Biology subjects

Carmody, M.

Publications and source records attributed to Carmody, M..

5 recordsLinked to original sources

bioRxiv2025

High-throughput screening and structure-guided design of small molecules enable modulation of SAL-PAP stress signaling

Chloroplasts sense environmental stress and activate chloroplast-to-nucleus retrograde signalling, reprogramming nuclear gene expression to drive plant acclimation. One such pathway is regulated by the chloroplastic phosphatase, SAL, which hydrolyses the nucleotide signal 3-phosphoadenosine 5-phosphate (PAP). In Arabidopsis thaliana, genetic loss of AtSAL1 elevates PAP and enhances stress tolerance but causes pleiotropic growth defects, complicating the interpretation of the role of PAP in cellular signalling. To uncouple stress signalling from genetic pleiotropy, we conducted a high-throughput in vitro screen of 13,000 small molecules and identified V20, a competitive inhibitor of AtSAL1 with three-fold greater potency than the known Li inhibitor. Structural analoguing of V20 and biochemical assays defined key pharmacophore features required for inhibition, including an ortho-halogenated aromatic ring, hydrogen-bond donor capacity at the triazole carboxamide, and electron-withdrawing substitutions that enhance {pi}-{pi} stacking with aromatic residues. Accelerated molecular dynamics simulations using a new high-resolution crystal structure revealed two previously uncharacterised V20 binding pockets adjacent to the catalytic site. V20 binding induces conformational changes which restrict substrate access to the catalytic site. Exogenous application of V20 to Arabidopsis led to increased PAP accumulation, activated PAP-responsive gene expression and enhanced oxidative tolerance, demonstrating cellular uptake and in vivo efficacy in the energy organelles, chloroplasts and mitochondria, where AtSAL1 is localised. Collectively, our findings reveal new insights into the regulatory domains of SAL enzymatic activity for control of PAP-mediated signalling and establish a proof-of-concept for targeted chemical modulation of SAL activity, which offers novel strategies to selectively manipulate chloroplast-to-nucleus retrograde signalling in plants. Significance StatementWe applied drug discovery strategies to test 13,000 small molecules for inhibition of AtSAL1, a key regulator of plant stress signalling from chloroplasts. Using biochemical assays, chemical analogues and molecular dynamic simulations, we defined the structural-activity relationship of a lead compound, V20, and elucidated its inhibitory mechanism. Application of V20 to plant leaves elevated the levels of AtSAL1s natural substrate, PAP (a chloroplast stress signalling molecule), activated stress-responsive genes and improved oxidative stress tolerance in Arabidopsis. This on-demand activation of SAL-PAP chloroplast signalling avoids pleotropic effects observed in loss-of-function mutants. Our approach provides a new strategy to study chloroplast-mediated cellular signaling pathways that are crucial for plant acclimation to stressful environmental conditions such as excessive sunlight.

plant biology↗

bioRxiv2025

Coumarins disrupt cell-cell communication for control of pathogenesis and virulence in ESKAPEEs and fungal opportunists

Intricate communication networks and sensing systems underpin the complexity of microbe-host interactions, enabling spatiotemporal control of optimised microbe-host consortia in a diverse range of ecosystems. A central component of these complex interactomes has been the signalling events that enable recognition of host and microbe, whether that niche be clinical or environmental, human or plant. Coumarins have emerged as significant plant derived signalling molecules shaping microbiome dynamics and pathogen behaviours from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in signal interference and control of pathogenesis in bacterial and fungal pathogens, uncovering an important hydroxylation-motif in the specific inhibition of two Pseudomonas aeruginosa interspecies and interkingdom communication molecules. Characterisation of the anti-biofilm activity of coumarins revealed changes in exopolysaccharide production independent of the initial attachment phenotype. Molecular modelling provides an insight into the receptor binding dynamics of three closely related natural coumarins, suggesting an intricate and highly specific host-microbe interaction at the species level. As the very real threat of antimicrobial resistance continues to shadow our horizons, phytochemicals such as coumarins have potential to deliver an ecological solution to dysbiosis in the host-microbe interaction. ImportanceNatural ecosystems rely on homeostatic interactions between the kingdoms of life to ensure sustainable and balanced communities can persist. Plant-derived coumarins have recently emerged as playing an important role in shaping microbial communities, presenting a remarkable chemical diversity that can influence the behaviour of bacteria and fungi. At the same time, one of the major challenges to human health continues to be the spread of antimicrobial resistance and the parallel absence of a concerted effort to source and produce new antibiotics at industrial scale. Therefore, new approaches to the control of infection are required, and an ecosystem-level lens may offer one such innovative intervention. Coumarins have the potential to neutralise the very mechanisms used by bacteria and fungi to cause infection leading to morbidity and mortality in hosts ranging from plant to animals. Here we present a mechanistic insight into how effective these molecules can be in targeting keystone pathogens termed the ESKAPEEs and their fungal counterparts.

microbiology↗

bioRxiv2023

Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages

Breast cancer is a heterogeneous disease, and treatment is guided by biomarker profiles representing distinct molecular subtypes. Breast cancer arises from the breast ductal epithelium, and experimental data suggests breast cancer subtypes have different cells of origin within that lineage. The precise cells of origin for each subtype and the transcriptional networks that characterize these tumor-normal lineages are not established. In this work, we applied bulk, single-cell (sc), and single-nucleus (sn) multi-omic techniques as well as spatial transcriptomics and multiplex imaging on 61 samples from 37 breast cancer patients to show characteristic links in gene expression and chromatin accessibility between breast cancer subtypes and their putative cells of origin. We applied the PAM50 subtyping algorithm in tandem with bulk RNA-seq and snRNA-seq to reliably subtype even low-purity tumor samples and confirm promoter accessibility using snATAC. Trajectory analysis of chromatin accessibility and differentially accessible motifs clearly connected progenitor populations with breast cancer subtypes supporting the cell of origin for basal-like and luminal A and B tumors. Regulatory network analysis of transcription factors underscored the importance of BHLHE40 in luminal breast cancer and luminal mature cells, and KLF5 in basal-like tumors and luminal progenitor cells. Furthermore, we identify key genes defining the basal-like (PRKCA, SOX6, RGS6, KCNQ3) and luminal A/B (FAM155A, LRP1B) lineages, with expression in both precursor and cancer cells and further upregulation in tumors. Exhausted CTLA4-expressing CD8+ T cells were enriched in basal-like breast cancer, suggesting altered means of immune dysfunction among breast cancer subtypes. We used spatial transcriptomics and multiplex imaging to provide spatial detail for key markers of benign and malignant cell types and immune cell colocation. These findings demonstrate analysis of paired transcription and chromatin accessibility at the single cell level is a powerful tool for investigating breast cancer lineage development and highlight transcriptional networks that define basal and luminal breast cancer lineages.

cancer biology↗

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2020

Genetic screen to saturate guard cell signaling network reveals a role of GDP-L-fucose metabolism in stomatal closure

Guard cells regulate plant gas exchange by controlling the aperture of stomatal pores. The process of stomatal closure involves a multi-input signaling network that governs the activity of ion channels, which in turn regulate guard cell turgor pressure and volume. Here we describe a forward genetic screen to identify novel components involved in stomatal movements. Through an ozone-sensitivity approach combined with whole-rosette gas exchange analysis, 130 mutants of established stomatal regulators and 76 novel mutants impaired in stomatal closure were identified. One of the novel mutants was mapped to MURUS1 (MUR1), the first enzyme in de novo GDP-L-fucose biosynthesis. Defects in synthesis or import of GDP-L-Fuc into the Golgi apparatus resulted in impaired stomatal closure to multiple stimuli. Stomatal phenotypes observed in mur1 were independent from the canonical guard cell signaling and instead could be related to altered mechanical properties of guard cell walls. Impaired fucosylation of xyloglucan, N-linked glycans and arabinogalactan proteins did not explain the aberrant function of mur1 stomata, however our data suggest that the stomatal phenotypes observed in mur1 can at least partially be attributed to defective dimerization of rhamnogalactouronan-II. In addition to providing the genetic framework for future studies on guard cell signaling, our work emphasizes the impact of fucose metabolism on stomatal movement.

plant biology↗