Biology subjects

Smith, B. C.

Publications and source records attributed to Smith, B. C..

15 recordsLinked to original sources

bioRxiv2026

TAOK3 inhibition constrains invasion, potentiates paclitaxel, and reprograms the tumor microenvironment toward anti-tumor immunity in cervical cancer

TAOK3 is a lesser-studied MAPK family serine/threonine kinase our group has shown to be targeted by HPV integration, suggesting a potential role in driving invasive cervical cancer (ICC). Here, we profiled TAOK3 expression in patient tumors, metastases, and cervical cancer models and localized TAOK3 within a tumor epithelial subpopulation by integrating two single-cell RNA-seq datasets. Functional consequences of TAOK3 loss were assessed with siRNA and CRISPRi in cell lines and 3D spheroids. In vivo effects were evaluated in intracervical xenografts with species-specific RNA-seq to resolve tumor versus microenvironmental responses. TAOK3 mRNA/protein were elevated in primary and metastatic ICC and primarily localized to a keratin-positive epithelial subset (T3epi) enriched for cadherin/S100 binding, vesicle/endocytic pathways, and leading-edge programs. TAOK3 silencing reprogrammed transcriptomes and proteomes toward reduced WNT/cell-cycle and motility signaling, altered endocytosis and cytoskeleton organization, and reshaped phospho-networks linked to chromatin remodeling and ERBB2-ERBB3/cytoskeletal kinase activity. Functionally, TAOK3 inhibition prolonged G2/M, suppressed invasion, and enhanced sensitivity to low dose paclitaxel. Prolonged inactivation induced methuosis-like cell death with extracellular ATP release. In xenografts, TAOK3 knockdown reduced tumor burden, downregulated KRT14--a leader cell marker--within the human tumor compartment, and enriched microenvironmental pathways for immune activation, with a specific decrease in CD206+ M2 macrophages. TAOK3 delineates an invasion-competent epithelial state in ICC and coordinates cell-cycle control, cytoskeleton-membrane dynamics, and tumor-immune crosstalk. Genetic or pharmacologic TAOK3 inhibition constrains tumor growth, potentiates paclitaxel, and remodels the microenvironment toward anti-tumor immunity, supporting TAOK3 as a potential therapeutic target and biomarker in ICC. Statement of SignificanceTAOK3 marks an invasion-competent epithelial subpopulation in cervical cancer. TAOK3 inhibition slows tumor growth, enhances chemoresponse, and reduces M2 macrophages, revealing TAOK3 as a potential therapeutic target and biomarker for patient stratification.

cancer biology↗

bioRxiv2026

BRD4 represses developmental and neuronal genes through interactions with polycomb complexes

BRD4 is best known as a transcriptional co-activator, yet heterozygous loss-of-function variants cause craniofacial and neurodevelopmental abnormalities through unclear mechanisms. Using human embryonic stem cells and neural organoids as an in vitro model of human embryonic brain development, we show that BRD4 represses polycomb-regulated developmental and neuronal genes. BRD4 co-occupies bivalent promoters with PRC1.6 and directly interacts with PCGF6 and RING1B through its C-terminal domain. H3K23ac contributes to BRD4 recruitment through BD2, linking acetylated chromatin recognition to polycomb-associated repression. Acute BRD4 depletion alters PRC1.6 occupancy, reduces ubiquitination of H2AK119 and rapidly increases developmental gene transcription. In neural organoids, BRD4 BD2 mutations alter neuronal cell composition and increase expression and chromatin accessibility associated with neuronal transcription-factor programmes. These findings establish BRD4 as a context-dependent chromatin regulator that prevents premature activation of developmental genes and provide mechanistic insight into BRD4-associated neurodevelopmental disorders.

molecular biology↗

bioRxiv2025

Gliomas phenocopy an inborn error of metabolism to drive neuronal activity and tumor growth

The metabolic hallmarks of high-grade glioma (HGG) are not fully understood. Human brain tissue metabolomics revealed that the creatine synthesis pathway intermediate guanidinoacetate (GAA) accumulated [~]100-fold in HGGs relative to controls, which was caused by imbalanced activities of enzymes in this pathway. Glioma cells secreted GAA rather than using it to produce creatine, implicating an unexpected function. GAA accumulates in GAMT deficiency, an inborn error of metabolism, and elevates neuronal excitability. Neuronal excitability is also increased in glioma and drives tumor growth through neuron-glioma interactions. We hypothesized that glioma-generated GAA excites surrounding neurons. Indeed, GAA induced neuronal hyperactivity by activating GABAA receptors and causing depolarizing GABA currents in glioma-associated neurons with dysregulated chloride homeostasis. Depleting tumoral GAA decreased electrochemical activity, neuron-glioma interactions, and tumor aggressiveness. Our findings unveil a new mechanism linking cancer metabolism with cancer neuroscience and leverage human genetics to nominate GAA synthesis as a target in gliomas.

cancer biology↗

bioRxiv2025

Nitrogen metabolism profiling reveals cell state-specific pyrimidine synthesis pathway choice

Conventional stable isotope tracing assays track one or several metabolites. However, cells use an array of nutrients to sustain nitrogen metabolic pathways. This incongruency hampers a system level understanding of cellular nitrogen metabolism. Therefore, we created a platform to simultaneously trace 30 nitrogen isotope-labeled metabolites. This platform revealed that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine despite expressing de novo pathway enzymes. This link between cell state and pyrimidine synthesis routes persisted in physiological contexts, including primary murine and human tissues and tumor xenografts. Mechanistically, we found that Ser1900 phosphorylation of CAD, the first enzyme of the de novo pathway, was enriched in primitive cells and that mimicking this modification in differentiated cells abrogated their preference for pyrimidine salvage. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.

cell biology↗

bioRxiv2024

BATF2 is a regulator of interferon-gamma signaling in astrocytes during neuroinflammation

Astrocytic interferon (IFN){gamma} signaling is associated with a reduction in neuroinflammation. We have previously shown that the benefits of astrocytic IFN{gamma} arise from a variety of mechanisms; however, downstream effectors responsible for regulating this protection are unknown. We address this by identifying a specific transcription factor that may play a key role in modulating the consequences of IFN{gamma} signaling. RNA-sequencing of primary human astrocytes treated with IFN{gamma} revealed basic leucine zipper ATF-like transcription factor (BATF)2 as a highly expressed interferon-specific gene. Primarily studied in the periphery, BATF2 has been shown to exert both inflammatory and protective functions; however, its function in the central nervous system (CNS) is unknown. Here, we demonstrate that human spinal cord astrocytes upregulate BATF2 transcript and protein in an IFN{gamma}-specific manner. Additionally, we found that BATF2 prevents overexpression of interferon regulatory factor (IRF)1 and IRF1 targets such as Caspase-1, which are known downstream pro-inflammatory mediators. We also show that Batf2-/- mice exhibit exacerbated clinical disease severity in a murine model of CNS autoimmunity, characterized by an increase in both CNS immune cell infiltration and demyelination. Batf2-/- mice also exhibit increased astrocyte-specific expression of IRF1 and Caspase-1, suggesting an amplified interferon response in vivo. Further, we demonstrate that BATF2 is expressed primarily in astrocytes in MS lesions and that this expression is co-localized with IRF1. Collectively, our results further support a protective role for IFN{gamma} and implicate BATF2 as a key suppressor of overactive immune signaling in astrocytes during neuroinflammation.

neuroscience↗

bioRxiv2023

CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

Macrophage efferocytosis, the process by which phagocytes engulf and remove apoptotic cells (ACs), plays a critical role in maintaining tissue homeostasis. Efficient efferocytosis prevents secondary necrosis, mitigates chronic inflammation, and impedes atherosclerosis progression. However, the regulatory mechanisms of efferocytosis under atherogenic conditions remain poorly understood. We previously demonstrated that oxidized LDL (oxLDL), an atherogenic lipoprotein, induces mitochondrial reactive oxygen species (mtROS) in macrophages via CD36. In this study, we demonstrate that macrophage mtROS facilitate continual efferocytosis through a positive feedback mechanism. However, oxLDL disrupts continual efferocytosis by dysregulating the internalization of ACs. This disruption is mediated by an overproduction of mtROS. Mechanistically, oxLDL/CD36 signaling promotes the translocation of cytosolic PKM2 to mitochondria, facilitated by the chaperone GRP75. Mitochondrial PKM2 then binds to Complex III of the electron transport chain, inducing mtROS production. This study elucidates a novel regulatory mechanism of efferocytosis in atherosclerosis, providing potential therapeutic targets for intervention. SUMMARYMacrophages clear apoptotic cells through a process called efferocytosis, which involves mitochondrial ROS. However, the atherogenic oxidized LDL overstimulates mitochondrial ROS via the CD36-PKM2 pathway, disrupting continual efferocytosis. This finding elucidates a novel molecular mechanism that explains defects in efferocytosis, driving atherosclerosis progression.

immunology↗

bioRxiv2023

A Multi-Layered Computational Structural Genomics Approach Enhances Domain-Specific Interpretation of Kleefstra Syndrome Variants in EHMT1

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretation of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within this domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism of variant dysfunction.

genomics↗

bioRxiv2023

Astrocyte interferon-gamma signaling dampens inflammation during chronic central nervous system autoimmunity via PD-L1

Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease of the central nervous system (CNS). Infiltrating inflammatory immune cells perpetuate demyelination and axonal damage in the CNS and significantly contribute to pathology and clinical deficits. While the cytokine interferon (IFN){gamma} is classically described as deleterious in acute CNS autoimmunity, we and others have shown astrocytic IFN{gamma} signaling also has a neuroprotective role. Here, we performed RNA sequencing and ingenuity pathway analysis on IFN{gamma}-treated astrocytes and found that PD-L1 was prominently expressed. Using a PD-1/PD-L1 antagonist, we determined that apoptosis was reduced in leukocytes exposed to IFN{gamma}-treated astrocytes in vitro. To further elucidate the role of astrocytic IFN{gamma} signaling on the PD-1/PD-L1 axis in vivo, we induced the experimental autoimmune encephalomyelitis (EAE) model of MS in Aldh1l1-CreERT2, Ifngr1fl/fl mice. Mice with conditional astrocytic deletion of IFN{gamma} receptor exhibited a reduction in PD-L1 expression which corresponded to increased infiltrating leukocytes, particularly from the myeloid lineage, and exacerbated clinical disease. PD-1 agonism reduced EAE severity and CNS-infiltrating leukocytes. Importantly, PD-1 is expressed by myeloid cells surrounding MS lesions. These data support that IFN{gamma} signaling in astrocytes diminishes inflammation during chronic autoimmunity via upregulation of PD-L1, suggesting potential therapeutic benefit for MS patients.

neuroscience↗

bioRxiv2023

NMR Resonance Assignment of the LA Motif of Human LA-Related Protein 1

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5 s terminal oligopyrimidine (5TOP) mRNAs as well as other mRNAs and binds to both the 5TOP motif and the 3-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

biophysics↗

bioRxiv2023

Beyond Structural Bioinformatics for Genomics with Dynamics Characterization of an Expanded KRAS Mutational Landscape

ABSTRACTCurrent capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation. Key PointsPlease provide 3 bullet points summarizing the manuscripts contribution to the field (100 characters max per point) 1) We functionally grouped 86 distinct KRAS mutations using MD scores, demonstrating scalability for genomics 2) MD-based groups explain experimental differences and mechanistic information about mutant proteins 3) Demonstrated added functional information from simulations for interpreting human genetic variation

genomics↗

bioRxiv2019

Regional astrocyte interferon-γ signaling regulates immunoproteasome-mediated protection during chronic autoimmunity

In early autoimmune neuroinflammation, interferon (IFN){gamma} and its upregulation of the immunoproteasome (iP) is pathologic. However, during chronic multiple sclerosis (MS), IFN{gamma} has protective properties and, the role of the iP remains to be fully elucidated. Here, we demonstrated that IFN{gamma} signaling in regional astrocytes induces the iP and protects the CNS during autoimmunity. In MS tissue, iP expression was enhanced in spinal cord compared to brainstem lesions, which correlated with a decrease in oxidative stress. In vitro, IFN{gamma} stimulation enhanced iP expression, reduced reactive oxygen species burden, and decreased oxidatively damaged and poly-ubiquitinated protein accumulation preferentially in human spinal cord astrocytes, which was abrogated with the use of the iP inhibitor, ONX 0914. During the chronic phase of an MS animal model, experimental autoimmune encephalomyelitis (EAE), ONX 0914 treatment exacerbated disease and led to increased oxidative stress and poly-ubiquitinated protein build-up. Finally, mice with astrocyte-specific loss of the IFN{gamma} receptor exhibited worsened chronic EAE associated with reduced iP expression, enhanced lesion size and oxidative stress and poly-ubiquitinated protein accumulation in astrocytes. Taken together, our data reveal a protective role for IFN{gamma} in chronic neuroinflammation and identify a novel function of the iP in astrocytes during CNS autoimmunity.

neuroscience↗

bioRxiv2019

ODORANT RECEPTOR INHIBITION IS FUNDAMENTAL TO ODOR ENCODING

Most natural odors are complex mixtures of many volatile components, competing to bind odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) of the nose. To date surprisingly little is known about how OR antagonism shapes neuronal representations in the periphery of the olfactory system. Here, we investigated its prevalence, the degree to which it disrupts OR ensemble activity, and its conservation across related ORs. Calcium imaging microscopy of dissociated OSNs revealed significant inhibition, often complete attenuation, of responses to indole, a commonly occurring volatile associated with both floral and fecal odors, by a set of 36 tested odorants. To confirm an OR mechanism for the observed inhibition, we performed single-cell transcriptomics on OSNs that exhibited specific response profiles to a diagnostic panel of odorants and identified the receptor Olfr743 which, when tested in vitro, recapitulated ex vivo responses. We screened ten ORs from the Olfr743 clade with 800 perfumery-related odorants spanning a range of chemical scaffolds and functional groups, over half of which (430) antagonized at least one of the ten ORs. Furthermore, OR activity outcomes were divergent rather than redundant, even for the most closely related paralogs. OR activity fitted a mathematical model of competitive receptor binding and suggests that normalization of OSN ensemble responses to odorant mixtures is the rule rather than the exception. In summary, we observed OR antagonism, inverse agonism and partial agonism occurring frequently and in a combinatorial manner. Thus, extensive receptor-mediated computation of mixture information appears to occur in the olfactory epithelium prior to transmission of odor information to the olfactory bulb.

neuroscience↗

bioRxiv2019

An online tool for calculating initial rates from continuous enzyme kinetic traces

A computer program was developed for semi-automated calculation of initial rates from continuous kinetic traces during the evaluation of Michaelis-Menten and EC50/IC50 kinetic parameters from high-throughput enzyme assays. The tool allows users to interactively fit kinetic traces using convenient browser-based selection tools, ameliorating tedious steps involved in defining linear ranges in general purpose programs like Microsoft Excel while still maintaining the simplicity of the \"ruler and pencil\" method of determining initial rates. As a test case, we quickly and accurately analyzed over 500 continuous enzyme kinetic traces resulting from experimental data on the response of Sirt1 to small-molecule activators. For a given titration series, our program allows simultaneous visualization of individual initial rates and the resulting Michaelis-Menten or EC50/IC50 kinetic model fit. In addition to serving as a convenient program for practicing enzymologists, our tool is also a useful teaching aid to visually demonstrate in real-time how incorrect initial rate fits can affect calculated steady-state kinetic parameters. For the convenience of the research community, we have made our program freely available online at https://continuous-enzyme-kinetics.herokuapp.com/continuous-enzyme-kinetics.

bioinformatics↗

bioRxiv2019

Nucleosome scaffolding by Brd4 tandem bromodomains in acetylation-dependent chromatin compartmentalization

Bromodomain binding of acetyl-lysine residues is a crucial step in many epigenetic mechanisms governing transcription. Nearly half of human bromodomains exist in tandem with at least one other bromodomain on a single protein. The Bromodomain and ExtraTerminal domain (BET) familyof proteins (BrdT, Brd2, Brd3 and Brd4) each encode two bromodomains at their N-termini and are important regulators of acetylation-dependent transcription in homeostasis and disease. Previous efforts have focused on identifying protein acetylation sites bound by individual bromodomains. However, the mechanisms through which tandem bromodomains act cooperatively on chromatin are largely unknown. Here, we first used small angle x-ray scattering combined with Rosetta ab initio modeling to explore conformational space available to BET tandem bromodomains. For Brd4, the flexible tandem bromodomain linker allows for distances between the two acetyl-lysine binding sites ranging from 15 to 157 [A]. Using a bioluminescence resonance energy transfer assay, we show a clear distance dependence for Brd4 tandem bromodomain bivalent binding of multiply acetylated histone H4 peptides. However, isothermal titration calorimetry studies revealed Brd4 binding affinity toward multiply acetylated peptides does not correlate with the potential for bivalent binding. We used sucrose gradient assays to provide direct evidence in vitro that Brd4 tandem bromodomains can simultaneously bind and scaffold multiple acetylated nucleosomes. Intriguingly, our bioinformatic analysis of deposited chromatin immunoprecipitation sequencing data indicates that Brd4 colocalizes with subsets of histone acetyl-lysine sites across transcriptionally active chromatin compartments. These findings support our hypothesis that scaffolding of acetylated nucleosomes by Brd4 tandem bromodomains contributes to higher-order chromatin architecture.

biochemistry↗

bioRxiv2019

Non-sedating benzodiazepines cause contractile paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports of cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines, given the known anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970s but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZs anti-parasitic target is likely distinct from the human receptors that drive sedation. Therefore, we screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, causing extensive vacuole formation beneath the apical membrane. The imidazobenzodiazepine compound series identified has a dramatically lower ([~]1 log) affinity for human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects.\n\nAuthor SummaryOver 200 million people are infected with schistosomiasis, yet there are limited therapeutic options available to treat this disease. The benzodiazepine meclonazepam is known to cure both intestinal and urinary schistosomiasis in animal and human studies, but dose-limiting sedation has been a barrier to its development. Little is known about the structure-activity relationship of meclonazepam and other benzodiazepines on schistosomes, or the identity of the parasite receptor for these compounds. However, schistosomes lack obvious homologs to the human GABAARs that cause sedation. This indicates that the parasite target of this drug is distinct from the host receptors that underpin dose-limiting side effects of meclonazepam, and raises the possibility that benzodiazepines with poor GABAAR affinity may still retain anti-parasitic effects. Here, we report an in vitro screen of various benzodiazepines against schistosomes, and the identification of hit compounds that are active against worms yet possess reduced affinity for the human GABAARs that cause sedation.

pharmacology and toxicology↗