Biology subjects

Lee, S.-J.

Publications and source records attributed to Lee, S.-J..

At least 19 recordsLinked to original sources

bioRxiv2026

TREM2 encodes plaque macrophage plasticity to mediate atherosclerosis regression

Cholesterol lowering therapies stabilize atherosclerotic plaque and reduce ischemic events, but the molecular mechanisms governing plaque remodeling or regression remain poorly defined. The lipid sensor TREM2 is a key modulator of plaque-associated foam cell development and was upregulated by plaque macrophages in atherosclerosis regression. We examined macrophage- or monocyte-specific Trem2 deletion during regression in mice and found that TREM2 expression on plaque macrophages, not newly recruited monocytes, was required for plaque regression. Trem2 deletion impeded necrotic core resolution, leading us to interrogate the efferocytic capacity of TREM2+ cells during lipid lowering. An in vitro model of cholesterol-experienced ex-lipid associated macrophages (Ex-LAMs) demonstrated that prior lipid priming enhanced efferocytic ability after unloading lipids and maintained expression of lysosomal and cytoskeletal remodeling genes. Augmented TREM2 signaling through genetic models or agonist treatment accelerated efferocytic ability in vitro and plaque regression in vivo. Thus, we posit that a TREM2-dependent ExLAM programming drives atherosclerosis resolution and facilitates beneficial plaque remodeling through enhanced efferocytosis. Overall, targeting the TREM2 pathway may be a viable strategy for stabilizing atherosclerotic plaques in patients treated with aggressive cholesterol control.

immunology↗

bioRxiv2026

MgATP/MgADP-dependent conformational dynamics and intrinsically disordered regions of vascular KATP channels revealed by cryoEM

Vascular smooth muscle KATP channels, composed of the pore-forming Kir6.1 and regulatory SUR2B subunits, control vascular tone, dysfunction of which causes systemic disease. Vascular KATP is regulated by Mg-nucleotides, but the underlying structural mechanism has remained elusive. Here, we determined cryoEM structures of these channels in the presence of MgATP and MgADP. Two key structures captured, one showing the SUR2B-nucleotide binding domains (NBDs) separated and one showing the SUR2B-NBDs dimerized, reveal conformation-specific organization of intrinsically disordered regions (IDRs) found in both Kir6.1 and SUR2B. In the NBD-separated conformation, the Kir6.1-N terminal IDR (KNt) sits within the central cleft of the ABC-core of SUR2B. In the NBD-dimerized conformation, KNt is excluded from the central cleft and instead forms contacts with an ED domain comprising 15 consecutive glutamate and aspartate residues within a SUR2B IDR, the N1-T2 linker connecting NBD1 (N1) to transmembrane domain 2 (T2). Moreover, within the N1-T2 linker a regulatory helix seen between the two NBDs in the NBD-separated conformation moves to outside the dimerized NBDs, interacting with the C-terminal residues unique to SUR2B, in the NBD-dimerized conformation. MD simulations further reveal that transient but frequent interactions mediated by the IDRs may facilitate Mg-nucleotide dependent conformational switch in vascular KATP channels.

biochemistry↗

bioRxiv2026

Human iPSCs and iPSC-Derived Endothelial Cells from PAD Patients and Healthy Donors Exhibit Comparable Characteristics and Potency: Implications for Autologous Cell Therapy in Peripheral Artery Disease

BACKGROUND: Peripheral artery disease (PAD) can lead to amputation in advanced cases, making cell therapy using human induced pluripotent stem cells (hiPSCs) a promising therapeutic option. hiPSC-derived endothelial cells (hiPSC-ECs) have shown favorable effects in treating experimental ischemic cardiovascular disease. An autologous approach for PAD patients is preferable to avoid immunological reactions. However, it is yet unknown whether hiPSCs and hiPSC-ECs derived from PAD patients have similar characteristics and potency compared to those derived from non-PAD donors. Therefore, we explored whether there are significant differences in the characteristics and potency of hiPSCs and hiPSC-ECs between non-PAD donors and PAD patients. METHODS: We generated hiPSCs from the blood of non-PAD donors and patients with PAD. We determined the pluripotency of hiPSCs using qRT-PCR, flow cytometry, immunostaining, RNA-seq, and teratoma formation assay. The genetic stability of hiPSCs was confirmed by G-banding karyotyping. We then differentiated hiPSCs into endothelial cells (ECs) in a clinically compatible manner. We compared the characteristics and therapeutic potency of hiPSC-ECs derived from non-PAD and PAD groups using qRT-PCR, flow cytometry, immunostaining, and RNA-seq. In vitro endothelial functions were determined by nitric oxide (NO) production and capillary network formation. Therapeutic potency and incorporation of engrafted hiPSC-ECs were evaluated in a hindlimb ischemia model. RESULTS: We successfully generated hiPSCs from the blood of seven non-PAD donors and eight PAD patients. Both non-PAD and PAD-derived hiPSCs exhibited similar expression levels of pluripotency markers. All hiPSCs, regardless of the group, formed teratomas and showed normal karyotypes. RNA-seq analyses revealed similar gene expression profiles between the groups. hiPSC-ECs derived from both non-PAD and PAD donors exhibited similar expression levels of EC markers at both the gene and protein levels. RNA-seq analyses showed no significant overall differences in gene expression profiles between the groups. Functional analyses demonstrated similar endothelial characteristics and function in hiPSC-ECs from both groups. Both groups showed similar perfusion recovery, limb salvage, and vessel-forming capacity. Engrafted hiPSC-ECs from both groups also exhibited similar angiogenic and vessel-forming capabilities. CONCLUSIONS: Our study demonstrated no significant differences in hiPSCs and hiPSC-ECs derived from non-PAD donors and PAD patients in terms of molecular and cell biological characteristics, therapeutic effects, and vessel-forming capability. Our study indicates that hiPSCs and hiPSC-ECs derived from PAD patients can serve as a novel platform for autologous cell therapy.

cell biology↗

bioRxiv2026

AI-Driven Design of Nanobinders Targeting the TSLPR Heterodimer Interface to Suppress Type 2 Inflammatory Signaling

Aberrant thymic stromal lymphopoietin (TSLP) signaling is a central driver of type 2 inflammatory diseases, yet the only approved TSLP-targeted therapy is a 150 kDa monoclonal antibody whose bulky format limits tissue penetration and precludes inhaled delivery. Here, we report an AI-driven framework for designing ultra-compact de novo nanobinders that suppress TSLP signaling by sterically disrupting assembly of the TSLPR-IL-7R heterodimer. We compare two structure-guided strategies, namely purely de novo helical bundle generation and interface-mimetic grafting of native binding motifs onto designed scaffolds. Although both yield nanomolar binders, only orthosteric mimicry of the native cytokine geometry blocks receptor heterodimerization, showing that functional antagonism is governed by precise epitope geometry rather than affinity alone. After library-based maturation, the lead nanobinder TRB5.1 is a hyper-stable monomer (Tm [~]97.4 {degrees}C) with single-digit nanomolar affinity (KD = 9.7 nM) and strict selectivity over related -chain interleukin receptors. TRB5.1 suppresses TSLP-induced JAK1 and STAT5 phosphorylation across multiple cellular models and drives a transcriptome-wide reversal of the pathogenic type 2 program. This work delivers a developable, potentially inhalable non-antibody lead and a scalable blueprint for antagonizing heterodimeric cytokine receptors.

biochemistry↗

bioRxiv2026

ProteinSketch translates spatial intuition into protein design with bimanual interaction in VR

Generative AI-based protein design can create diverse structures for desired functions but offers human designers limited control over three-dimensional architectures. We developed ProteinSketch, a bimanual virtual reality platform in which immersive backbone sketches and volumetric envelopes are directly created and translated into constraints for diffusion-based protein generation. RFdiffusion-based real-time refinement enabled interactive exploration and construction of user-specified protein topologies. Volumetric conditioning of sketched envelopes enabled high-fidelity control of anisometric geometries, confirmed by cryo-electron microscopy. These user-defined volumetric constraints enabled functional-binder design and extension of pre-existing minibinders to surfaces otherwise difficult to access within complex molecular environments using conventional design approaches. This collaborative human-AI framework integrates human spatial reasoning with generative AI for spatially directed, shape-controlled design of protein structure and function. One-sentence summaryImmersive ProteinSketch embeds human spatial reasoning in AI-guided design of protein topology, shape, and function.

bioengineering↗

bioRxiv2025

Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

This study investigates the transcriptional impact of Q901, a highly selective CDK7 inhibitor in clinical development. Q901 primarily disrupted MYC and E2F-dependent transcription program, downregulating cell cycle control and DNA damage repair pathways. CDK7 binding at the promoter-proximal regions was dramatically stabilized by Q901, leading to reduced occupancy of MYC, E2F, and RNA Polymerase II (RNAPII). These findings offered a novel therapeutic strategy to enhance cancer susceptibility to TOP1-DNA protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors. Resistance to TOP1 inhibitors arises through activation of DNA repair pathway when elongating RNAPII encounters TOP1-DPCs. By suppressing RNAPII transition from initiation to elongation and DNA repair pathways, Q901 stabilizes TOP1-DPCs and sensitizes tumor to TOP1 inhibitors. Preclinical studies demonstrated enhanced tumor suppression when combining Q901 with TOP1 inhibitor-based antibody-drug conjugates (TOP1i-ADCs), highlighting its potential as a therapeutic option for cancers resistant to TOP1i-ADC therapy. TeaserOvercoming ADC cross-resistance through combination approach.

cancer biology↗

bioRxiv2025

Stay or Stray: Lpar1 regulates neutrophil retention and epidermal homeostasis in early zebrafish development

Neutrophils are the most abundant myeloid cells in the vertebrate innate immune system and play a crucial role in host defense. Dysregulated release from hematopoietic tissue or excessive recruitment to inflamed sites is associated with immunopathologies, including chronic inflammation and recurrent infections. Lysophosphatidic acid (LPA) is a bioactive lipid that signals through a family of G protein-coupled receptors (LPAR1-6). Among these, LPA-LPAR1 signaling is activated in inflamed tissues and is known to modulate neutrophil infiltration during inflammatory responses. However, its role in neutrophil dynamics during early development remains unclear. Here, we report a novel function of Lpar1 in regulating neutrophil behavior during early zebrafish development. In Lpar1-deficient embryos, we observed a significant increase in neutrophil dispersal from the caudal hematopoietic tissue (CHT) at 3 days post-fertilization (dpf), with most dispersed neutrophils infiltrating the skin and contributing to elevated inflammatory signaling. Prior to dispersal, Lpar1-deficient embryos exhibited increased apoptosis of superficial epidermal cells and reduced expression of cxcl12a, a key retention signal for neutrophils in the CHT. Together, these findings highlight an essential role for Lpar1 in maintaining neutrophil retention and epidermal homeostasis during early development, thereby limiting inappropriate inflammation. SUMMARY STATEMENTThis work identifies a new role for a known inflammatory receptor in coordinating immune cell retention and skin stability during early development.

developmental biology↗

bioRxiv2025

Differential DNA damage response to WRN inhibition identifies a targetable vulnerability in ARID1A-mutated cancers

ARID1A, a key subunit of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancers. However, effective clinical treatments for patients with this mutation are limited, highlighting a demand for new therapeutic strategies. Here, we establish Werner syndrome ATP-dependent helicase (WRN) as a critical vulnerability target in ARID1A-mutated cancers. Upon genetic and pharmacological inhibition of WRN, ARID1A-mutated cells had defective Chk1-mediated DNA damage signaling, resulting in compensatory Chk2 activation, leading to G1-phase arrest and apoptosis, whereas ARID1A-proficient cells underwent Chk1-dependent G2/M arrest. Additional p21 inhibition, combined with WRN suppression, drove G1-arrested cells to re-enter the cell cycle, triggering mitotic catastrophe. The anti-tumor efficacy of WRN inhibition was validated using in vivo cell lines and patient-derived xenograft mouse models. Our findings define WRN as a selective therapeutic target in ARID1A-mutated cancers and suggest a combinatorial strategy of WRN and p21 inhibition as a therapeutic approach.

cancer biology↗

bioRxiv2025

DNA polymerase actively and sequentially displaces single-stranded DNA-binding proteins

Single-stranded DNA-binding proteins (SSBs) play a crucial role in stabilizing and protecting transiently exposed single-stranded DNA (ssDNA), yet the mechanisms governing their displacement by DNA polymerase (DNAp) during replication remain largely unexplored. Using bacteriophage T7 DNAp and its SSB, T7 gp2.5, we investigated the molecular mechanisms and visualized the dynamic process underlying SSB displacement. Our single-molecule force spectroscopy demonstrates that T7 SSB modulates DNA replication in an ssDNA conformation-dependent manner, regulated by tension applied to the DNA template. By integrating dual-color single-molecule imaging, we observe that T7 SSB remains stationary as DNAp approaches, indicating that SSB molecules are sequentially displaced rather than pushed forwards. Molecular dynamics (MD) simulations revealed reduced energy barriers for SSB dissociation in the presence of DNAp. This finding, combined with the detected FRET signals when DNAp approaches an SSB-bound ssDNA region and observations of faster replication rates compared to relative slow intrinsic SSB dissociation, collectively support an active displacement mechanism. Using both ensemble and single-molecule analyses, we demonstrated that SSB saturation of ssDNA is critical for optimal replication efficiency, with each SSB molecule contributing positively to the process. Taken together, the uncovered spatial-temporal coordination between SSB and DNAp is necessary for resolving molecular collisions during DNA replication, and may represent a universal strategy employed by other DNA translocating motors to ensure genomic integrity.

biophysics↗

bioRxiv2024

Common mycelial network mediated inter-plant signals modulate plant biotic stress responses and defence against foliar pathogens

Arbuscular mycorrhizal fungi (AMF) are ubiquitous root symbionts that form common mycelial networks (CMN), linking multiple plants underground. CMN are hypothesized to play a role for information exchange between plants for neighbour-primed defences. However, the key transcriptomic and metabolome responses in receiver plant associated with inter-plant CMN connections remain yet to be elucidated. Additionally, the confounding effects of hyphal damage from CMN disconnection have not been clearly resolved. To uncover the contribution of CMN integrity to neighbour-primed plant defences, we used model AMF Rhizophagus irregularis to inter-connect two Medicago truncatula plants and explored the effect of sender wounding and flg22 elicitation on receiver plants leaf responses and pathogen tolerance. For the first time, we demonstrate that changes in receivers biotic stress and defence signalling pathways rely on CMN-mediated inter-plant signals, not on mycelial network damage. This response was associated with distinct leaf isoprenoid production, including volatile monoterpenes and triterpene saponins. Furthermore, CMN-mediated signals from stressed senders enhanced receiver resistance to Fusarium sporotrichoides whilst simultaneously increasing susceptibility to Botrytis cinerea. Our findings highlight the critical role of CMN in inter-plant signalling for pathogen-specific susceptibility and resistance which can be a key for understanding plant community-level defence in nature and agroecosystems.

plant biology↗

bioRxiv2024

Replication of DNA containing trinucleotide repeats by the bacteriophage T7 replisome

Trinucleotide repeats in the human genome are implicated in various neurodegenerative diseases. The tendency of these repetitive DNA sequences to form non-B DNA structures can cause abnormal replication, leading to genomic instability. This instability contributes to disease progression, though the underlying mechanisms are not fully understood. We investigated the replication of DNA containing CAG and CTG trinucleotide repeats using individual components of the T7 bacteriophage replication machinery, as well as the complete replisome. Our results show that repeats in linear single-stranded DNA (ssDNA) inhibit the activity of T7 DNA polymerase and ssDNA-binding proteins, with a more pronounced effect observed in CTG repeats compared to CAG repeats. Minicircle templates containing CTG repeats exhibited robust DNA synthesis on both the leading and lagging strands, though synthesis was not enhanced by the T7 gene 2.5 ssDNA-binding protein. The lagging strand products generated from the CTG repeat minicircle were significantly longer than those from random sequence templates, and their lengths were not extended by the presence of T7 gene 2.5 protein. When the repeated sequences were incorporated into the T7 phage genome, heterogeneity was observed downstream of the repeats, depending on their length. We propose that aberrant extension occurs predominantly in the lagging strand, driven by dynamic interactions between the repeated sequences and the DNA replisome. This study may provide a foundation for understanding the mechanisms underlying the extension or deletion of repetitive genomic regions.

biochemistry↗

bioRxiv2023

Targeted protein degradation systems to enhance Wnt signaling

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETSTM) to drive tissue-specific degradation of ZNRF3/RNF43 E3-ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1 and ASGR1/2 specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate binding site. Both antibodies enhanced Wnt-activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

synthetic biology↗

bioRxiv2023

Myostatin is a major endocrine driver of follicle-stimulating hormone synthesis

Myostatin is a paracrine myokine that regulates muscle mass in a variety of species, including humans. Here, we report a functional role for myostatin as an endocrine hormone directly promoting pituitary follicle-stimulating hormone (FSH) synthesis and thereby ovarian function. Previously, this FSH-stimulating role was attributed to other members of the transforming growth factor {beta} family, the activins. The results both challenge activins eponymous role in FSH synthesis and establish an endocrine axis between skeletal muscle and the pituitary gland. The data also suggest that efforts to antagonize myostatin to treat muscle wasting disorders may have unintended consequences on fertility. One-Sentence SummaryHormone synthesis and reproduction depend on crosstalk between skeletal muscle and the pituitary gland.

physiology↗

bioRxiv2023

BRAIDing receptors for cell specific targeting

Systemic toxicity is a major challenge in the development of therapeutics. Consequently, cell-type-specific targeting is needed to improve on-target efficacy while reducing off-target toxicity. Here, we describe a cell-targeting system we have termed BRAID (BRidged Activation by Intra/intermolecular Division) whereby an active molecule is divided into two inactive or less active parts that are subsequently brought together via a so-called bridging receptor on the target cell. This concept was validated using the WNT/{beta}-catenin signaling system, demonstrating that a multivalent WNT agonist molecule divided into two inactive components assembled from different epitopes via the hepatocyte receptor {beta}Klotho induces signaling specifically on hepatocytes. These data provide proof-of-concept for this cell-specific targeting strategy and in principle, this may also allow activation of multiple signaling pathways where desirable. This approach has broad application potential for other receptor systems.

synthetic biology↗

bioRxiv2023

Clots reveal anomalous elastic behavior of fiber networks

The mechanical properties of many soft natural and synthetic biological materials are relevant to their function. The emergence of these properties from the collective response of the structural components of the material to external stress as well as to intrinsic cell traction, remains poorly understood. Here, we examine the nonlinear elastic behavior of blood clots by combining microscopy and rheological measurements with an elastic network model that accounts for the stretching, bending, and buckling of constituent fibrin fibers. We show that the inhibition of fibrin crosslinking reduces fiber bending stiffness and introduces an atypical fiber buckling-induced softening regime at intermediate shear, before the well-characterized stiffening regime. We also show that crosslinking and platelet contraction significantly alter force propagation in the network in a strain-dependent manner. Our mechanics-based model, supported by experiments, provides a framework to understand the origins of characteristic and anomalous regimes of non-linear elastic response not only in blood clots, but also more generally in active biopolymer networks.

biophysics↗

bioRxiv2023

Label-free histological analysis of retrieved thrombi in acute ischemic stroke using optical diffraction tomography and deep learning

For patients with acute ischemic stroke, histological quantification of thrombus composition provides evidence for determining appropriate treatment. However, the traditional manual segmentation of stained thrombi is laborious and inconsistent. In this study, we propose a label-free method that combines optical diffraction tomography (ODT) and deep learning (DL) to automate the histological quantification process. The DL model classifies ODT image patches with 95% accuracy, and the collective prediction generates a whole-slide map of red blood cells and fibrin. The resulting whole-slide composition displays an average error of 1.1% and does not experience staining variability, facilitating faster analysis with reduced labor. The present approach will enable rapid and quantitative evaluation of blood clot composition, expediting the preclinical research and diagnosis of cardiovascular diseases.

bioengineering↗

bioRxiv2022

Therapeutic modulation of the blood-brain barrier and ischemic stroke by a bioengineered FZD4-selective WNT surrogate

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimers disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/{beta}-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we developed L6-F4-2, a non-lipidated, FZD4-specific surrogate with significantly improved sub-picomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reversed neonatal retinal angiogenesis deficits, but also restored BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduced BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with general applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

physiology↗

bioRxiv2022

Sex-Specific Role of Myostatin Signaling in Neonatal Muscle Growth, Denervation Atrophy, and Neuromuscular Contractures

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders.

developmental biology↗