Biology subjects

Barlow, C. K.

Publications and source records attributed to Barlow, C. K..

8 recordsLinked to original sources

bioRxiv2026

A mixer tap mechanism in Mycobacterium tuberculosis regulates a critical metabolic node in response to nutrient availability

Mycobacterium tuberculosis persists within the host by exploiting metabolic flexibility underpinned by the tricarboxylic acid (TCA) cycle-glyoxylate shunt junction, where isocitrate dehydrogenase (ICD) and isocitrate lyase (ICL) compete to direct carbon flux between energy production and carbon conservation. Yet, how nutrient availability regulates flux partitioning at this junction is unknown. We show that among the four gatekeeping isoforms of isocitrate lyase (ICL1/2) and isocitrate dehydrogenase (ICD1/2), only icl1 transcription substantially responds to carbon substrate switching. Systematic metabolite screening revealed that pyruvate, oxaloacetate, and glyoxylate are novel allosteric activators of ICD2, while concurrently inhibiting ICL1/2 at their physiological concentrations. To resolve how these regulatory layers integrate, we developed an experimental 1H NMR-based flux assay that combines recombinant enzymes with metabolomics-informed metabolite conditions to quantify flux partitioning under defined states. This systems-level framework reveals that flux partitioning emerges from coordinated balancing of enzyme abundance and protein-metabolite effector combinations that shift in a time-dependent, nutrient-responsive manner. This dynamic, multi-layered regulatory framework, analogous to a mixer-tap mechanism, offers new strategies for therapeutic disruption of M. tuberculosis metabolism.

biochemistry↗

bioRxiv2026

Generation of a novel Slc7a9G105R mutant mouse identifies new biomarkers for cystinuria

Cystinuria is a rare inherited disease characterized by increased urinary cystine levels resulting in the formation of cystine stones in the urinary tract. Mutations in the genes encoding the cystine transporter complex, SLC3A1 and SLC7A9, are the primary drivers of the disease. Current mouse models used to study cystinuria rely on gene deficiency or spontaneous mutations in mice that do not accurately reflect the pathogenic mutations found in humans. To overcome this limitation, we generated novel Slc7a9G105R mice carrying the most common pathogenic single-point mutation in the SLC7A9 gene. Both male and female Slc7a9G105R mice developed a cystinuria phenotype by 9 weeks of age, characterized by substantial cystine stone formation and increased urinary cystine, lysine, arginine, and ornithine. Slc7a9G105R mice displayed distinct serum and urinary metabolite profiles mapped to dibasic amino acid pathways. Fecal metagenomics revealed that Slc7a9G105R mice had a heterogeneous microbiota with altered functional pathways, including increased L-cysteine biosynthesis. Depletion of the microbiota with antibiotics did not impact cystine stone burden but reduced urinary tract inflammation. Prophylactic or therapeutic dietary supplementation with alpha-lipoic acid reduced stone burden and inflammation, but it also caused damage to the urothelium. Untargeted metabolomics analysis following alpha-lipoic acid supplementation identified metabolites that can increase cystine solubility, reduce inflammation, and damage epithelial cells. Correlation analysis revealed novel serum biomarkers of stone burden, including blood urea nitrogen, 2-hydroxybutyric acid, 2-amino-2-thiazoline-4-carboxylic acid, and indole-3-acetylglycine. Collectively, the Slc7a9G105R mutant mouse model offers a precise, rapid-onset, and translational platform for investigating cystinuria pathogenesis and evaluating potential therapeutic strategies. Translational statementThe development of a novel knock-in mouse model carrying the most common pathogenic point mutation in the human SLC7A9 gene provides a clinically relevant and translationally valuable platform for investigating cystinuria pathogenesis and testing emerging therapies. This model represents the closest possible approximation of human SLC7A9-mediated cystinuria, enabling rigorous preclinical evaluation of small molecules and gene therapies. It has also facilitated the identification of candidate biomarkers for cystine stone burden and treatment response, which are urgently needed to improve disease monitoring and clinical decision-making. The next critical step is to validate these biomarkers in human cystinuria cohorts to support their clinical translation.

pathology↗

bioRxiv2024

Quinone extraction drives atmospheric carbon monoxide oxidation in bacteria

Diverse bacteria and archaea use atmospheric carbon monoxide (CO) as an energy source during long-term survival. This process enhances the biodiversity of soil and marine ecosystems globally and removes 250 million tonnes of a toxic, climate-relevant pollutant from the atmosphere each year. Bacteria use [MoCu]-carbon monoxide dehydrogenases (Mo-CODH) to convert CO to carbon dioxide, then transfer the liberated high-energy electrons to the aerobic respiratory chain. However, given no high-affinity Mo-CODH has been purified, it is unknown how these enzymes oxidise CO at low concentrations and interact with the respiratory chain. Here we resolve these knowledge gaps by analysing Mo-CODH (CoxSML) and its hypothetical partner CoxG from Mycobacterium smegmatis. Kinetic and electrochemical analyses show purified Mo-CODH is a highly active high-affinity enzyme (Km = 139 nM, kcat = 54.2 s-1). Based on its 1.85 [A] resolution cryoEM structure, Mo-CODH forms a CoxSML homodimer similar to characterised low-affinity homologs, but has distinct active site coordination and narrower gas channels that may modulate affinity. We provide structural, biochemical, and genetic evidence that Mo-CODH transfers CO-derived electrons to the aerobic respiratory chain via the membrane-bound menaquinone-binding protein CoxG. Consistently, CoxG is required for CO-driven respiration, extracts menaquinone from mycobacterial membranes, and binds quinones in a hydrophobic pocket. Finally, we show that Mo-CODH and CoxG genetically and structurally associate in diverse bacteria and archaea. These findings reveal the basis of a biogeochemically and ecologically important process, while demonstrating that the newly discovered process of long-range quinone transport is a general mechanism of energy conservation, which convergently evolved on multiple occasions.

biochemistry↗

bioRxiv2023

Hypoxic injury triggers maladaptive repair in human kidney organoids

Acute kidney injury (AKI) is a common clinical disorder linked to high rates of illness and death. Ischemia is a leading cause of AKI, which can result in chronic kidney disease (CKD) through maladaptive repair marked by impaired epithelial regeneration, inflammation, and metabolic dysregulation. There are no targeted therapies for AKI or to prevent progression to CKD and insight into human disease mechanisms remains limited. Here we show that human kidney organoids recapitulate key molecular and metabolic signatures of AKI and maladaptive repair in response to hypoxic injury. Transcriptional, proteomic, and metabolomic profiling revealed tubular injury, cell death, cell cycle arrest and metabolic reprogramming in organoids exposed to hypoxia. Following return to normoxic conditions, injured organoids had increased signatures of TNF and NF-{kappa}B signalling pathways and S100A8/9, associated with maladaptive repair. Single cell RNA sequencing localized AKI and maladaptive repair markers including GDF15, MMP7, ICAM1, IL32, SPP1, C3 and CCN1 to injured tubules. Metabolic phenotypes linked to CKD were also evident, including dysregulated gluconeogenesis, altered amino acid metabolism and lipid peroxidation. iPSC-derived macrophages incorporated into organoids displayed a robust activation and inflammatory response to hypoxia. Spatial transcriptomics revealed a shift from a tissue resident-like to inflammatory macrophage states and localized effects on tubular injury and inflammation. This multi-omic analysis defines conserved mechanisms of human ischemic AKI and maladaptive repair, highlighting new opportunities to test therapeutics and model immune-mediated interactions.

systems biology↗

bioRxiv2023

A defined diet for pre-adult Drosophila melanogaster

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that improves larval growth, development rate, and rescues survival. We demonstrate its use for exploring pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder.

genetics↗

bioRxiv2021

The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation

The enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/467024v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@153dd35org.highwire.dtl.DTLVardef@dfeb43org.highwire.dtl.DTLVardef@ce8148org.highwire.dtl.DTLVardef@189d8da_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma. C_LIO_LIThe N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner. C_LIO_LIEZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2s tetramerization-mediated activity independently of EZH2 methyltransferase. C_LIO_LIEZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase. C_LIO_LISappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells. C_LI

cancer biology↗

bioRxiv2020

Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria

F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, and product bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance.

biochemistry↗

bioRxiv2019

Restriction of essential amino acids dictates the systemic response to dietary protein dilution

Dietary protein dilution (DPD) promotes metabolic remodelling and health but the precise nutritional components driving this response remain elusive. Here we demonstrate that dietary amino acids (AA) are sufficient and necessary to drive the response to DPD. In particular, the restriction of dietary essential AA (EAA) supply, but not non-EAA, drives the systemic metabolic response to total AA deprivation. Furthermore, systemic deprivation of Thr and Trp, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Thr is also potentially limiting in low-protein diet fed humans, and dietary Thr restriction (DTR) retarded the development of obesity-associated metabolic dysfunction in mice. Liver-derived fibroblast growth factor 21 was required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of Thr biosynthetic capacity reversed the systemic response to DTR. Taken together, our studies demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

physiology↗