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Koo, B.-K.

Publications and source records attributed to Koo, B.-K..

At least 19 recordsLinked to original sources
bioRxiv2025

Structural basis of cyclic phytocytokine recognition by the HSL3/NUT receptor

Plant receptor kinases perceive diverse peptide signals to coordinate stress responses and developmental programs. The HAESA-LIKE 3 (HSL3/NUT) receptor recognizes CTNIP/SCREW phytocytokines--disulfide-stabilized cyclic peptides that regulate immune signaling and stress adaptation. However, how HSL3 distinguishes these structurally constrained cyclic peptides from linear signaling molecules remains unknown. Here we report near-atomic resolution cryo-EM structures of HSL3 in apo and CTNIP448-70-bound states at [~]2.6 [A], using Arabidopsis CTNIP4 as a representative family member, revealing distinct mechanisms for cyclic peptide recognition. The conserved CTNIP motif occupies a negatively charged pocket in HSL3s C-terminal region through a combination of polar contacts, hydrogen bonds, salt bridges, and van der Waals interactions. The receptor employs a two-step recognition mechanism--electrostatic steering followed by motif anchoring--that enables rapid ligand capture and release, consistent with the transient nature of stress signaling. Notably, an N-glycan at Asn449 directly contacts the CTNIP4 peptide, establishing glycosylation as an active participant in ligand recognition. Structure-guided mutagenesis combined with reactive oxygen species (ROS) burst assays confirmed the functional importance of key binding interfaces. N-terminal truncation experiments revealed a minimal active fragment: CTNIP451-70 supported both rapid ROS production and sustained seedling growth inhibition, whereas the shorter CTNIP454-70 variant retained ROS activity but failed to trigger long-term seedling growth inhibition. Structure-guided coevolutionary analysis across plant lineages reveals patterns of both conserved and variable receptor-ligand interfaces, highlighting evolutionary flexibility while preserving core features of recognition. These conserved recognition principles, mediated by receptor glycosylation and evolutionary plasticity, enable specificity in peptide signaling with implications for engineering stress-resilient crops.

plant biology↗
bioRxiv2025

WNK kinase regulates plasma membrane levels of the WNT inhibitor RNF43

The E3 ubiquitin ligases RNF43 and ZNRF3 are key negative regulators of canonical WNT signaling, promoting turnover of the WNT receptors FRIZZLED and LRP5/6 at the plasma membrane. While their mechanism of action is well established, how RNF43/ZNRF3 themselves are regulated remains unclear. Here, we identify WNK kinases as novel upstream regulators of RNF43 through proximity labeling proteomics. Using gain- and loss-of-function approaches, we show that WNKs control RNF43 surface localization and thereby its ability to ubiquitinate and downregulate WNT receptors. Pharmacological inhibition of WNKs increases RNF43 membrane abundance and enhances WNT suppression - an effect abolished in RNF43/ZNRF3 double knockout cells and organoids. Mechanistically, WNK inhibition alters RNF43 trafficking and ubiquitination, revealing a role for WNKs in regulating its plasma membrane distribution. These findings define a new regulatory axis linking the pro-WNT activity of WNKs to RNF43/ZNRF3-mediated feedback inhibition. Targeting WNK now offers a novel therapeutic strategy to restore WNT pathway control in cancers with RSPO fusions or RNF43 mutations.

biochemistry↗
bioRxiv2025

The Stmn1-lineage contributes to acinar regeneration but not to neoplasia upon oncogenic Kras expression

BACKGROUND & AIMSThe exocrine pancreas has a limited regenerative capacity, but to what extent all acinar cells are involved in this process is unclear. Nevertheless, the heterogenous nature of acinar cells suggests that cells exhibiting higher plasticity might play a more prominent role in acinar regeneration. In that regard, Stmn1-expressing acinar cells have been identified as potential facultative progenitor-like cells in the adult pancreas. Here, we studied Stmn1-progeny under physiological conditions, during regeneration, and in the context of KrasG12D expression. METHODSWe followed the fate of Stmn1-progenies both under baseline conditions, following caerulein-induced acute or chronic pancreatitis, pancreatic duct ligation, and in the context of KrasG12D expression. RESULTSThe Stmn1-lineage contributes to baseline acinar cell turnover under physiological conditions. Furthermore, these cells rapidly proliferate and repopulate the acinar compartment in response to acute injury in an ADM-independent manner. Moreover, acinar regeneration during chronic pancreatitis progression is in conjunction with a decline in the proliferative capacity of the Stmn1-lineage. Interestingly, newly generated acinar cells display increased susceptibility to additional injury during recurrent acute pancreatitis (RAP). Finally, given their inability to form ADMs, the Stmn1-lineage fails to form PanINs upon oncogenic Kras expression. CONCLUSIONSOur findings establish the Stmn1-lineage as a pivotal subpopulation for acinar regeneration. The ability of these cells to restore acinar tissue in an ADM-independent manner distinguishes them as a critical regenerative population. This study presents a new paradigm for acinar regeneration and repair in the context of pancreatitis and neoplasia.

cell biology↗
bioRxiv2024

APOBEC3A, not APOBEC3B, drives deaminase mutagenesis in human gastric epithelium

Cancer genomes frequently carry APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-associated DNA mutations, suggesting APOBEC enzymes as innate mutagens during cancer initiation and evolution. However, the pure mutagenic impacts of the specific enzymes among this family that are responsible for APOBEC-associated mutagenesis remain unclear in human normal cell lineage, particularly the comparative mutagenic activities of APOBEC3A and APOBEC3B. Here, we investigated the mutagenic contributions of these enzymes through whole-genome sequencing of human normal gastric organoid lines carrying doxycycline-inducible APOBEC3A or APOBEC3B cassettes. Our findings demonstrated that transcriptional APOBEC3A upregulation led to the acquisition of a massive number of genomic mutations in a few cell cycles. By contrast, APOBEC3B upregulation did not generate a substantial number of mutations in gastric epithelium. APOBEC3B-associated mutagenesis remained insignificant even after a combined inactivation of TP53. Based on the spectrum of acquired mutations after APOBEC3A upregulation, we further analyzed APOBEC3A-associated mutational signatures, encompassing indels mainly composed of 1bp deletions, characteristics of clustered mutations, and selective pressures operative on cells carrying the mutations. Our observations provide a clear foundation for understanding the mutational impact of APOBEC enzymes in human cells.

genomics↗
bioRxiv2023

Long-term three-dimensional high-resolution imaging of live unlabeled small intestinal organoids using low-coherence holotomography

The prevailing challenges in live unlabeled high-resolution imaging of native organoids stem from technical issues like complex sample handling and optical scattering in three-dimensional architectures. In this study, we introduce low-coherence holotomography as an advanced, label-free, quantitative imaging modality, designed to overcome related technical obstacles for long-term live imaging of 3D organoids. We successfully captured high-resolution morphological intricacies and dynamic events within mouse small intestinal organoids at a subcellular resolution. Furthermore, this method provides a unique advantage in differentiating between viable and non-viable organoids, thereby expanding its potential applications in organoid-based research.

developmental biology↗
bioRxiv2023

NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as preeclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of its epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator Mastermind-like 1 (MAML1). Doxycyclin-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, the differentiated hormone-producing cell type of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development promoting self-renewal of CTB progenitors.

developmental biology↗
bioRxiv2023

Acquired epithelial WNT secretion drives niche independence of developing gastric cancer

Recent studies have shed light on the signaling pathways required for gastric tissue maintenance and how aberrations in these key pathways lead to gastric cancer development. Although it has been shown that the WNT pathway is important for gastric epithelial homeostasis, the identity and source of the responsible canonical WNT ligands remain unknown. Furthermore, it is unclear how gastric cancer acquires WNT niche independence - an important early step in tumorigenesis. Using human and mouse gastric organoids and in vivo mouse models, we found that mesenchymal WNT2B and WNT7B maintain gastric epithelium in homeostasis. Next, mouse genetic studies and single-cell multi-omics analyses revealed that activation of MAPK signaling induces secretion of WNT7B in the epithelium itself. We further confirmed that in human gastric cancer, MAPK pathway activation through HER2 overexpression or copy number gains of WNT2 confers WNT independence. Importantly, the epithelium-intrinsic WNT expression could be therapeutically inhibited. Taken together, our results reveal that normal gastric epithelial turnover relies on WNT ligands secreted by niche mesenchymal cells, while transformation involves acquisition of a WNT secretory phenotype in the epithelium - representing a potential target for therapeutic interventions.

cancer biology↗
bioRxiv2023

Red2Flpe-SCON: A Versatile, Multicolor Strategy for Generating Mosaic Conditional Knockout Mice

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system, Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a new mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We have developed the Red2Flpe mouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (Short Conditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We used the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which revealed that the stem cell gene, Sox2, is not essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.

genetics↗
bioRxiv2023

Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt signaling by Daam1/2

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by actively cycling stem cells residing at the crypt bottom1,2. Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt33. With 19 family members, different Wnt ligands can selectively activate {beta}-catenin dependent (canonical) or independent (non-canonical) signaling4,5. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis.

cell biology↗
bioRxiv2022

Exploration of mechanisms of drug resistance by polyaneuploid cancer cells in a microfluidic device and patient tissues

Chemoresistance is a major cause of treatment failure in many cancers. However, the life cycle of cancer cells as they respond to and survive environmental and therapeutic stress is understudied. In this study, we utilized a microfluidic device to induce the development of doxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11 days by generating gradients of DOX and medium. In vivo chemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis all showed that chemoresistance arose from failed epigenetic control of the nuclear protein-1 (NUPR1)/histone deacetylase 11 (HDAC11) axis, and high Nupr1 expression correlated with poor clinical outcomes. These results suggest that the chip can rapidly induce resistant cells that increase tumor heterogeneity and chemoresistance, highlighting the need for further studies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

cancer biology↗
bioRxiv2021

Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration

While the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Upon damage, IL-1{beta} signalling-dependent modulation of Jag1/2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives reprogramming and acquisition of differentiation plasticity. We identify a core role for the transcription factor Fosl2/Fra2 in secretory cell fate conversion to alveolar type 2 (AT2) cells retaining the distinct genetic and epigenetic signatures of secretory lineages. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate the functional role of a IL-1{beta}-Notch-Fosl2 axis for the fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration.

cell biology↗
bioRxiv2021

SCON - A Short Conditional intrON for conditional knockout with one-step zygote injection

The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables rapid generation of conditional alleles via one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

genetics↗
bioRxiv2021

Mutational impact and signature of ionizing radiation

Whole-genome sequencing (WGS) of human tumors and normal cells exposed to various carcinogens has revealed distinct mutational patterns that provide deep insights into the DNA damage and repair processes. Although ionizing radiation (IR) is conventionally known as a strong carcinogen, its genome-wide mutational impacts have not been comprehensively investigated at the single-nucleotide level. Here, we explored the mutational landscape of normal single-cells after exposure to the various levels of IR. On average, 1 Gy of IR exposure generated [~]16 mutational events with a spectrum consisting of predominantly small nucleotide deletions and a few characteristic structural variations. In [~]30% of the post-irradiated cells, complex genomic rearrangements, such as chromoplexy, chromothripsis, and breakage-fusion-bridge cycles, were resulted, indicating the stochastic and chaotic nature of DNA repair in the presence of the massive number of concurrent DNA double-strand breaks. These mutational signatures were confirmed in the genomes of 22 IR-induced secondary malignancies. With high-resolution genomic snapshots of irradiated cells, our findings provide deep insights into how IR-induced DNA damage and subsequent repair processes operate in mammalian cells.

genomics↗
bioRxiv2020

Loss of RNF43/ZNRF3 predisposes to Hepatocellular carcinoma by impairing liver regeneration and altering liver fat metabolism

The homologous E3 ubiquitin ligases RNF43/ZNRF3 negatively regulate WNT signalling activation. Recently, both genes have been found mutated in several types of cancers. Specifically, loss-of-function mutations result in adenoma formation in mouse small intestine. However, their role in liver cancer has not been explored yet. Here we describe that hepatocyte-specific deletion of both Rnf43/Znrf3 results in altered lipid metabolism and a non-alcoholic steatohepatitis (NASH) phenotype in mouse, in the absence of exogenous fat supplementation. The effect is cell-autonomous, as evidenced by the intracellular lipid accumulation detected in mutant liver organoids. Upon chronic liver damage, Rnf43/Znrf3 deletion results in impaired hepatocyte regeneration, subsequent to an imbalance between hepatocyte differentiation and proliferation, which leads to hepatocellular carcinoma. Remarkably, hepatocellular carcinoma patients with mutations in ZNRF3 also present altered lipid metabolism and poorer survival. Our findings imply that Wnt activation through the RNF43/ZNRF3 module predisposes to liver cancer by altering the liver lipid metabolic ground-state and impairing liver regeneration, which combined, facilitate the progression towards malignancy. Our results highlight the requirement for personalized therapeutic or dietary interventions for those RNF43/ZNRF3 mutated individuals at risk of developing steatosis, NASH and/or liver cancer.

cancer biology↗
bioRxiv2020

Reconstitution of Alveolar Regeneration via novel DATPs by Inflammatory Niches

Tissue regeneration involves a multi-step process composed of diverse cellular hierarchies and states that are also implicated in tissue dysfunction and pathogenesis. Here, we leveraged single-cell RNA sequencing analysis in combination with in vivo lineage tracing and organoid models to fine-map trajectories of alveolar lineage cells during injury repair and regeneration. We identified Damage-Associated Transient Progenitors (DATPs) as a distinct AT2-lineaged population arising during alveolar regeneration. Specifically, we found that IL-1{beta}, secreted by interstitial macrophages, primes a subset of Il1r1+AT2 cells for conversion into DATPs, via a Hif1a-mediated glycolysis pathway, that are functional mediators for mature AT1 cell differentiation. Importantly, we show that chronic inflammation mediated by IL-1{beta} prevents differentiation into AT1 cells, leading to aberrant accumulation of DATPs and impaired alveolar differentiation. Our step-wise fine-mapping of cell fate transitions demonstrates how the inflammatory niche impedes alveolar regeneration by directing stem cell fate behavior.

cell biology↗
bioRxiv2020

Sox2 modulation increases naive pluripotency plasticity

Induced pluripotency provides a tool to explore the molecular mechanisms underlying the establishment, maintenance and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, Sox2-low cells also differentiate towards the trophoblast lineage both in vitro and in vivo. At the single-cell level self-renewing Sox2-low nPSCs exhibit a homogeneous naive molecular signature. However, they also display a basal trophoblast molecular signature and decreased ability of Oct4 to bind naive-associated regulatory sequences compared to control cells. These features underlie observed enhanced cell potency upon the removal of self-renewing cues. In sum, this work defines Sox2 as a restrictor of developmental potential and suggests perturbation of the naive pluripotent network as an underlying cause of increased cell potency. HighlightsO_LILow Sox2 expression is sufficient for naive pluripotent stem cell self-renewal C_LIO_LILow Sox2 expression does not impair neurectoderm differentiation in vitro C_LIO_LILow Sox2 expression impairs Oct4 genomic occupancy C_LIO_LILow Sox2 expression increases naive pluripotent cell plasticity in vitro and in vivo C_LI

developmental biology↗
bioRxiv2019

RNF43 truncating mutations mediate a tumour suppressor-to-oncogene switch to drive niche-independent self-renewal in cancer

Wnt/{beta}-catenin signalling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumours that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that strongly induce {beta}-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulating activity. Mechanistically, these RNF43 mutants trap Casein Kinase (CK)1 at the plasma membrane, which prevents {beta}-catenin turnover and propels ligand-independent Wnt target gene transcription. When introduced in human colon stem cells, these oncogenic RNF43 mutants cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Importantly, onco-RNF43 mutations, unlike conventional LOF RNF43 mutations, confer resistance to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.

cancer biology↗