractions with the nucleosome are mediated by arginine-rich motifs in the IDR. C_LIO_LIThe IDR properly orients KDM5A on the nucleosome to enable catalysis. C_LI

biochemistry↗

bioRxiv2024

Diverse and Location-Specific Roles of PlexinA2, PlexinA4, and NCAM in Developing Hippocampal Mossy Fibers

Mossy fibers (MFs) originate from dentate granule cells and innervate area CA3 of the hippocampus. Upon entry of CA3, MFs partition into two prominent axon bundles, the suprapyramidal tract (SPT) and infrapyramidal tract (IPT) and form lamina specific synaptic contacts in the stratum lucidum (SL) and stratum oriens (SO), respectively. Here we employed new mouse lines to dissect the function of Sema6A and its receptors, PlexinA2 (PlxnA2) and PlxnA4, in developing MFs. In Sema6a-/- mice, MF partitioning into SPT and IPT bundles is incomplete and IPT axons in the SO are overextended, while the SPT correctly innervates the SL. Loss of neuronal Sema6a results in defective MF patterning and we show that this involves Sema6A reverse signaling. Plxna4 controls MF partitioning, SPT axon bundling and laminar targeting to the SL, as well as IPT length. Many of these defects are replicated in mice deficient for PlxnA4 GAP catalytic activity, underscoring the importance of this GAP domain. MFs are tightly fasciculated in Plxna2-/- mice and fail to separate into SPT and IPT bundles, and defects are significantly reduced in PlxnA2 GAP mutants, highlighting the involvement of GAP-independent signaling events. To further explore the molecular basis of aberrant axon fasciculation, we employed anti-PlxnA2 dependent proximity biotinylation and identified several PlxnA2-associated Ig-CAM family members. We observed a genetic interaction between Plxna2 and Ncam1, but not Plxna4 and Ncam1, for SPT and IPT formation and positioning in CA3. Together, our studies provide insights into the multifaceted and overlapping, yet distinct, functions of PlxnA family members in orchestrating specific guidance decisions in developing MFs.

developmental biology↗

bioRxiv2024

The VGCC auxiliary subunit α2δ1 is an extracellular GluA1 interactor and regulates LTP, spatial memory, and seizure susceptibility

Activity-dependent synaptic accumulation of AMPA receptors (AMPARs) and subsequent long-term synaptic strengthening underlie different forms of learning and memory. The AMPAR subunit GluA1 amino-terminal domain is essential for synaptic docking of AMPAR during LTP, but the precise mechanisms involved are not fully understood. Using unbiased proteomics, we identified the epilepsy and intellectual disability-associated VGCC auxiliary subunit 2{delta}1 as a candidate extracellular AMPAR slot. Presynaptic 2{delta}1 deletion in CA3 affects synaptic AMPAR incorporation during long-term potentiation, but not basal synaptic transmission, at CA1 synapses. Consistently, mice lacking 2{delta}1 in CA3 display a specific impairment in CA1-dependent spatial memory, but not in memory tests involving other cortical regions. Decreased seizure susceptibility in mice lacking 2{delta}1 in CA3 suggests a regulation of circuit excitability by 2{delta}1/AMPAR interactions. Our study sheds light on the regulation of activity-dependent AMPAR trafficking, and highlights the synaptic organizing roles of 2{delta}1. Significance statementActivity-dependent accumulation of AMPA receptors (AMPARs) at excitatory synapses and subsequent synaptic strengthening underlies long-term potentiation (LTP), forms of learning and memory, and some epilepsies. The "slot model" posits that postsynaptic scaffolding contain "slots" for AMPAR complexes, and that increased synaptic activity augments the availability of slots to accommodate more receptors, thereby strengthening synapses and enabling LTP. The presence of the GluA1 AMPAR subunit amino-terminal domain (ATD) has recently emerged as an additional requirement for LTP. Here we identify the auxiliary voltage-gated calcium channel subunit 2{delta}1 as a GluA1 ATD interacting protein and provide evidence supporting a role for 2{delta}1 as an extracellular AMPAR slot regulating activity dependent synaptic AMPAR clustering, excitability, and cognitive function.

neuroscience↗

bioRxiv2024

Liver CYP4A autophagic-lysosomal degradation (ALD): A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored Type I endoplasmic reticulum (ER)-proteins, that are inducible by hypolipidemic drugs and other "peroxisome proliferators". They are engaged in the {omega}-/{omega}-1-oxidation of various fatty acids including arachidonic acid, prostaglandins and leukotrienes and in the biotransformation of some therapeutic drugs. Herein we report that of the mammalian liver CYPs 4A, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation (ERLAD). Consequently, these proteins are stabilized both as 1%Triton X100-soluble and -insoluble species in mouse hepatocytes and HepG2-cells deficient in the autophagic initiation ATG5-gene. Although these proteins exhibit surface LC3-interacting regions (LIRs) that would target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the Cyp4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization both as Triton X100-soluble and -insoluble species. Consistently, p62Mut mouse liver microsomes exhibit enhanced {omega}- and {omega}-1-hydroxylation of arachidonic acid to its physiologically active metabolites 19- and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that any disruption of CYP4A ERLAD results in functionally active P450 protein and consequent production of proinflammatory metabolites on one hand, and insoluble aggregates on the other, which may contribute to pathological aggregates i.e. Mallory-Denk bodies/inclusions, hallmarks of many liver diseases.

cell biology↗

bioRxiv2023

Reversible histone deacetylase activity catalyzes lysine acylation

Starvation and low carbohydrate diets lead to the accumulation of the ketone body, {beta}-hydroxybutyrate (BHB), whose blood concentrations increase more than 10-fold into the millimolar range. In addition to providing a carbon source, BHB accumulation triggers lysine {beta}-hydroxybutyrylation (Kbhb) of proteins via unknown mechanisms. As with other lysine acylation events, Kbhb marks can be removed by histone deacetylases (HDACs). Here, we report that class I HDACs unexpectedly catalyze protein lysine modification with {beta}-hydroxybutyrate (BHB). Mutational analyses of the HDAC2 active site reveal a shared reliance on key amino acids for classical deacetylation and non-canonical HDAC-catalyzed {beta}-hydroxybutyrylation. Also consistent with reverse HDAC activity, Kbhb formation is driven by mass action and substrate availability. This reverse HDAC activity is not limited to BHB but also extends to multiple short-chain fatty acids. The reversible activity of class I HDACs described here represents a novel mechanism of PTM deposition relevant to metabolically-sensitive proteome modifications.

molecular biology↗

bioRxiv2023

Contrasting synaptic roles of MDGA1 and MDGA2

Neurodevelopmental disorders are frequently linked to mutations in synaptic organizing molecules. MAM domain containing glycosylphosphatidylinositol anchor 1 and 2 (MDGA1 and MDGA2) are a family of synaptic organizers suggested to play an unusual role as synaptic repressors, but studies offer conflicting evidence for their localization. Using epitope-tagged MDGA1 and MDGA2 knock-in mice, we found that native MDGAs are expressed throughout the brain, peaking early in postnatal development. Surprisingly, endogenous MDGA1 was enriched at excitatory, but not inhibitory, synapses. Both shRNA knockdown and CRISPR/Cas9 knockout of MDGA1 resulted in cell-autonomous, specific impairment of AMPA receptor- mediated synaptic transmission, without affecting GABAergic transmission. Conversely, MDGA2 knockdown/knockout selectively depressed NMDA receptor-mediated transmission but enhanced inhibitory transmission. Our results establish that MDGA2 acts as a synaptic repressor, but only at inhibitory synapses, whereas both MDGAs are required for excitatory transmission. This nonoverlapping division of labor between two highly conserved synaptic proteins is unprecedented. TeaserMDGAs 1 and 2 independently localize to and modulate excitatory and inhibitory hippocampal synapses by different mechanisms.

neuroscience↗

bioRxiv2022

O-GlcNAc glycosylation orchestrates fate decision and niche function of bone marrow stromal progenitors

In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked {beta}-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBP{beta}-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche.

physiology↗

bioRxiv2021

Molecular characterization of AMPA receptor trafficking vesicles

Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPAR trafficking vesicles (ATVs) from whole mouse brains via immunoprecipitation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified several proteins on ATVs that were previously found to play a role in AMPAR trafficking, including SNARES (including synaptobrevin 2), Rabs, the SM protein Munc18-1, a calcium-sensor (synaptotagmin-1), as well as several new markers, including synaptophysin and synaptogyrin on ATV membranes. Additionally, we identified two populations of ATVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ATVs and larger transferrin-receptor-, GluA1-, GluA2-, GluA3-containing ATVs. The smaller population of ATVs likely represents a trafficking vesicle whose fusion is essential for LTP. These findings reveal the important role of AMPAR sorting into fusion-competent trafficking vesicles that are implicated in synaptic strength modification and reveal candidates of putative effectors and regulators of AMPAR trafficking.

neuroscience↗

bioRxiv2021

Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K+ channels

Neuronal ankyrins cluster and link membrane proteins to the actin and spectrin-based cytoskeleton. Among the three vertebrate ankyrins, little is known about neuronal Ankyrin-R (AnkR). We report AnkR is highly enriched in Pv+ fast-spiking interneurons in mouse and human. We identify AnkR-associated protein complexes including cytoskeletal proteins, cell adhesion molecules (CAMs), and perineuronal nets (PNNs). We show that loss of AnkR from forebrain interneurons reduces and disrupts PNNs, decreases anxiety-like behaviors, and changes the intrinsic excitability and firing properties of Pv+ fast-spiking interneurons. These changes are accompanied by a dramatic reduction in Kv3.1b K+ channels. We identify a novel AnkR-binding motif in Kv3.1b, and show that AnkR is both necessary and sufficient for Kv3.1b membrane localization in interneurons and at nodes of Ranvier. Thus, AnkR regulates Pv+ fast-spiking interneuron function by organizing ion channels, CAMs, and PNNs, and linking these to the underlying {beta}1 spectrin-based cytoskeleton.

neuroscience↗

bioRxiv2020

TSC1 loss-of-function increases risk for tauopathy by inducing tau acetylation and preventing autophagy-mediated tau clearance

Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions, including Alzheimers disease (AD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), are collectively known as tauopathies. The vast majority of human tauopathies accumulate non-mutant tau rather than mutant forms of the protein, yet cell and animal models for non-mutant tauopathies are lacking. We previously linked a monoallelic mutation in the TSC1 gene to tau accumulation and FTLD. Now, we have identified new variants in TSC1 that predisposed to other tauopathies such as AD and PSP. These new TSC1 risk variants significantly decreased the half-life of TSC1/hamartin in vitro. Cellular and murine models of TSC1 haploinsufficiency (TSC1+/-) accumulated tau protein that exhibited aberrant acetylation on six lysine residues. Tau acetylation hindered its lysosomal degradation via chaperone-mediated autophagy leading to neuronal tau accumulation. Enhanced tau acetylation in TSC1+/- models was achieved through both an increase in p300 acetyltransferase activity and a decrease in SIRT1 deacetylase levels. Pharmacological modulation of either enzyme restored tau levels. Together, these studies substantiate TSC1 as a novel tauopathy risk gene and advance TSC1 haploinsufficiency as a new genetic model for tauopathy. In addition, these results promote acetylated tau as a rational target for diagnostic and therapeutic modalities in multiple tauopathies.

neuroscience↗

bioRxiv2020

PTBP1 Regulates Injury Responses and Sensory Pathways in Adult Peripheral Neurons

Polypyrimidine Tract Binding Protein 1 (PTBP1) is expressed only at embryonic stages in central neurons. Its downregulation triggers neuronal differentiation in precursor and non-neuronal cells, an approach recently used to generate neurons de novo for amelioration of neurodegenerative disorders. Moreover, PTBP1 is replaced by its paralog PTBP2 in mature central neurons. Surprisingly, we found both proteins co-expressed in adult sensory and motor neurons, with PTBP2 restricted mainly to the nucleus, while PTBP1 shows strong axonal localization. Levels of axonal PTBP1 increased markedly after peripheral nerve injury, and its cargos include mRNAs involved in axonal growth and regeneration, such as importin {beta}1 and RhoA. Perturbation of PTBP1 affects neuronal injury responses, axon outgrowth and sensation in vivo. Thus, PTBP1 has roles in sensory function and regenerative capacity of adult sensory neurons. These findings suggest that caution may be required before considering targeting PTBP1 for therapeutic purposes.

neuroscience↗

bioRxiv2019

Role for Merlin/NF2 in transcription elongation through interaction with the PAF complex

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing events and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions at the cell cortex and nucleus and is a key mediator of contact inhibition. Here we provide a direct link between nuclear Merlin and transcription elongation controlled by cell-cell adhesion. Merlin interacts with the PAFC in a cell density-dependent manner and tumour-derived inactivating mutations in both Merlin and CDC73 mutually disrupt their interaction. Growth suppression by Merlin requires CDC73 and Merlin regulates PAFC association with chromatin in a subset of genes. We also identify by CDC73 affinity-proteomics a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, nuclear Merlin is part of a tumour suppressor network which coordinates postinitiation steps of the transcription cycle of genes mediating in contact inhibition.

cancer biology↗

bioRxiv2019

Targeted phosphoproteomics of the Ras signaling network reveal regulatory mechanisms mediated by oncogenic KRAS

BackgroundKRAS mutations are present in up to 30% of lung adenocarcinoma cases and are associated with poor survival. No effective targeted therapy against KRAS is currently available, and novel strategies to counteract oncogenic KRAS signaling are needed.\n\nResultsWe used targeted proteomics to monitor abundance and site-specific phosphorylation in a network of over 150 upstream and downstream effectors of KRAS signaling in H358 cells (KRAS G12C). We compared patterns of protein regulation following sustained signaling blockade in the RAS/ERK module at two different levels, KRAS and MEK. Network-based analysis demonstrated complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Among 85 most regulated proteins in the network, only 12 proteins showed concordant regulation in response to signaling blockade at both KRAS and MEK levels, while the remainder were either specifically regulated in response to KRAS knockdown or MEK inhibition or showed orthogonal regulation in both conditions. Dephosphorylation of DNA methyltransferase 1 (DNMT1) at S714 was identified among the changes unique to KRAS knockdown, and here we elucidate the role of this phosphorylation in KRAS-dependent transcriptional silencing of tumor suppressor genes.\n\nConclusionsNetwork-based analysis of the Ras signaling has shown complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Our work illustrates a targeted proteomics approach to functional interrogation of complex signaling networks focused on identification of readily testable hypotheses. These methods are widely applicable to diverse questions in tumor biology and other signaling paradigms.

cancer biology↗

bioRxiv2019

A NOVEL MECHANISM FOR NF-κB-ACTIVATION VIA IκB-AGGREGATION: IMPLICATIONS FOR HEPATIC MALLORY-DENK-BODY INDUCED INFLAMMATION

Background & AimsMallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-{kappa}B activation. However, the precise mechanism that links protein aggregation to NF{kappa}B-activation and inflammatory response remains unclear.\n\nMethodsHerein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an I{kappa}B-loss with consequent NF-{kappa}B activation. We characterized the underlying mechanism in detail using hepatocytes from various knockout mice and MEF cell lines and multiple approaches including immunoblotting, EMSA, RT-PCR, confocal immunofluorescence microscopy, affinity immunoprecipitation, and protein solubility assays. Additionally, we performed rigorous proteomic analyses to identify the proteins aggregating upon PPIX treatment and/or co-aggregating with I{kappa}B.\n\nResultsFour known mechanisms of I{kappa}B-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this I{kappa}B-loss was due to its sequestration along with I{kappa}B{beta} into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with I{kappa}B through direct interaction or proximity. Of these ZnPPIX-aggregation targets, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as likely mediators of I{kappa}B-nuclear import.\n\nConclusionWe discovered a novel mechanism of inflammatory NF-{kappa}B activation through I{kappa}B-sequestration into insoluble aggregates along with interacting aggregation-prone proteins. This mechanism may account for the protein aggregate-induced inflammation observed in MDB-associated liver diseases, thereby identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.\n\nLay SummaryMallory-Denk-bodies (MDBs) are hepatic protein aggregates commonly featured in many liver diseases. MDB-presence is associated with the induction of inflammatory responses both clinically and in all MDB-inducing models. Similar protein aggregation in neurodegenerative diseases is also known to trigger inflammation and NF{kappa}B pathway activation via an as yet to be characterized non-canonical mechanism. Herein using a MDB-inducing cell model, we uncovered a novel mechanism for NF{kappa}B activation via cytosolic I{kappa}B-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with I{kappa}B. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.

cell biology↗

bioRxiv2019

A role for liquid-liquid phase separation in ESCRT-mediated nuclear envelope reformation.

At mitotic exit, microtubule arrays are dismantled in concert with the reformation of the nuclear envelope. We show how the inner nuclear membrane protein, LEM2, exploits liquid-liquid phase separation to direct microtubule remodeling and nuclear envelope sealing via the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway. LEM2 tethers membrane to chromatin disks through direct binding between its LEM motif and the chromatin-associated barrier-to-autointegration factor (BAF). Concurrently, a low-complexity domain within LEM2 undergoes liquid-liquid phase separation to coat spindle microtubule bundles. Spatially restricted, LEM2s winged helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7. Together LEM2 and CHMP7 copolymerize around microtubule bundles to form a molecular \"O-ring\" that promotes nuclear compartmentalization and initiates downstream ESCRT factor recruitment. These results demonstrate how multivalent interactions of a transmembrane protein, including those that mediate phase separation, coordinate localized ESCRT polymerization, mitotic spindle disassembly, and membrane fusion. Defects in this pathway compromise spindle disassembly, nuclear integrity, and genome stability.

cell biology↗

bioRxiv2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biophysics↗