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Kim, A.-R.

Publications and source records attributed to Kim, A.-R..

At least 19 recordsLinked to original sources

ABF2 and bZIP2 remodel root system architecture under combined phosphate deficiency and salinity in Arabidopsis

Climate change is degrading arable soils and imposing chronic abiotic stress on plants. As the primary interface with the soil, the root system is especially vulnerable, and plants respond by remodelling their root system architecture (RSA). Most studies have examined single stresses in isolation and so fail to capture the multifactorial environments in which plants actually grow. Rising salinity impairs development by compromising cellular integrity and triggering cytotoxic responses, and it simultaneously restricts uptake of phosphorus, a macronutrient required for nucleic acid and protein synthesis. Combined stresses can amplify these effects, yet how plants adjust RSA to cope with them remains poorly understood. Here we performed a meta-analysis of public transcriptomic datasets for salinity and phosphate deficiency in Arabidopsis thaliana. Integrating these data with protein-protein interaction modelling and DNA-binding (DAP-seq) analyses, we identified two basic leucine zipper transcription factors, ABF2 and bZIP2, as candidate integrators of the combined-stress response. Characterization of abf2, bzip2 and double mutants revealed RSA alterations and allowed us to identify downstream targets of each factor. Our findings offer new insight into root adaptation under combined stress and represent one of the few comprehensive analyses of how multiple abiotic stressors jointly shape root development and plant fitness.

plant biology↗

The Drosophila Secretome

Secreted proteins are synthesized within the cell and actively transported outside, where they contribute to development, physiology, immunity, and cellcell communication. In Drosophila melanogaster, many secreted signaling pathways are evolutionarily conserved, making this species a powerful model for studying human development, cancer, neurobiology, and immune regulation. To assemble the Drosophila secretome, we systematically analyzed all fly protein sequences with computational tools, integrating FlyBase, UniProt and Gene Ontology annotations, and incorporating large-scale proteomics datasets. We identified 4,831 genes encoding putative secreted proteins, assigned confidence scores based on the type and strength of supporting evidence, and generated an online resource (www.flyrnai.org/apps/fly_secretome/) for exploring these data. Comparison with the human secretome shows that 54% of Drosophila secretome genes are conserved in the human genome and of these, 83% are annotated as secreted in human. In addition, comparison with Drosophila single-cell transcriptomic data revealed that secreted proteins are more tissue-specific than other genes. Finally, we demonstrate the utility of this resource by analyzing changes in expression of genes encoding putative secreted proteins during aging based on snRNA-seq datasets from the Aging Fly Cell Atlas.

genetics↗

NECAP antagonizes light-induced Rhodopsin-1 internalization to promote photoreceptor homeostasis

AP-2 is a key mediator of clathrin-mediated endocytosis (CME) that internalizes cargo from the plasma membrane. NECAP proteins physically bind phosphorylated AP-2 during CME, but their roles during endocytosis remain unresolved, with conflicting reports about their function. Here, we report that Drosophila NECAP is dispensable for development, but antagonizes light-dependent Rhodopsin-1 (Rh1) internalization, a process that occurs through AP-2-mediated endocytosis. Specifically, loss of Drosophila NECAP causes excessive light-dependent Rh1 internalization and an age-related retinal degeneration that can be rescued by photoreceptor-specific expression of a wild-type transgenic NECAP. A Drosophila NECAP mutant transgene, equivalent to a canine NECAP1 variant associated with retinal atrophy, failed to rescue the NECAP loss-of-function phenotype in the eye. Furthermore, overexpression of wild-type NECAP suppressed massive Rh1 internalization in a distinct Drosophila model of light-dependent Rh1 endocytosis and retinal degeneration. These results establish NECAP as a negative regulator of light-dependent Rh1 internalization essential for photoreceptor survival.

cell biology↗

LIVIA: a browser-based tool for assessing and visualizing predicted protein interactions

As protein structure prediction tools become widely adopted across biology, there is a growing need for accessible methods to assess and visualize predicted protein-protein interactions (PPIs). Here we present LIVIA (Local Interaction Visualization and Analysis), a browser-based tool that computes local PPI confidence metrics across multiple prediction platforms, identifies predicted interface residues, embeds an interactive Mol* 3D viewer, and generates visualization scripts for ChimeraX and PyMOL. The tool automatically detects prediction formats; all parsing and computation occur locally on the users machine. LIVIA is freely available at https://flyark.github.io/LIVIA.

bioinformatics↗

Genome-wide CRISPR knockout cell screening platform for the disease vector tick species Ixodes scapularis

The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.

genetics↗

FlyPredictome: A structural atlas of predicted protein-protein interactions in Drosophila

Protein-protein interactions (PPIs) are fundamental to cellular function. Yet most Drosophila PPIs remain structurally uncharacterized despite the wealth of genetic and biochemical data available for this organism. Here we present FlyPredictome, a structural interactome based on 1.5 million pairwise AlphaFold-Multimer predictions. Using a local confidence metric performing robustly on interactions involving flexible and disordered proteins, we systematically assess experimentally reported Drosophila PPIs and predict direct binding interfaces at residue-level resolution. Testing their functional relevance, we find that phenotype-associated missense mutations are enriched at predicted interaction interfaces. Building on these predictions, we construct an evidence-supported PPI network, revealing modular organization from signaling pathways to individual protein complexes. We further predict higher-order complexes for nearly 400 of these modules with AlphaFold3, matching available cryo-EM structures and extending to unresolved assemblies. Fly-Predictome is available as an open, interactive database that maps interactions to residue-level binding surfaces, providing a structural foundation for interaction discovery in Drosophila.

bioinformatics↗

Phage display-mediated immuno-PCR to detect low-abundance secreted proteins in Drosophila

Circulating hormones, that mediate communications across organs to maintain physiological balance, are commonly detected and quantified using enzyme-linked immunosorbent assays (ELISAs). However, while ELISA is well-suited for organisms where sample blood can be readily obtained, its application is considerably more challenging in smaller organisms, particularly Drosophila, which has gained widespread use in recent years for physiological studies. Here, we present sensitive phage display-mediated immuno-PCR (PD-iPCR) to detect Drosophila hemolymph proteins via two approaches: 1) by identifying high-affinity nanobodies through phage-display library screening and subsequent affinity maturation and 2) by generating a knock-in fly line producing secreted proteins tagged with tandem NanoTags composed of VHH05 and 127D01. Using these approaches, we successfully established PD-iPCR to detect insulin-binding ImpL2 protein in fly hemolymph. Notably, the tandem NanoTag-based sandwich PD-iPCR enabled highly sensitive detection of tagged antigens, allowing us to quantify elevated ImpL2 levels in the hemolymph of starved flies and those bearing Yki-induced gut tumors. Collectively, our results demonstrate that PD-iPCR enables detection of endogenous, low-abundance circulating hormones in Drosophila, providing a powerful tool for studying interorgan communication. Significance statementHormones and other secreted factors orchestrate organism-wide physiology, yet their routine quantification in Drosophila has been limited by the limited volume of hemolymph available for assays like enzyme-linked immunosorbent assay (ELISA). Here, we present phage display-mediated immuno-PCR (PD-iPCR) as a novel sensitive platform for quantifying secreted proteins in flies in vivo. Using ImpL2 as an example, we successfully detected nanomolar level of circulating ImpL2 and monitored its physiological changes during starvation and tumorigenesis using PD-iPCR. This approach can be readily expanded to multiplexed quantification of secreted proteins in vivo by leveraging the multiple available nanobodies and the vast collection of epitope-tagged Drosophila lines. This work opens the door to systematic endocrine phenotyping across developmental stages and diverse physiological conditions.

genetics↗

A Structure-Guided Kinase-Transcription Factor Interactome Atlas Reveals Docking Landscapes of the Kinome

AbstractProtein kinases orchestrate cellular processes through phosphorylation, yet the structural basis for their specific binding partner interactions remains largely unmapped. Here, we present a structure-guided atlas of the human and Drosophila kinome, built by applying a new interface-aware scoring framework (iLIS) to AlphaFold-Multimer predictions. The resulting atlas recapitulates hallmark sequence preferences, confirms previously reported and functionally related protein-protein interactions, and uncovers unrecognized docking interactions. Notably, our analysis predicts a potentially widespread docking motif on homeodomain transcription factors that mediates interactions with basophilic kinases. Furthermore, we map putative allosteric interaction hotspots across the kinome and provide proof-of-concept evidence that targeting these surfaces can inhibit kinase activity. Finally, we demonstrate the physiological utility of the atlas by identifying a novel regulatory mechanism between Sgg/GSK3 and Hnf4 that controls lipid metabolism in vivo. This resource provides a blueprint for dissecting signaling networks and for the rational design of docking-site-specific kinase modulators.

bioinformatics↗

A family of lethal exotoxins defined by cell entry via the Attractin receptor

Although bacterial genomes encode numerous potential toxins, it is unclear how evolution drives the specificity of these important virulence factors. Using an insect CRISPR screen, we identified the transmembrane protein Attractin (ATRN) as the receptor for Nigritoxin (Ntx), a Vibrio toxin that causes seasonal shrimp pandemics. We found that Ntxs effector "warhead" inhibits translation via a previously uncharacterized mechanism. Moreover, we show that two related toxins require ATRN for entry but possess unrelated effector domains. One has a Rho-GTPase AMPylation function and the other an actin-targeting/proteolysis function. Our findings reveal the mechanism of Ntx entry and toxicity and show that the ATRN-targeting domain can deliver disparate effector domains, strongly indicating that this class of exotoxins can evolve as modular proteins using a common entry domain.

microbiology↗

Hepatic ceramide synthesis links systemic inflammation to organelle dysfunction in cancer

Paraneoplastic syndromes arise when tumor-derived cytokines reprogram distant organs. Although mediators such as Interleukin-6 have been implicated, how these signals impair host organ function remains incompletely defined. Here, we identify a cytokine-lipid axis that drives hepatic autophagy dysfunction. Specifically, in Drosophila, the gut tumor-derived interleukin-like cytokine Upd3 induces the expression of the triglyceride lipase CG5SCC, which we named "cancer-associated lipid mobilizer (calm)", and the ceramide synthase schlank in the fat body. This upregulation rewires fat body lipid metabolism, resulting in an autophagic-flux blockade. Genetic reduction of either CG5SCC or schlank restores organelle homeostasis and mitigates paraneoplastic phenotypes. This mechanism is conserved in mammals: in mice, IL-6 upregulates the lipoprotein lipase Lpl and ceramide synthases which in turn trigger a hepatic autophagy-flux blockade; in humans, hepatic LPL and ceramide synthases expression correlates with poorer survival in hepatocellular carcinoma. Our findings position hepatic lipid metabolism rewiring, especially ceramide synthesis as a critical, conserved node coupling systemic inflammation to organelle dysfunction, and suggest this pathway as a possible therapeutic entry point for cancer-associated liver disorders.

genetics↗

A Genome-wide CRISPR Screen Reveals ZDHHC8-Dependent Gαq Palmitoylation as a Key Regulator of GPCR Signaling

G protein-coupled receptors (GPCRs) that couple to the Gq signaling pathway control diverse physiological processes, yet the full complement of cellular regulators for this pathway remains unknown. Here, we report the first genome-wide CRISPR knockout screen targeting a Gq-coupled GPCR signaling cascade. Using a Drosophila model of adipokinetic hormone receptor (AkhR) signaling, we identified CG34449 (Zdhhc8), encoding a palmitoyl acyltransferase and its adapter protein CG5447, as a top hit required for robust Gq-mediated GPCR signaling. We show that Zdhhc8 enhances GPCR signaling through palmitoylation of Gq, which promotes its membrane localization and function. Loss of Zdhhc8 markedly reduces palmitoylation of Gaq resulting in attenuation of AkhR/Gq signaling and a reduction in receptor stability. Mechanistically, Zdhhc8 is necessary for palmitoylation of Gq. These findings uncover Zdhhc8-dependent Gq palmitoylation as a pivotal regulatory mechanism in GPCR signal transduction and highlight palmitoyl transferase as potential modulators of GPCR pathways.

cell biology↗

Characterizing the regulatory logic of transcriptionalcontrol at the DNA sequence level by ensembles ofthermodynamic models

Understanding how the genome encodes the regulatory logics of transcription is a main challenge of the post-genomic era to be overcome with the aid of customized computational tools. We report an automatic framework for analyzing an ensemble of fittings to data of a thermodynamics-based sequence-level model for transcriptional regulation. The fittings are clustered accordingly with their intrinsic regulatory logics. A multiscale analysis enables visualization of quantitative features resulting from the deconvolution of the regulatory profile provided by multiple transcription factors interacting with the locus of a gene. Quantitative experimental data on reporters driven by the whole locus of the even-skipped gene in blastoderm of Drosophila embryos was used for validating our approach. A few clusters of highly active DNA binding sites within the enhancers collectively modulate even-skipped gene transcription. Analysis of variable enhancers length shows the importance of bound protein-protein interactions for transcriptional regulation.

systems biology↗

The epigenetic factor Zrf1 regulates intestinal stem cell proliferation during midgut regeneration

Stem cells are essential for tissue maintenance and regeneration, balancing self-renewal and differentiation to support homeostasis and repair. Through an RNAi screen in the Drosophila midgut, we identified the epigenetic factor Zrf1 as a critical regulator of intestinal stem cell (ISC) proliferation. Functional analyses reveal that Zrf1 integrates inputs from multiple signaling pathways and interacts with components of the RNA-induced silencing complex (RISC). Zrf1 mutants exhibit elevated expression of transposable elements (TEs) and chromatin disruption, highlighting a broader role in genome stability. Single-nuclei RNA sequencing (snRNA-seq) further demonstrated the influence of Zrf1 on chromatin organization and TE repression, particularly within stem cell progenitors. Our findings suggest that Zrf1 is potentially a key chromatin regulator necessary for maintaining stem cell proliferation and genome integrity, enhancing our understanding of the molecular controls underlying stem cell function and chromatin dynamics.

genetics↗

Phage-displayed synthetic library and screening platform for nanobody discovery

Nanobodies, single-domain antibodies derived from camelid heavy-chain antibodies, are known for their high affinity, stability, and small size, which make them useful in biological research and therapeutic applications. However, traditional nanobody generation methods rely on camelid immunization, which can be costly and time-consuming, restricting their practical feasibility. In this study, we present a phage-displayed synthetic library for nanobody discovery. To validate this approach, we screened nanobodies targeting various Drosophila secreted proteins. The nanobodies identified were suitable for applications such as immunostaining and immunoblotting, supporting the phage-displayed synthetic library as a versatile platform for nanobody development. To address the challenge of limited accessibility to high-quality synthetic libraries, this library is openly available for non-profit use.

biochemistry↗

Inducing a synergistic anti-obesity effect by increasing the bioavailability of the flavonoid rutin with a L. plantarum species

Flavonoids, plant-derived compounds, are broadly categorized into glycoside (sugar-bonded) and aglycone (sugar-removed) forms and are predominantly found as glycosides in nature. Rutin, a glycoside, is a widely recognized flavonoid with significant potential as an anti-obesity agent. However, its low bioavailability in the body presents challenges in obesity treatment. We aimed to enhance the bioavailability and anti-obesity effect of rutin through microbial involvement. Lactiplantibacillus plantarum HAC03, one of the candidates, demonstrated the ability to hydrolyze rutin into isoquercetin and quercetin in vitro. In a diet-induced obesity mouse model, the combination of rutin and the L. plantarum strain resulted in significant weight loss, reduced adipocyte size and lowered obesity-related biomarkers in the blood, decreased fat-synthesis related gene expression, and increased fatty acid {beta}-oxidation related gene expression compared to other test groups. This includes groups treated with rutin or quercetin alone or in combination with a different species from the same Lactobacillus genus, known for its anti-obesity effect but lacking the ability to hydrolyze rutin. This synergistic combination also alleviated insulin resistance and reduced fat in the liver. Gut microbiota analysis revealed localization of L. plantarum in the ileum and beneficial changes in disrupted microbiota in the intestine. These findings provide insights into underlying mechanisms causing the synergistic effect and suggest a novel combination that is as safe as microbial monotherapies with L. plantarum or L. rhamnosus but more effective in anti-obesity treatment.

microbiology↗

Characterization of Newly Discovered Polyester Polyurethane-degrading Methylobacterium Aquaticum Strain A1

In this study, we present Methylobacterium aquaticum A1, a novel strain capable of degrading polyester polyurethane (PE-PUR). The attachment of M. aquaticum A1 to PE-PUR and its degradation capabilities were verified using Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). Analysis of the reference genome of M. aquaticum revealed genes encoding enzymes with potential PE-PUR degrading activity, including esterases, lipase, proteases and amidase such as tesA, pgpB, aes, aprE, lon, degQ, and gatA. An esterase activity assay using p-nitrophenyl acetate (p-NPA) showed increased ester bond-cleaving activity when M. aquaticum A1 was exposed to polyurethane diol (PU-diol), suggesting inducible enzymatic activity involved in PE-PUR degradation. These findings highlight the potential of M. aquaticum A1 as a promising biocatalyst for PE-PUR degradation. IMPORTANCEMicrobial biodegradation is increasingly recognized as a sustainable approach to addressing microplastic pollution. This study introduces M. aquaticum A1, a newly isolated strain capable of adhering to and degrading polyester polyurethane (PE-PUR), one of the most widely utilized plastics. To our knowledge, this is the first report of PE-PUR degradation by a member of the Methylobacterium genus. This paper provides a detailed characterization of M. aquaticum A1 and identifies several enzyme candidates--tesA, pgpB, aes, aprE, lon, degQ, and gatA--as potentially involved in the degradation process. Given that Methylobacterium species are known to be ecologically beneficial and inhabit diverse environments, the capacity of M. aquaticum A1 to degrade PE-PUR presents a promising strategy for mitigating microplastic pollution across a range of ecosystems.

microbiology↗

Identification of high sugar diet-induced dysregulated metabolic pathways in muscle using tissue-specific metabolic models in Drosophila

Individual tissues perform highly specialized metabolic functions to maintain whole-body metabolic homeostasis. Although Drosophila serves as a powerful model for studying human metabolic diseases, modeling tissue-specific metabolism has been limited in this organism. To address this gap, we reconstruct 32 tissue-specific genome-scale metabolic models (GEMs) by integrating a curated Drosophila metabolic network with pseudo-bulk single-nuclei transcriptomics data, revealing distinct metabolic network structures and subsystem coverage across tissues. We validate enriched pathways identified through tissue-specific GEMs, particularly in muscle and fat body, using metabolomics and pathway analysis. Moreover, to demonstrate the utility in disease modeling, we apply muscle-GEM to investigate high sugar diet (HSD)-induced metabolic dysregulation. Constraint-based semi-quantitative flux and sensitivity analyses identify altered NAD(H)-dependent reactions and distributed control of glycolytic flux, including GAPDH. This prediction is further validated through in vivo 13C-glucose isotope tracing study. Notably, decreased glycolytic flux, including GAPDH, is linked to increased redox modifications. Finally, our pathway-level flux analyses identify dysregulation in fructose metabolism. Together, this work establishes a quantitative framework for tissue-specific metabolic modeling in Drosophila, demonstrating its utility for identifying dysregulated reactions and pathways in muscle in response to HSD.

systems biology↗

Enhanced Protein-Protein Interaction Discovery via AlphaFold-Multimer

Accurately mapping protein-protein interactions (PPIs) is critical for elucidating cellular functions and has significant implications for health and disease. Conventional experimental approaches, while foundational, often fall short in capturing direct, dynamic interactions, especially those with transient or small interfaces. Our study leverages AlphaFold-Multimer (AFM) to re-evaluate high-confidence PPI datasets from Drosophila and human. Our analysis uncovers a significant limitation of the AFM-derived interface pTM (ipTM) metric, which, while reflective of structural integrity, can miss physiologically relevant interactions at small interfaces or within flexible regions. To bridge this gap, we introduce the Local Interaction Score (LIS), derived from AFMs Predicted Aligned Error (PAE), focusing on areas with low PAE values, indicative of the high confidence in interaction predictions. The LIS method demonstrates enhanced sensitivity in detecting PPIs, particularly among those that involve flexible and small interfaces. By applying LIS to large-scale Drosophila datasets, we enhance the detection of direct interactions. Moreover, we present FlyPredictome, an online platform that integrates our AFM-based predictions with additional information such as gene expression correlations and subcellular localization predictions. This study not only improves upon AFMs utility in PPI prediction but also highlights the potential of computational methods to complement and enhance experimental approaches in the identification of PPI networks.

bioinformatics↗