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Lee, W.

Publications and source records attributed to Lee, W..

At least 37 records · Page 2Linked to original sources
bioRxiv2025

Deep learning-enabled scaffolding of spatial arrays of PfCSP epitopes

Malaria is a leading cause of disease in developing countries. The licensed malaria vaccine RTS,S/AS01 confers partial protection in part due to the elicitation of circumsporozoite protein (CSP) antibodies, of which those to the CSP repeat and junctional regions offer the most potent protection. Anti-repeat region antibodies, including the protective antibody L9, frequently develop mutations that promote inter-Fab contacts when bound to CSP in "spiral" quaternary structures. As a first step toward the design of immunogens that elicit L9-like antibodies, we utilized generative deep learning models to design epitope-scaffolds that incorporated up to three junctional repeat epitopes with structural conformations and relative spatial orientations matching those of the multivalent complex of CSP bound to three copies of L9. Affinity and structural studies demonstrated accurate scaffolding of two epitopes with the intended relative orientation, and less accurate positioning of the third epitope. This study demonstrates proof of principle for design of multi-epitope scaffolds with pre-determined relative epitope spatial positioning. The study also represents an initial step toward development of multi-epitope immunogens to elicit antibodies that utilize homotypic interactions to bind pathogen in multivalent clusters.

immunology↗
bioRxiv2025

Role of Uba7-UFD in E1-E2 ISGylation: NMR-Based Insights into E2 Binding Dynamics

The ISGylation pathway, a key post-translational modification, plays a pivotal role in the innate immune response by covalently attaching ISG15 to target proteins. This cascade involves a series of enzymatic steps, including activation by the E1 enzyme Uba7, conjugation by the E2 enzyme UbcH8, and ligation by an E3 ligase. Central to this process is the ubiquitin-fold domain (UFD) of Uba7, which facilitates the transfer of ISG15 to UbcH8. However, the structural and mechanistic details of this interaction remain poorly understood. In this work, we present the solution NMR structure and functional analysis of the ubiquitin-fold domain of human Uba7, the E1 enzyme in the ISGylation cascade. Through detailed NMR titration experiments and mutational studies, we mapped the interaction surface of Uba7-UFD with UbcH8, identifying key residues and their contributions to the E1-E2 interaction. We found that Uba7-UFD is flexible in its free form; this flexibility is conserved across ubiquitin-like systems but shows a regional shift that may play an important role in correct E2 and Ubl selection. Chemical shift perturbation and mutational analysis further demonstrate the importance of specific residues, particularly Cys996, in maintaining UFDs structural integrity and binding capacity. Additionally, mutations designed to alter the flexibility and length of the loop region between UFD and UbcH8 show significant effects on binding, indicating that these regions are crucial for efficient E2 recruitment. These findings provide new insights into the mechanistic basis of E2 enzyme selection in ISGylation and underscore the functional relevance of dynamic structural transitions in E1-E2 complex formation. Statement of SignificanceISGylation is a key ubiquitin-like modification pathway essential for antiviral defense, immune regulation, and protein quality control. However, the molecular principles that govern communication between the E1 and E2 enzymes in this pathway remain poorly defined. Here, we present the first NMR structure of the human Uba7 ubiquitin-fold domain (UFD) and its interaction with UbcH8. Our findings reveal how structural flexibility within the UFD is crucial for specific and efficient E2 recognition, providing fundamental insight into the dynamic mechanism of ISGylation and advancing our understanding of ubiquitin-like enzyme cascades relevant to cellular regulation and disease.

biochemistry↗
bioRxiv2025

Learning the cellular origins of cancer using single-cell chromatin landscapes

Deciphering the cell of origin (COO) of different cancers is critical for understanding tumor development and improving diagnostic and therapeutic strategies in oncology. Previous studies demonstrated that the COO chromatin accessibility landscape shapes the genomic distribution of cancer somatic mutations. We leveraged machine learning, 559 single-cell chromatin accessibility cellular profiles, and 2,734 whole genome sequencing patient samples to predict the COO of 36 cancer subtypes with high robustness and accuracy, confirming both the known anatomical and cellular origins of numerous human cancers, often at cell subset resolution. Importantly, our data-driven approach predicts that basal cells give rise to small cell lung cancers, challenging the traditional view of neuroendocrine COO. Our study also highlights distinct cellular trajectories during cancer development of different histological subtypes and uncovers an intermediate metaplastic state during tumorigenesis for multiple gastrointestinal cancers, which have important implications for cancer prevention, early detection, and treatment stratification.

cancer biology↗
bioRxiv2025

Rapid changes in cholinergic signaling, myelination and thyroid signaling pathway gene expression in amygdala subnuclei in response to social status maintenance and reorganization

Male CD-1 mice form linear social hierarchies and can rapidly reform them following social reorganization. Through Tag-based sequencing in the medial amygdala (MeA), we identified several genes regulating cholinergic signaling, myelination, and thyroid signaling that rapidly shift expression 70 minutes after animals change social status. Here, we further characterize the expression patterns of individual genes within these pathways in both stable and reorganized hierarchies. We find that genes related to cholinergic signaling show higher expression in the MeA of dominant males in stable hierarchies as well as when reestablishing dominance in reorganized hierarchies. Dominant males also show higher levels of myelination related genes than socially descending males when reestablishing their social status during social reorganization but less so in stable groups. Conversely, thyroid signaling genes show higher expression in the MeA in subordinate males and previously dominant males who are socially descending. Using RNAscope, we were able to demonstrate broadly similar patterns of gene expression immediately following social reorganization across the MeA, basolateral, and central amygdala for 7 genes of interest (chat, slc5a7, ache, mbp, mog, crym, mybpc1). High levels of co-expression of cholinergic signaling and myelination gene expression in dominant males suggest that these processes work together to promote resilience to the social challenge and promote dominance. In summary, we demonstrate that rapid changes in amygdala gene expression in each pathway are associated with the formation and maintenance of dominance and subordinate social status in stable and reorganized environments.

neuroscience↗
bioRxiv2025

A Domain-Specific Language Model Approach for Identifying Immunogenic Epitopes from Publicly Available Data: EpitopeMiner

Personalized cancer vaccines represent a transformative approach in immunotherapy, leveraging tumor-specific antigens, such as neoantigens, to stimulate durable and targeted immune responses. However, identifying immunogenic neoantigen epitopes through conventional approaches - relying on in silico predictions followed by experimental validation - remains a significant challenge due to limitations of computational tools and scattering of experiment data. To address these challenges, we introduce EpitopeMiner, a domain-specific large language model (LLM) enhanced with a Retrieval-Augmented Generation (RAG) framework, specifically designed to identify immunogenic epitopes predicted by existing computational tools. EpitopeMiner utilizes a custom database of MHC-I-associated epitope-related literature to provide domain-specific knowledge, enhancing the precision and relevance of LLM-generated responses. EpitopeMiner offers three key features: First, the ability to identify epitopes with similar sequences and potentially similar immunogenic effects, which is especially valuable for neoantigens that are patient-specific and rarely found in public datasets. Second, it supports multiple epitope searches with structured outputs, enhancing scalability. Third, it provides original text chunks and paper identifiers, significantly simplifying validation and further exploration of the retrieved knowledge. Applying EpitopeMiner to well-characterized MHC class I epitopes demonstrates its ability to consistently retrieve relevant papers, efficiently providing targeted insights on T-cell response and immunogenicity, outperforming a commercial AI-powered literature tool. Notably, when applied to lymphoma patient-derived neoantigens, EpitopeMiner successfully retrieves immune response information for a few similar epitopes, despite operating with a relatively smaller database compared to the benchmark tool. All in all, EpitopeMiner bridges the gap between computational prediction and experimental validation, providing a scalable solution for extracting knowledge from public data, fostering cross-study synergies, and accelerating the development of personalized cancer vaccines.

cancer biology↗
bioRxiv2025

LARP6 regulates the mRNA translation of fibrogenic genes in liver fibrosis

Metabolic syndrome and excessive alcohol consumption result in liver injury and fibrosis, which is characterized by increased collagen production by activated Hepatic Stellate Cells (HSCs). LARP6, an RNA-binding protein, was shown to facilitate collagen production. However, LARP6 expression and functionality as a regulator of fibrosis development in a disease relevant model remains elusive. By using snRNA-sequencing, we show that LARP6 is upregulated mainly in HSCs of liver fibrosis patients. Moreover, LARP6 knockdown in human HSCs suppresses fibrogenic gene expression. By integrating eCLIP analysis and ribosome profiling in HSCs, we show that LARP6 interacts with mature mRNAs comprising over 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. Furthermore, LARP6 knockdown in HSC attenuates fibrosis development in human liver spheroids. Altogether, our results suggest that targeting LARP6 in human HSCs may provide new strategies for anti-fibrotic therapy. HighlightsO_LILARP6 is upregulated in liver fibrosis, mainly in HSCs. C_LIO_LILARP6 knockdown in human HSCs reduces liver fibrosis development. C_LIO_LIOf the hundreds of gene targets, LARP6 interacts most with collagen mRNAs. C_LIO_LILARP6 regulates mRNA translation via interaction with 5UTRs. C_LI

cell biology↗
bioRxiv2024

Decoding task representations that support generalization in hierarchical task

Task knowledge can be encoded hierarchically such that complex tasks can be built by associating simpler tasks. This associative organization supports generalization to facilitate learning of related but novel complex tasks. To study how the brain implements generalization in hierarchical task learning, we trained human participants on two complex tasks that shared a simple task and tested them on novel complex tasks whose association could be inferred via the shared simple task. Behaviorally, we observed faster learning of the novel complex tasks than control tasks. Using electroencephalogram (EEG) data, we decoded constituent simple tasks when performing a complex task (i.e., EEG association effect). Crucially, the shared simple task, although not part of the novel complex task, could be reliably decoded from the novel complex task. This decoding strength was correlated with EEG association effect and behavioral generalization effect. The findings demonstrate how task learning can be accelerated by associative inference. Significance StatementHumans can generalize knowledge of existing tasks to accelerate the learning of new tasks. We hypothesize that this generalization is achieved by building complex tasks that associate simple (sub)tasks that can be reused. Using electroencephalogram (EEG) data, we showed that constituent simple tasks can be decoded from the EEG data of humans learning new complex tasks. Crucially, when participants represent complex tasks as associations between multiple simple tasks, the simple tasks can be decoded from the new complex task, even when they are not part of the new complex task. These findings demonstrate the importance of the reinstatement of simple tasks in task learning through generalization.

neuroscience↗
bioRxiv2024

TEAD-targeting small molecules induce a cofactor switch to regulate the Hippo pathway

TEAD proteins are the main transcriptional effectors of the Hippo signaling pathway and a pharmacological target in oncology. Most TEAD-targeting small molecules act by disrupting interaction with the oncogenic transcriptional activators YAP and TAZ. Here, we describe an alternative mechanism for TEAD lipid pocket binding molecules. We report that select sulfonamide-containing compounds promote TEAD interaction with the transcriptional repressor VGLL4 to induce a small molecule-mediated cofactor switch from YAP to VGLL4. Chemically induced VGLL4-TEAD complexes counteract YAP activity at chromatin to repress pro-growth gene networks, including genes involved in cellular proliferation and mechanosignaling. VGLL4 is required for an anti-proliferative response to these select compounds, and genetic deletion of VGLL4 causes resistance to these molecules in vitro and in vivo. Our data reveal a category of molecules that facilitate the repressive VGLL4-TEAD interaction and open up new understandings for curbing the oncogenic activity of Hippo pathway deregulation.

cancer biology↗
bioRxiv2024

Fibroblastic FLT3L supports lymph node dendritic cells in the interfollicular niche

Dendritic cell (DC) homeostasis is maintained in secondary lymphoid organs (SLOs) by Fms-like tyrosine kinase 3 ligand (FLT3L). The specific niche providing this DC growth factor within human and mouse SLOs is unclear. Here, we show that Gremlin1 (Grem1)-expressing lymph node fibroblastic reticular cells (FRCs) support DC homeostasis via provision of FLT3L. Grem1 FRCs co-localize with DCs and express FLT3L in human and mouse lymph nodes. Using a new genetic model, we provide evidence that FLT3L produced by GREM1 FRCs maintains lymph node preDCs, cDCs, and plasmacytoid DCs (pDCs). Spatial transcriptomics and cytofluorometry reveal that Grem1 FRC-derived FLT3L supports not only proliferation, but also survival of lymph node cDCs within the interfollicular zone (IFZ). Functionally, loss of Grem1 FRC-derived FLT3L impairs cDC priming of antigen-specific T cell responses. These findings provide key mechanistic insights underlying stromal cell support of DC homeostasis and function.

immunology↗
bioRxiv2024

Mechano-adaptation: Exercise-driven Piezo1 & Piezo2 augmentation and chondroprotection in articular cartilage

Chondrocytes in adult joints are mechanosensitive post-mitotic quiescent cells with robustly expressed both Piezo1 and Piezo2 ion channels. Here, we examined the mechano-adaptation and Piezo modulations in articular chondrocytes using a mouse exercise model. We first found differential expression patterns of PIEZO1 and PIEZO2 in articular chondrocytes of healthy knee joints; chondrocytes in tibial cartilage (T) exhibit significantly higher PIEZO1 and PIEZO2 than femoral chondrocytes (F). Interestingly, a few weeks of exercise caused both PIEZO1 and PIEZO2 augmentation in F and T compared to the sedentary control group. Despite the increased expression levels of these mechanosensors, chondrocytes in exercised cartilage exhibit significantly reduced mechanical susceptibility against 1mJ impact. PIEZO1 modulation was relatively more rapid than PIEZO2 channels post-exercise. We tested the exercise-induced effect using Piezo1-conditional knockout (Pz1-cKO; Agc1CreERT2;Piezo1fl/fl). Pz1-cKO mice exhibit diminished exercise-driven chondroprotection against 1mJ impact, suggesting essential roles of Piezo1-mediated mechanotransduction for physiologic-induced cartilage matrix homeostasis. In addition, using a mouse OA model, we further found the modulated PIEZO1 in chondrocytes, consistent with reports in Ren et al., but without PIEZO2 modulations over OA progression. In summary, our data reveal the distinctly tuned Piezo1 and Piezo2 channels in chondrocytes post-exercise and post-injury, in turn modulating the mechanical susceptibility of chondrocytes. We postulate that Piezo1 is a tightly-regulated biphasic biomarker; Piezo1 antagonism may increase cellular survival post-injury and Piezo1 (with Piezo2) agonism to promote cartilage ECM restoration.

bioengineering↗
bioRxiv2024

Elucidating Interactome Dynamics of the A2A Adenosine Receptor

Polydeoxyribonucleotide (PDRN) is a tissue regeneration substance that resembles human DNA and is present in human cells, mullets, salmons, and flatfish. It stimulates physiological regeneration and metabolic activity. The regenerative and metabolic effects of PDRN are attributed to the activation of Adenosine A2A receptors (ADORA2A), which increases the production of angiogenesis factors and growth factors. Activation of ADORA2A leads to the activation of ADORA2A-interacting proteins with similar functions. To investigate the changes and dynamics of proteins in the presence of PDRN, we conducted selected reaction monitoring-mass spectrometry (SRM-MS) to quantify 491 proteins, 3,852 peptides. Through peptide-level analysis, we founded 374 proteins and 1,193 peptides demonstrating both up-regulation and down-regulation in expression. We conducted gene ontology (GO) analysis and physical network analysis and discovered a novel network of proteins related to neuronal differentiation among the ADORA2A-interacting protein. Based on network analysis, we found indirect interactions with MAPK1 and MP2K1, which are known to influence neuronal cell differentiation and suggest the formation of a network involving MAPK signaling transduction. Finally, using AlphaFold multimer, we were able to predict interaction sites among ADORA2A-interacting proteins in the network associated with neuronal cell differentiation. Specifically, we predicted five interaction sites between ADORA2A and NTRK1 (which interacts with ADORA2A), forming the edge. Thus, we provided indicators for further research using ADORA2A present in robust network and highlighted the potential of PDRN to impact neuronal differentiation.

systems biology↗
bioRxiv2024

Rapid changes in plasma corticosterone and medial amygdala transcriptome profiles during social status change reveal molecular pathways associated with a major life history transition in mouse dominance hierarchies

Social hierarchies are a common form of social organization across species. Although hierarchies are largely stable across time, animals may socially ascend or descend within hierarchies depending on environmental and social challenges. Here, we develop a novel paradigm to study social ascent and descent within male CD-1 mouse social hierarchies. We show that mice of all social ranks rapidly establish new stable social hierarchies when placed in novel social groups with animals of equivalent social status. Seventy minutes following social hierarchy formation, previously socially dominant animals exhibit higher increases in plasma corticosterone and vastly greater transcriptional changes in the medial amygdala (MeA), which is central to the regulation of social behavior, compared to previously subordinate animals. Specifically, social descent is associated with reductions in MeA expression of myelination and oligodendrocyte differentiation genes. Maintaining high social status is associated with high expression of genes related to cholinergic signaling in the MeA. Conversely, social ascent is related to relatively few unique rapid changes in the MeA. We also identify novel genes associated with social transition that show common changes in expression when animals undergo either social descent or social ascent compared to maintaining their status. Two genes, Myosin binding protein C1 (Mybpc1) and -Crystallin (Crym), associated with vasoactive intestinal polypeptide (VIP) and thyroid hormone pathways respectively, are highly upregulated in socially transitioning individuals. Further, increases in genes associated with synaptic plasticity, excitatory glutamatergic signaling and learning and memory pathways were observed in transitioning animals suggesting that adaptations in these processes may support rapid social status changes. Author SummaryWe investigate how male CD-1 mice behaviorally, physiologically, and molecularly respond to changes in their social hierarchies. We found that mice rapidly reform social hierarchies when placed into novel social groups. Formerly dominant mice experience the greatest increase in stress hormone levels and the largest shifts in gene expression in the medial amygdala, a brain region crucial for regulating social behavior. Specifically, we observed reduced expression of genes related to myelin production and maintenance as well as cholinergic functioning in formerly dominant males who socially descended. In contrast, formerly subordinate males who socially ascended displayed fewer genomic changes. Moreover, we identified a set of genes that change expression when animals transition their social status regardless of whether they rise or fall in rank. These social transition genes provide new insights into the biological mechanisms underpinning social plasticity. Our findings demonstrate the distinct molecular mechanisms that are shared between socially ascending versus descending mice, as well as those that are unique to each type of social transition.

neuroscience↗
bioRxiv2024

Zebrafish and cellular models of SELENON-Related Myopathy exhibit novel embryonic and metabolic phenotypes

SELENON-Related Myopathy (SELENON-RM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology commonly includes multiminicores or a dystrophic pattern but is often non-specific. The SELENON gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency causes SELENON-RM are undetermined. A hurdle is the lack of cellular and animal models that show assayable phenotypes. Here we report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression between selenon and genes involved in glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit changes in glutathione and redox homeostasis, suggesting a direct relationship with SelN function. We report changes in metabolic function abnormalities in SelN-null myotubes when compared to WT. These results suggest that SelN has functional roles during zebrafish early development and myoblast metabolism.

molecular biology↗
bioRxiv2023

Misexpression of inactive genes in whole blood is associated with nearby rare structural variants

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and over half of inactive genes. Using 2,821 paired whole genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions and gene inversion. Overall, we develop misexpression as a novel type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression.

genetics↗
bioRxiv2023

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway in human cells. Here, we identified RNF146 as a ubiquitin ligase (E3) of TEADs, which negatively regulates their stability in cells through proteasome-mediated degradation. We show that RNF146-mediated TEAD ubiquitination is dependent on the TEAD PARylation state. We further validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Despite the RNF146 and proteasome-mediated degradation mechanisms, TEADs are stable proteins with a long half-life in cells. We demonstrate that degradation of TEADs can be greatly enhanced pharmacologically with heterobifunctional chemical inducers of protein degradation (CIDEs). These TEAD-CIDEs can effectively suppress activation of YAP/TAZ target genes in a dose-dependent manner and exhibit significant anti-proliferative effects in YAP/TAZ-dependent tumor cells, thus phenocopying the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates that the ubiquitin-proteasome system plays an important role in regulating TEAD functions and provides a proof-of-concept demonstration that pharmacologically induced TEAD ubiquitination could be leveraged to target YAP/TAZ-driven cancers.

cancer biology↗
bioRxiv2023

eQTLs identify regulatory networks and drivers of variation in the individual response to sepsis

Sepsis is a clinical syndrome of life-threatening organ dysfunction caused by a dysregulated response to infection, for which disease heterogeneity is a major obstacle to developing targeted treatments. We have previously identified gene expression-based patient subgroups (Sepsis Response Signatures: SRS) informative for outcome and underlying pathophysiology. Here we aimed to investigate the role of genetic variation in determining the host transcriptomic response and to delineate regulatory networks underlying SRS. Using genotyping and RNA-seq data on 638 adult sepsis patients, we report 16,049 independent expression (eQTLs) and 32 co-expression module (modQTLs) quantitative trait loci in this disease context. We identified significant interactions between SRS and genotype for 1,578 SNP-gene pairs, and combined transcription factor (TF) binding site information (SNP2TFBS) and predicted regulon activity (DoRothEA) to identify candidate upstream regulators. These included HIF1A and CEBPB, which were associated with progenitor and immature neutrophil subsets respectively, further implicating glycolysis and emergency granulopoiesis in SRS1. Overall, these approaches identified putative mechanistic links between host genetic variation, cell subtypes, and the individual transcriptomic response to infection. Understanding the regulatory networks underlying patient heterogeneity provides additional information for developing immunomodulatory treatments and a personalised medicine approach to treating sepsis.

genomics↗
bioRxiv2023

Distinct modulation of calcium-activated chloride channel TMEM16A by a novel drug-binding site

TMEM16A is a calcium-activated chloride channel with significant role in multiple cellular processes. Several TMEM16A inhibitors have been identified; however, their binding sites and inhibitory mechanisms remain unclear. Using magnolol and honokiol, the two regioisomeric inhibitors, as chemical probes, we have identified a novel drug-binding site distinct from the pore region, in TMEM16A, which is described here. With electrophysiology, unbiased molecular docking and clustering, molecular dynamics simulations, and experimental validation with mutant cycle analysis, we show that magnolol and honokiol utilize different drug-binding sites, pore and non-pore pockets. The pore blocker utilizes amino acids crucial for chloride passage, whereas the non-pore blocker allosterically modulates the pore residues to hinder ion permeation. Among 17 inhibitors tested, 11 were pore blockers and six were non-pore blockers, indicating the importance of this newly identified non-pore pocket. Our study provides insights into drug-binding mechanism in TMEM16A together with a rationale for future drug development.

physiology↗