Biology subjects

Muir, T. W.

Publications and source records attributed to Muir, T. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Release of the Staphylococcus aureus autoinducing peptide is enabled by active export of the biosynthetic intermediate

Staphylococcus aureus is a species of Gram-positive bacteria that uses the accessory gene regulator (agr) quorum sensing system to regulate many aspects of cell behavior such as virulence and toxin production. Quorum sensing is activated in dense S. aureus populations by the extracellular accumulation of a signaling molecule called the autoinducing peptide (AIP). While the biosynthetic enzymes that produce AIP have been elucidated in certain subgroups of S. aureus, the mechanism of AIP secretion is unknown. Here, we show that the biosynthetic intermediate known as AgrD (1-32) thiolactone is actively exported into the surrounding environment in certain S. aureus subgroups. Using a high-throughput transposon mutagenesis screen, we identify the integral membrane transporters, AbcA and PmtCD, as being involved in the biosynthesis of certain AIPs. Subsequent genetic complementation and biochemical experiments reveal that these proteins actively transport AgrD (1-32) thiolactone out of the cell. This enables the production of AIP directly on the cell surface following the action of MroQ, an integral membrane protease demonstrated here to possess an active site facing the extracellular milieu. Furthermore, we show that media supplementation with catalytically-active MroQ nanodiscs rescues AIP production in an mroQ knockout strain. This observation suggests the possibility that some proportion of AgrD (1-32) thiolactone exported by one cell may travel to neighboring cell surfaces for further processing into the diffusive AIP molecule. Collectively, our studies shed light on a poorly understood step in the agr quorum sensing pathway with clear implications for the design of novel therapeutic strategies.

biochemistry↗

bioRxiv2026

KMT2E recruitment by H3 serotonylation regulates neurodevelopmental chromatin dynamics

Histone H3 Gln 5 serotonylation (H3Q5ser) is a recently identified epigenetic modification in brain that modulates reader interactions with adjacent H3 Lys 4 trimethylation (H3K4me3) to promote transcriptional permissiveness. However, whether H3K4me3Q5ser and its associated binding proteins regulate neurodevelopmental gene expression programs remains unknown. Here, we identified the catalytically inactive Lysine methyltransferase 2E (KMT2E) as a reader of combinatorial H3K4me3Q5ser. KMT2E preferentially binds H3K4me3Q5ser over H3K4me3 alone, and enriches at broad chromatin domains marking actively transcribed neurodevelopmental loci. Notably, heterozygous variants in KMT2E have been implicated in ODLURO syndrome, a recently characterized neurodevelopmental disorder (NDD). To identify the molecular mechanisms underlying ODLURO syndrome, we generated a Kmt2e transgenic mouse model that reproduces behavioral, physiological, and cellular endophenotypes associated with this and other NDDs. Furthermore, we observed that KMT2E mediates these effects by recruiting the NCoR/HDAC3 repressor complex to H3K4me3Q5ser-marked loci to restrict spreading of co-localized H3 Lys 9 acetylation (H3K9ac). Inhibition of aberrant H3K9ac spreading was sufficient to rescue transcriptional dysregulation in Kmt2e haploinsufficient neurons. These findings thus establish KMT2E as a critical reader of H3 serotonylation during neurodevelopment and provide mechanistic insights into the pathogenesis of ODLURO syndrome.

neuroscience↗

bioRxiv2026

Quinone reductase 2 reads H3 serotonylation to support neuronal maturation

Histone H3 Gln5 serotonylation (H3Q5ser) is a recently described posttranslational modification1 that plays important roles in guiding transcriptional permissiveness in brain and peripheral systems2-5. H3Q5ser has been implicated in diverse physiological and pathological processes ranging from neural differentiation1 to sensory processing6, circadian rhythmicity7, stress responsivity8, placental gene regulation9, and tumorigenesis10-19. Since H3Q5ser can occur in combination with H3 Lys4 trimethylation (H3K4me3), most mechanistic studies to date have focused on H3Q5sers roles in modulating H3K4me3 reader interactions, where it has been shown to potentiate TAF3/TFIID binding to H3K4me31,20,21 and inhibit the recruitment of K4me3 demethylases21; however, whether H3 serotonylation functions as an autonomous chromatin signaling mark through dedicated reader proteins has remained unknown. Here, using a combination of proteomic-, structural-, molecular-, epigenomic-, and cellular-based approaches, we demonstrate that the Quinone reductase 2 (QR2) enzyme reads H3Q5ser independently of H3K4me3. CRISPR-Cas9-mediated disruption of H3 serotonylation or QR2s binding to the mark in human induced pluripotent stem cell-derived neurons impairs the establishment of neuronal transcriptional programs, alters synaptic connectivity, and disrupts electrophysiological maturation. These findings thus uncover an H3 serotonylation-dependent chromatin signaling axis that is essential for human neurodevelopment.

molecular biology↗

bioRxiv2026

Traceless protein semi-synthesis in cells using the promiscuous ultra-fast split intein NrdJ-1

Chemistry-driven approaches that allow proteins to be manipulated in ways not permitted by standard genetics are indispensable in the basic and applied biomedical sciences. Of the various platform technologies in common use, those that exploit intein-mediated protein splicing have proven especially powerful owing to compatibility with both in vitro and in vivo applications. Here, we provide detailed biochemical characterization of the split intein, NrdJ-1. We demonstrate that the rapid splicing kinetics associated with this system are largely independent of splice junction primary sequence context. The promiscuity and high efficiency of this split intein makes it well suited to the generation of chemically modified proteins in vitro and in living cells, as demonstrated by the traceless generation of semisynthetic chromatin harboring various post-translational modifications. These studies highlight the utility of NrdJ-1 for traceless protein editing and suggest it is a superior choice for many intracellular biochemical applications.

biochemistry↗

bioRxiv2026

Caenorhabditis elegans avoids Todstoff, a novel nociceptive necrotaxis cue

To survive in hazardous environments, organisms must navigate and avoid myriad threats. Many species across phyla detect warning cues in the remains of dead or injured conspecifics, allowing them to enact defensive strategies and avoid a similar fate. Here, we demonstrate that Caenorhabditis elegans senses a novel aversive cue (which we term Todstoff, or death substance) present in the remains of dead worms that induces living conspecifics to perform negative necrotaxis behaviors. Todstoff is distinct from previously-identified social signals in C. elegans, including ascaroside and alarm pheromones, and our biochemical analysis has revealed that Todstoff may be a protein-associated amino acid derivative less than 1.35 kDa in size. We determined that Todstoff is sensed by the ASH polymodal nociceptive neurons, and further, that the necrotaxis signal is transduced via the activity of G-protein coupled receptor (GPCR) signaling, the Gi/o-like protein ODR-3, TRPV channels OSM-9 and OCR-2, and glutamatergic synaptic transmission to downstream AIB interneurons. Taken together, our work illuminates a post-mortem inter-animal chemical signaling pathway that promotes death avoidance, and thus, survival.

genetics↗

bioRxiv2024

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including {gamma}CaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that {gamma}CaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

neuroscience↗

bioRxiv2024

Protein Editing using a Concerted Transposition Reaction

Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this protein transposition reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multi-subunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular cut and paste on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

biochemistry↗

bioRxiv2023

Cell surface sculpting using logic-gated protein actuators

Cell differentiation and tissue specialization lead to unique cellular surface landscapes and exacerbated or loss of expression patterns can result in further heterogenicity distinctive of pathological phenotypes1-3. Immunotherapies and emerging protein therapeutics seek to exploit such differences by engaging cell populations selectively based on their surface markers. Since a single surface antigen rarely defines a specific cell type4,5, the development of programmable molecular systems that integrate multiple cell surface features to convert on-target inputs to user-defined outputs is highly desirable. Here, we describe an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive fragments. We show that this protein actuator platform can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. Due to its intrinsic modularity and tunability, this technology is expected to be compatible with different types of inputs, targeting modalities and functional outputs, and as such will have broad application in the synthetic biology and biotechnology areas.

synthetic biology↗

bioRxiv2023

Electrostatic encoding of genome organization principles within single native nucleosomes

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

biophysics↗

bioRxiv2023

Community behaviors of bacteria in flow: Divergent roles for transport and collective morphology

Fluid flows can impact bacterial behaviors in unexpected ways (1-3). The high shear rate in heart valves should reduce colonization, but in endocarditis, valves are often counter-intuitively colonized by Staphylococcus aureus and Enterococcus faecalis (4, 5). Here we discover bacteria-specific mechanisms for preferential surface colonization in higher shear rate environments. This behavior enables bacteria that are outcompeted in low flow to dominate in high flow. Flow-dependent colonization by S. aureus and E. faecalis are mediated by distinct mechanisms that depend on each species microcolony morphologies: transport of a dispersal signaling molecule for clustered S. aureus and mechanical forces for linear chains of E. faecalis. These results suggest that microcolony morphologies have previously unappreciated costs and benefits in different environments, like those introduced by flow. One-Sentence SummaryBacterial surface colonization in high fluid flow depends upon the species clustered or chained microcolony morphologies.

microbiology↗

bioRxiv2023

Distinct phases of cellular signaling revealed by time-resolved protein synthesis

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here, we deploy a proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from pre-made parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions BCR/ABL and DNAJB1/PRKACA where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as novel therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

biochemistry↗

bioRxiv2022

Histone monoaminylation dynamics are regulated by a single enzyme and promote neural rhythmicity

Histone H3 monoaminylations at glutamine(Q) 5 represent an important family of epigenetic markers in neurons that play critical roles in the mediation of permissive gene expression (1, 2). We previously demonstrated that H3Q5 serotonylation(ser) and dopaminylation(dop) are catalyzed by the Transglutaminase 2 (TGM2) enzyme and alter both local and global chromatin states (3, 4). Here, we found that TGM2 additionally functions as an "eraser" of H3 monoaminylations that is capable of "re-writing" these epigenetic marks in cells, including a new class of this modification, H3Q5 histaminylation(his), which displays dynamic diurnal expression in brain and contributes to neural rhythmicity. We found that H3Q5his inhibits binding of the MLL1 complex to the H3 N-terminus and attenuates its methyltransferase activity on H3 lysine(K) 4. We determined that H3Q5 monoaminylation dynamics are dictated by local monoamine concentrations, which are utilized by TGM2. Taken together, we present here a novel mechanism through which a single chromatin regulatory enzyme is capable of sensing chemical microenvironments to affect the epigenetic states of cells. One sentence summaryA single enzyme, TGM2, bidirectionally controls H3 monoaminylation dynamics, which, in turn, facilitate neural rhythmicity.

molecular biology↗

bioRxiv2022

A genetically encoded photo-proximity labeling approach for mapping protein territories

Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein interactomes to be captured using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to local generation of reactive radical species within cell-permeable probes. When combined with quantitative proteomics, the system generates snapshots of protein interactions with high temporal resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein (MVP). The technology should be broadly useful in the biomedical area.

biochemistry↗

bioRxiv2021

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and post-translational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that MroQ, an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specifiy groups -I and -II, but not group-III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

microbiology↗

bioRxiv2021

Tracking chromatin state changes using μMap photo-proximity labeling

Interactions between biomolecules, particularly proteins, underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects.1,2 Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health.1 However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study.3 Chromatin remodelers, modifying enzymes, interactors, and transcription factors can all be redirected by subtle changes to the microenvironment, causing global changes in protein expression levels and subsequent physiology. Here, we describe the Chroma-Map method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment for analysis via quantitative chemoproteomics.4 We demonstrate this concept on nine different nuclear proteins with varied function and in each case, elucidating their microenvironments. Additionally, we show that this short-range proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. Chroma-Map improves our fundamental understanding of nuclear protein-protein interactions, as well as the effects that small molecule therapeutics have on the local chromatin environment, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

cancer biology↗

bioRxiv2019

PRC2 bridges non-adjacent nucleosomes to establish heterochromatin

Polycomb repressive complex 2 (PRC2) maintains transcriptionally silent heterochromatin by installing and spreading repressive histone methylation marks along nucleosome arrays. Despite extensive research, the mechanism by which PRC2 engages with chromatin remains incompletely understood. Here we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the interactions of PRC2 with polynucleosome substrates. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides interacting with bare DNA, mononucleosomes, and neighboring nucleosome pairs, PRC2 is also found to bridge non-adjacent nucleosomes, an activity associated with chromatin compaction. Furthermore, the distribution and stability of these PRC2-chromatin interaction modes are differentially modulated by accessory PRC2 cofactors, oncogenic histone mutations, and the methylation state of chromatin. Overall, this work provides a paradigm for understanding the physical basis of epigenetic maintenance mediated by Polycomb group proteins.

biophysics↗

bioRxiv2019

JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, like dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1s targeting to chromatin depends on a new PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). The JASPer-JIL-1 (JJ)-complex is the major form of the kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes. Put in place, the complex modulates the transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identified many new interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation, but also coordinates chromatin-based regulation in the transcribed part of the genome.

molecular biology↗

bioRxiv2019

PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Polycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors- the K27M oncohistone and the EZHIP oncohistone-mimic- that dysregulate gene silencing to promote tumorigenesis.

cancer biology↗