Biology subjects

Conway, S. J.

Publications and source records attributed to Conway, S. J..

12 recordsLinked to original sources

bioRxiv2026

Redox-dependent lipophilicity of phenazine metabolites is modulated by intramolecular hydrogen bonds and controls their biological distribution

Phenazines are redox-active microbial metabolites produced and secreted in diverse ecological contexts from soils to chronic infections. In these disparate environments phenazines can function variously as antibiotics, extracellular electron shuttles, and nutrient scavengers. Key to understanding the impact of these functions is a robust expectation of phenazine retention or diffusion in a given context. But predicting phenazine fate and transport is difficult because of the chemical complexity of their local microenvironments. To address this challenge, we measured the octanol water distribution coefficient (LogD) as a proxy for lipophilicity of three naturally occurring phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa: phenazine-1-carboxylic acid, phenazine-1-carboxamide, and pyocyanin. We investigated the behavior of both oxidized and reduced forms of these phenazines across broad ionic strength and pH conditions. While the ionic context exerts only small effects, the pH and redox state contribute strongly and independently to changes in phenazine lipophilicity. The pH trends are expected, but the observed redox dependence is generally missed by existing lipophilicity calculation methods. Additional LogD measurements with 1-hydroxyphenazine and unsubstituted phenazine, together with density functional theory modeling of phenazines in their reduced and oxidized forms, reveal that intramolecular hydrogen bonding contributes significantly to the increased lipophilicity of reduced phenazines that possess H-bond accepting substituents in the 1-position. These results explain phenazine behavior in a biological context: redox state alone significantly alters retention of pyocyanin in planktonic P. aeruginosa cells, with the reduced species being predominantly retained by membranes. We propose that the modulation of phenazine lipophilicity in response to the local redox environment has evolved to give a competitive advantage to bacteria by retaining or dispersing these bioactive molecules. Beyond improving our understanding of natural phenazine fate in diverse microbial contexts, our results emphasize an oft-overlooked theme relevant to rational drug and electrochemical shuttle design: redox state matters.

biochemistry↗

bioRxiv2026

Discovery of a phenazine thiol conjugase from sparse data using genome-informed machine learning

Machine learning has enabled powerful biological discoveries using models trained on large datasets. However, for many important biological questions, such as identifying enzymes that transform understudied substrates, sparsity of training data is often a major bottleneck. Here, using phenazine natural products as a case study, we show that integrating genome-informed data augmentation with contrastive learning in protein language space enables identification of phenazine-interacting proteins starting from only 14 known phenazine modifying sequences. Applying this framework led to the discovery of PTC (Phenazine-Thiol Conjugase), the first enzyme known to catalyze phenazine thioconjugation, a phenazine modification reaction long observed but previously presumed to occur only through non-enzymatic chemistry. In silico simulation and experimental measurements demonstrate that PTC binds to both phenazine and glutathione as substrates. Recombinant expression and biochemical characterization reveal that PTC promotes glutathione-dependent modification of phenazines, yielding distinct reaction outcomes that depend on substrate identity. Although thiol-conjugated phenazine products exhibit reduced toxicity to bacterial cells, deletion of the gene encoding PTC does not confer a strong fitness disadvantage, illustrating how direct learning of sequences can uncover relevant enzymes that might evade phenotype-based genetic screens. Together, these results demonstrate that coupling comparative genomics with protein machine learning can convert "small data" typically outside the scope of machine learning into actionable predictive power, thereby facilitating enzyme discovery. SignificanceMachine learning excels when large, well-labeled datasets are available, yet many biologically important problems lack sufficient experimental data to support such approaches to discovery. This limitation is particularly acute for identifying enzymes acting on rare or understudied substrates. Here, we show that genomic organization can be leveraged as an additional source of biological information to address data sparsity. Starting with only 14 enzymes experimentally shown to modify phenazines, we developed a model identifying phenazine-interacting enzymes by integrating genome-informed data augmentation with protein machine learning. Guided by the model, we discovered the first enzyme known to catalyze thioconjugation modifications of phenazines, demonstrating a simple yet powerful strategy for extracting predictive insight from sparse biological knowledge.

microbiology↗

bioRxiv2026

A transcriptional code controlling fluid shear stress-induced gene expression

The formation and health of the vascular system is dependent on fluid shear stress (FSS), a hemodynamic force exerted onto endothelium by flowing blood. FSS strongly induces the endothelial expression of Kruppel-like factor 2 (KLF2), an atheroprotective TF essential for vascular development and homeostasis. Despite its early and crucial role in the cascade of cardiovascular events triggered by FSS, the transcriptional mechanisms by which FSS regulates KLF2 expression remain unclear, although they are known to involve the widely expressed MEF2 proteins. Here, we identified and characterized two FSS-dependent enhancers for KLF2 which collectively recapitulate endogenous endothelial KLF2 expression, and determined the TFs contributing to their regulation. This analysis identified an essential and precisely spaced MEF2-TBP double motif also shared by the FSS-sensitive KLF2 promoter. MEF-TBP double motifs are extremely rare across the genome but were also found within regulatory elements of three other FSS-induced KLF genes, including KLF4. Although normally part of the basal transcriptional machinery, TBP specifically bound all KLF elements at the MEF-TBP double motifs in a FSS-dependent manner. Collectively, this work demonstrates a specific and targetable requirement for combined MEF2-TBP binding during FSS-induced gene activation. Significance statementBlood flow induces a force known as fluid shear stress (FSS) which is required for vascular development and for the health of the mature arterial system. One of the first endothelial responses to FSS is the induction of Kruppel-like transcription factors (KLFs). However, the mechanisms by which FSS activates KLF gene expression are incompletely understood. In this paper, we characterized all regulatory elements involved in driving FSS-induced expression of KLF2. This identified an essential MEF2-TBP double motif that was extremely rare across the genome, yet found within regulatory elements for multiple FSS-responsive KLF genes including KLF2 and KLF4. This MEF2- and TBP-bound motif therefore enables blood flow to specifically activate the cascade of cardiovascular responses necessary for atheroprotective gene expression.

developmental biology↗

bioRxiv2025

Nitric oxide tunes secreted metabolite bioactivity

The radical nitric oxide ({middle dot}NO) is short-lived but has imprinted itself on many aspects of physiology and disease. {middle dot}NOs rapid production and consumption, coupled with its intrinsic reactivity, drive its biological importance; thus, defining mechanisms and targets of {middle dot}NO reactivity is necessary to assess its fate and impact. Cellular small molecules are a major class of {middle dot}NO-reactive targets, possessing a variety of molecular functionalities that can react with {middle dot}NO. Yet the capacity for secreted small molecules to react with {middle dot}NO, as well as the biological consequences of such reactivity, have received little attention. Here, we explore the reactivity of {middle dot}NO with phenazine metabolites, microbially-derived secreted small molecules that possess antibiotic properties and can modulate their microenvironment. Using Pseudomonas aeruginosa as a model phenazine producer, we find that {middle dot}NO reacts with specific phenazines to yield stable, chemically-distinct products. These chemical transformations significantly attenuate phenazine antibiotic properties, including against the phenazine nonproducer Staphylococcus aureus, a competitor with P. aeruginosa for niches in the context of infection. By contrast, P. aeruginosa experiences rapid loss in viability when phenazines and {middle dot}NO react. This toxicity occurs even in the presence of S. aureus, which displays resistance to nitrosylated phenazines, implicating a specific toxicity dependent on the formation of the phenazine-NO adduct. These findings highlight the capacity of {middle dot}NO to transform metabolite activity and suggest that {middle dot}NO can tune microbial interactions in complex environments by a mechanism of action hitherto unappreciated.

microbiology↗

bioRxiv2025

Deficient Cardiolipin Remodeling Alters Muscle Fiber Composition and Neuromuscular Connectivity in Barth Syndrome

BackgroundBarth syndrome (BTHS) is a rare X-linked mitochondrial disorder caused by mutations in the TAFAZZIN gene, which disrupts cardiolipin (CL) remodeling and mitochondrial function. While cardiac manifestations of BTHS are well characterized, the mechanisms underlying skeletal muscle weakness and fatigability are poorly understood. MethodsWe investigated neuromuscular and mitochondrial alterations in a novel murine model (TazPM) carrying a patient-derived D75H point mutation in Tafazzin. This mutation preserves protein abundance but abolishes enzymatic activity. Skeletal muscle function was assessed via weightlifting and hanging tests. Muscle fiber composition and neuromuscular junction (NMJ) integrity were evaluated using immunofluorescence, western blotting, and in vivo electrophysiology. Mitochondrial morphology was examined by transmission electron microscopy, and bioenergetics were quantified using ultra-performance liquid chromatography. Stress signaling was assessed by western blotting. ResultsMale TazPM mice exhibited elevated monolysocardiolipin and reduced mature CL levels, confirming deficient transacylase activity. These mice exhibited lower muscle strength and endurance, smaller muscle fibers of all types, and a shift toward fast-twitch type 2B fibers, which are more susceptible to fatigue. Electrophysiological analysis revealed a 60% reduction in motor unit number and an increase in average single motor unit potential, indicating motor neuron remodeling. NMJ protein analysis showed decreased MUSK and DOK7 and increased CHRNA1, suggesting impaired NMJ integrity. Despite mitochondrial structural abnormalities and reduced expression of key mitochondrial proteins (NDUFB8, MCU, TMEM65), resting ATP, phosphocreatine, and adenine nucleotide ratios were unchanged in both glycolytic and oxidative muscles. However, stress signaling pathways were markedly activated, including phosphorylation of eIF2, increased CHOP, DELE1, p53 expression, and altered Wnt/{beta}-catenin signaling components. ConclusionsDeficiency of Tafazzin enzymatic activity in skeletal muscle is sufficient to result in widespread neuromuscular remodeling, including fiber size/type shifts, motor unit loss, NMJ dysregulation, and stress pathway activation, without overt energetic failure at rest. These findings suggest that myopathy in BTHS arises not solely from mitochondrial ATP insufficiency but rather from cumulative structural and signaling disruptions.

cell biology↗

bioRxiv2025

Hypoxia-activated fluorescent probes as markers of oxygen levels in plant cells and tissues

O_LILow oxygen signalling in plants is important in development and stress responses. Measurement of oxygen levels in plant cells and tissues is hampered by a lack of chemical tools with which to reliably detect and quantify endogenous oxygen availability. We have exploited hypoxia-activated fluorescent probes to visualise low oxygen (hypoxia) in plant cells and tissues. C_LIO_LIWe applied 4-nitrobenzyl (4NB-) resorufin and methyl-indolequinone (MeIQ-) resorufin to Arabidopsis thaliana whole cells and seedlings exposed to hypoxia (1% O2) and normoxia (21% O2). Confocal microscopy and fluorescence intensity measurements were used to visualise regions of resorufin fluorescence. C_LIO_LIBoth probes enter A.thaliana whole cells and are activated to fluoresce selectively in hypoxic conditions. Similarly, incubation with A.thaliana seedlings resulted in hypoxia-dependent activation of both probes and observation of fluorescence in hypoxic roots and leaf tissue. MeIQ-Resorufin was used to visualise endogenous hypoxia in lateral root primordia of normoxic A.thaliana seedlings. C_LIO_LIOxygen measurement in plants until now has relied on invasive probes or genetic manipulation. Use of these chemical probes to detect applied and endogenous hypoxia has the potential to facilitate a greater understanding of oxygen dynamics in plant cells and tissues, allowing correlation of oxygen concentrations with adaptive and developmental responses to hypoxia. C_LI

plant biology↗

bioRxiv2024

Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation. One-Sentence SummaryEngineered zinc-finger-based degrons enable targeted protein degradation induced by selective molecular glues.

biochemistry↗

bioRxiv2023

Bromodomain Factor 5 as a Target for Antileishmanial Drug Discovery

Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarisation assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarisation and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC-CBP30, bromosporine and I-BRD9, moreover, SGC-CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a drug target in Leishmania and provide a foundation for the future development of optimised antileishmanial compounds targeting this epigenetic reader protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/554125v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@16be162org.highwire.dtl.DTLVardef@19a1cf3org.highwire.dtl.DTLVardef@e29c7forg.highwire.dtl.DTLVardef@190564d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Quantitative live-imaging of Aquilegia floral meristems reveals distinct patterns of floral organ initiation and cell-level dynamics of floral meristem termination

In-depth investigation of any developmental process in plants requires knowledge of both the underpinning molecular networks and how they directly determine patterns of cell division and expansion over time. Floral meristems (FM) produce floral organs, after which they undergo floral meristem termination (FMT), and precise control of organ initiation and FMT is crucial to reproductive success of any flowering plant. Using a live confocal imaging, we characterized developmental dynamics during floral organ primordia initiation and FMT in Aquilegia coerulea (Ranunculaceae). Our results have uncovered distinct patterns of primordium initiation between stamens and staminodes compared to carpels, and provided insight into the process of FMT, which is discernable based on cell division dynamics preceding carpel initiation. To our knowledge, this is the first quantitative live imaging of meristem development in a system with numerous whorls of floral organs as well as an apocarpous gynoecium. This study provides crucial information for our understanding of how the spatial-temporal regulation of floral meristem behavior is achieved in both an evolutionary and developmental context.

developmental biology↗

bioRxiv2020

Naa12 rescues embryonic lethality in Naa10-Deficient Mice in the amino-terminal acetylation pathway

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralogue with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

developmental biology↗

bioRxiv2020

Developmental Expression of Transforming Growth Factor Induced Protein Promotes NF-Kappa-B Mediated Angiogenesis During Postnatal Lung Development

RationalePulmonary angiogenesis is a key driver of alveolarization. Our prior studies showed that nuclear factor kappa-B (NF{kappa}B) promotes pulmonary angiogenesis during early alveolarization. However, the mechanisms regulating temporal-specific NF{kappa}B activation in the pulmonary vasculature are unknown. ObjectivesTo identify mechanisms that activate pro-angiogenic NF{kappa}B signaling in the developing pulmonary vasculature. MethodsProteomic analysis of the lung secretome was performed using 2D-DIGE. NF{kappa}B activation and angiogenic function was assessed in primary pulmonary endothelial cells (PEC) and TGFBI-regulated genes identified using RNA-sequencing. Alveolarization and pulmonary angiogenesis was assessed in WT and TGFBI null mice exposed to normoxia or hyperoxia. Lung TGFBI expression was determined in premature lambs supported by invasive and noninvasive respiratory support. Measurements and Main ResultsSecreted factors from the early alveolar, but not the late alveolar or adult lung, promoted proliferation and migration in quiescent, adult PEC. Proteomic analysis identified transforming growth factor beta-induced protein (TGFBI) as a protein highly expressed by myofibroblasts in the early alveolar lung that promoted PEC migration by activating NF{kappa}B via v{beta}3 integrins. RNA-sequencing identified Csf3 as a TGFBI-regulated gene that enhances nitric oxide production in PEC. Loss of TGFBI in mice exaggerated the impaired pulmonary angiogenesis induced by chronic hyperoxia, and TGFBI expression was disrupted in premature lambs with impaired alveolarization. ConclusionsOur studies identify TGFBI as a developmentally-regulated protein that promotes NF{kappa}B-mediated angiogenesis during early alveolarization by enhancing nitric oxide production. We speculate that dysregulation of TGFBI expression may contribute to diseases marked by impaired alveolar and vascular growth.

developmental biology↗

bioRxiv2018

LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged.

plant biology↗