Biology subjects

Lee, H. K.

Publications and source records attributed to Lee, H. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Lipoproteins modulate the uptake and biological functionof cationic cell-penetrating peptides across the animal lineage.

Cationic cell-penetrating peptides (+CPPs) are pervasive killer peptides produced by all animals and found in innate immune systems, venoms and neurodegenerative diseases. Despite their ubiquity, the mechanisms underlying their uptake remain opaque and intensely debated. Here, we interrogate +CPPs spanning 600 million years of animal evolution and identify endocytosis as a conserved/convergent uptake mechanism across cell types and organisms, at physiological concentrations. By combining multiplex imaging, genetic screening, cryo-electron tomography, and biophysical methods, we uncover that +CPPs seemingly universally enter eukaryote cells via 'hitchhiking' on lipoproteins. We further show that the interaction of +CPPs with lipoproteins modulates their antimicrobial function. Combined, our findings establish a unified molecular framework describing a pan-eukaryote cell penetrance mechanism. As hyperlipidemia is a common comorbidity, our findings have direct implications for human health. Lastly, the insights gleaned from this work highlight design principles that may inform the future engineering of peptide-based therapeutics and delivery vehicles.

cell biology↗

bioRxiv2026

DHHC3-dependent S-Acylation of CRY1 regulates its subcellular localization and repressor function in the circadian clock

The circadian clock is essential for maintaining cellular homeostasis and physiological fitness. At the molecular level, core clock proteins function via transcriptional-translational feedback loops in the cellular oscillator, and are highly regulated by post-translational modifications. Our unbiased screening of core clock proteins revealed that Cryptochrome 1 (CRY1), the central transcriptional repressor in the circadian clock, undergoes a novel post-translational modification known as S-acylation. We show that this reversible lipidation of CRY1 is required for its nuclear import and interaction with key clock components. Further, we mapped four cysteine residues as CRY1 S-acylation sites and identified DHHC3 as the primary protein acyltransferase for CRY1. Importantly, loss of CRY1 S-acylation, either via cysteine mutagenesis or genetic deletion of DHHC3, impaired CRY1 repressor function and consequently cellular circadian rhythms, suggesting that dynamic S-acylation couples cytoplasmic regulation of CRY1 and its transcriptional repressor function in the nucleus. Together, our findings identify S-acylation as a previously unknown post-translational modification of CRY1 critical for circadian clock function and establish DHHC3 as a pivotal circadian regulatory enzyme. Targeting CRY1 S-acylation or its regulatory enzymes may constitute an innovative therapeutic approach against clock-associated diseases.

biochemistry↗

bioRxiv2026

YAP activation reverses aging-related visual dysfunction caused by impaired cell-matrix adhesion

Cellular senescence of retinal pigment epithelium (RPE) cells drives age-related visual decline, particularly in the pathology of age-related macular degeneration (AMD). While genome-wide association studies (GWAS) have identified genetic risk factors underlying AMD, the molecular mechanisms governing RPE senescence remain unclear. Here, single-cell RNA sequencing of young and old mouse RPE revealed dysregulated cell-matrix adhesion as a key feature of senescence, consistent with transcriptional changes in AMD patients. Hydrogel-based experiments confirmed that impaired integrin-mediated adhesion induces RPE senescence. Yes-associated protein 1 (YAP), a crucial mechanotransducer, mediated the protective effects of cell-matrix adhesion, and its activation alone reversed aging phenotypes in senescent RPE cells. Notably, treatment with TRULI, a small-molecule YAP activator, significantly improved visual function in AMD and naturally aged mice. These findings highlight the integrin-YAP mechanotransduction pathway as a fundamental regulator of RPE senescence and a potential therapeutic target for AMD.

physiology↗

bioRxiv2025

Heritability and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family

The Janus Kinase (JAK) and Signal Transducers and Activators of Transcription (STAT) pathway regulate a range of biological processes, including immune response and hematopoiesis. While a major research focus has been on somatic human mutations in disease, less is known about the heritability of germline variants and their physiological impact. Here we identify the rare JAK3P151R, JAK3R925S, STAT5AV494L and STAT6Q633H variants in an extended family spanning three generations and integrate in silico analyses, AlphaFold 3 structural predictions and investigate the immune transcriptomes in probands carrying one or more variants. All four variants are inherited through the germline without any evident clinical or physiological manifestations in the carriers. As individual variants, not all persons carrying a specific variant showed the same immune transcriptome. The presence of activated basal transcriptomes was limited to some, but not all, individuals carrying the above variants above. A next step in understanding the role of germline variants will be to understand how and why other factors including both other germline variants as well as environmental and developmental factors influence the likelihood of expression of an activated basal transcriptome.

genomics↗

bioRxiv2025

Disease-associated mutations in the STAT5B SH2 domain reprogram hepatic cholesterol and lipid metabolism

Growth hormone (GH) signaling through STAT5B is a central regulator of hepatic metabolism, yet the functional consequences of disease-associated STAT5B variants remain poorly understood. Here, we analyzed mice carrying STAT5BY665F (gain-of-function) and STAT5BY665H (loss-of-function) variants and dissect their impact on metabolic regulation. STAT5BY665F mice developed hepatic lipid accumulation, hypercholesterolemia, and enhanced insulin sensitivity, whereas STAT5BY665H mice displayed reduced body weight and impaired insulin responsiveness. Transcriptomic analyses revealed that STAT5BY665F activated lipid, cholesterol, and immune transcriptional programs, while STAT5BY665H failed to induce these pathways. Notably, STAT5BY665F substantially feminized male liver gene expression, inducing 77% of female-biased genes while repressing 51% of male-biased genes, thereby mimicking the persistent STAT5B activation characteristic of female livers. ChIP-seq demonstrated extensive STAT5BY665F enhancer occupancy at metabolic and immune loci, contrasting with the minimal chromatin engagement of STAT5BY665H. Beyond the liver, STAT5BY665F broadly reprogrammed adipose tissue gene expression, activating lipid metabolism and immune regulatory networks, whereas STAT5BY665H exerted more restricted effects. Together, these findings illustrate how alterations in STAT5B activity affect enhancer activation and can lead to changes in metabolic function and hepatic sexual dimorphism.

genomics↗

bioRxiv2025

Umbilical cord structure shapes feto-maternal heat exchange across mammals

The umbilical cord exhibits striking structural diversity across placental mammals; however, the consequences of such variation on physiological function are poorly understood. Combining comparative anatomy across 130 mammal species with ancestral-trait reconstruction, multimodal imaging, and physics-based transport modelling, we show that the human umbilical cord structure is uncommon in other mammals and that the ancestral umbilical cord was unspiralled, with three vessels and an allantoic duct. Across species, vessel number and cord length correlate with birth weight: four-vessel cords and the persistence of an allantoic duct occur in species with heavier neonates, while two-vessel cords are confined to small muroid rodents. Mathematical modelling predicts that oxygen delivery remains near maximal across all cord architectures, but placental heat removal depends strongly on umbilical flow, and moderately on cord length and coiling. Scaling of umbilical flow with birth weight implies that larger species approach near-maximal heat-removal capacity, while smaller species are less efficient. Finally, three-dimensional imaging of the human cord reveals a triple-helix configuration of umbilical vessels, rather than arteries simply twisting around a central vein. This, and other observed cross-sectional vessel configurations in mammals and atypical human cords, aligns with theoretically predicted arrangements that minimize inter-vessel shunting, supporting an adaptive role for cord geometry in thermal regulation function. Together, these findings reframe the umbilical cord as a heat-exchange system and link its structural diversity to evolutionary history, neonatal mass, and maternal investment, with implications for fetal resilience to environmental stress.

physiology↗

bioRxiv2025

Glycan recognition by a plant sentinel immune receptor

Pathogens target and degrade the extracellular matrix surrounding plant cells. A central question is how cell wall-derived damage-associated molecular patterns (DAMPs) are recognized and integrated to trigger immune responses. We address this question by determining the structure of the multidomain receptor IGP1 in both apo form and bound to the cellulose-derived DAMP cellotriose. Structural analyses reveal that constitutive Leucine Rich Repeat-malectin interactions preconfigure IGP1 for ligand recognition and that the receptor features a highly specific sugar-binding pocket in the LRR domain capable of distinguishing fine variations in glycan structures. By directly sensing cello-oligomers, IGP1 acts as a cell wall sentinel that links pathogen-induced wall degradation to immune alerting, equipping plants to mount rapid and robust defense responses.

plant biology↗

bioRxiv2025

High-fidelity CRISPR genome editing of single-nucleotide mutation with near-complementary guide RNA via enhanced target binding kinetics

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.

biochemistry↗

bioRxiv2025

Pathogenic DVL frameshifting variants in Robinow syndrome disrupt WNT signaling and cellular dynamics

Robinow syndrome (RS) is a genetically heterogeneous rare disorder involving six genes in the WNT/planar cell polarity (PCP) signaling pathway. Frameshifting variants in DVL genes that introduce a novel basic C-terminus are a common cause of autosomal dominant RS, accounting for [~]33% of individuals without ROR2 variants. Here, we review ClinVar and literature variants affecting DVL paralogs resulting in RS and investigate the cellular effects of pathogenic DVL frameshift variants with mutant tails replacing at least 82 amino acids. In silico analysis of the new C-termini revealed altered intrinsically disordered regions (IDRs), charge distribution, and predicted protein structures. To explore potential altered biological mechanisms caused by novel C-termini, we generated wild-type (WT), frameshift, and truncated constructs of DVL1-3, and analyzed their behavior in a transfection-based in vitro systems. DVL proteins normally polymerize into cytoplasmic puncta that redistribute upon WNT stimulation. Immunocytochemistry showed that mutant DVL proteins failed to change their localization in response to WNT ligands, in contrast to WT alleles--a consistent observation across all three DVLs. In line with this, TOPFlash reporter assays demonstrated that mutant DVL1 and DVL3 failed to activate canonical WNT signaling, while WT proteins induced strong activation. Additionally, the mutant C-terminal tail interfered with CSNK1E-induced phosphorylation, offering a potential mechanism underlying the impaired WNT response. Our results provide further understanding of the cellular consequences of pathogenic DVL frameshifting variants and offers insights into the effects of such alleles on WNT signaling, and the perturbations thereof, that may lead to developmental phenotypes observed in RS.

genetics↗

bioRxiv2025

Deep contrastive feature compression with classical machine learning enables ligand discovery through efficient triage of large chemical libraries

Improving in silico compound-protein interaction (CPI) predictability is critical for productive drug discovery. Current deep learning approaches largely rely on end-to-end models trained on limited labeled CPI datasets, overlooking the representational power of large-scale biochemical foundation models. We present COMRADE (Contrastive Multirepresentation Accelerated Docking Engine), a hybrid virtual screening framework that accelerates docking by triaging compounds using CE-Screen (Contrastive Embedding-Screen). CE-Screen leverages seven high-dimensional pretrained representations - including those from protein language models and molecular transformers, along with an original physics-based interaction potential encoding - for rapid first-pass screening [~]100x faster than docking. Its contrastive compression neural network maps these inputs onto a single compact, discriminative representation optimized for CPI prediction via a lightweight ensemble classifier. CE-Screen outperforms state-of-the-art end-to-end models by up to 111.11% on retrospective benchmarks and is successfully used to triage [~]10.8 million compounds against five targets, yielding novel hits for each one - including a new scaffold for the branched-chain ketoacid dehydrogenase kinase (BCKDK), an understudied yet high-value target in metabolic disease and oncology.

bioinformatics↗

bioRxiv2025

Targeting the ferroptosis pathway: A novel compound, AZD1390, protects the brain after ischemic stroke

BackgroundFerroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. MethodsWe investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo, and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. ResultATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo. ConclusionsThis study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

neuroscience↗

bioRxiv2025

Y665F variant of mouse Stat5b protects against acute kidney injury through transcriptomic shifts in renal gene expression

The impact of single nucleotide polymorphisms (SNP) on physiology is often underestimated. One amino acid change can result in a variety of phenotypes apparent only in response to disease or injury. Even known pathogenic SNPs have widespread effects that are currently unaccounted for. In this study, we investigated the impact of the known activating and pathogenic STAT5BY665F mutation in a renal injury context in mice carrying this variant. Using ischemia-reperfusion model of acute kidney injury, immunohistochemistry, RNA-seq and ChIP-seq, we establish the protective role of STAT5B activation in renal epithelium and showcase the shifts in transcriptomic landscape in a tissue not associated with the usual human phenotype of the Stat5bY665F mutation. Our data indicate new links between the JAK/STAT pathway and known kidney injury markers, contribute to the understanding of the sexual dimorphism of renal disease, and provide new potential targets for JAK inhibitor- and amino acid transport modulation-based therapies.

genetics↗

bioRxiv2024

STAT5B leukemic mutations, altering SH2 tyrosine 665, have opposing impacts on immune gene programs

STAT5B is a vital transcription factor for lymphocytes. Here, function of two STAT5B mutations from human T cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5BY665F), the other with histidine (STAT5BY665H) was interrogated. In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro STAT5BY665F showed gain-of-function while STAT5BY665H demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of-function Stat5bY665F mutation resulted in accumulation of CD8+ effector and memory and CD4+ regulatory T-cells, altering CD8+/CD4+ ratios. In contrast, STAT5BY665H knock-in mice showed diminished CD8+ effector and memory and CD4+ regulatory T cells. In contrast to wild-type STAT5, the STAT5BY665F variant displayed greater STAT5 phosphorylation, DNA binding and transcriptional activity following cytokine activation while the STAT5BY665H variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of function variant that does not directly induce hematopoietic malignancy.

genetics↗

bioRxiv2024

Combined inhibition of S100A4 and TIGIT suppresses late-stage breast cancer metastasis to the lung by activating T and NK cells

Cancer metastasis is responsible for approximately 90% of cancer-related deaths, but very few treatment options exist currently. While the role of S100A4 in promoting metastasis has been known for decades, this knowledge has not been translated in the clinic. Here, we report that a novel monoclonal antibody against the human S100A4 protein (S1004-11) effectively suppresses breast cancer metastasis in two different mouse models. Importantly, a novel combination of ant-TIGIT and S100A4-11 can suppress lung metastases even in late-stage disease after the lung premetastatic niche (PMN) has already been established similar to a stage when many breast cancer patients are diagnosed. Mechanistically, S1004-11 mAb treatment block the formation of PMN by suppressing neutrophil infiltration and activating natural killer (NK) and T cells in the lung. Single-cell RNA-sequencing and cell:cell communication analyses indicate that TIGIT signaling suppresses NK cells, which is reversed in S100A4-11 treated PMN. In summary, this study provides compelling evidence for a novel mechanism of S100A4 function in PMN formation and the feasibility of using S100A4-11 monoclonal antibody to suppress metastases at different stages of breast cancer progression.

cancer biology↗

bioRxiv2024

Differential Responses of Lung and Intestinal Microbiota to SARS-CoV-2 Infection: A Comparative Study of the Wuhan and Omicron Strains in K18-hACE2 tg Mice

The COVID-19 pandemic, caused by SARS-CoV-2, has led to the emergence of viral variants with distinct characteristics. We investigated the differential effects of the original Wuhan strain and the emergent Omicron variant of SARS-CoV-2 using a K18-hACE2 transgenic mouse model. We compared the mortality rates, viral loads, and histopathological changes in lung and tracheal tissues, as well as alterations in the lung and intestinal microbiota following infection. We observed significant differences in disease severity, with the Wuhan strain causing higher mortality and more severe lung damage than the Omicron variant. Furthermore, microbiome analyses revealed distinct shifts in microbiota associated with infection by each variant, suggesting that microbiome-related mechanisms might influence disease outcomes. This comprehensive comparison enhances our understanding of COVID-19 pathogenesis and highlights the importance of microbiome dynamics in viral infections, providing insights for future therapeutic and preventive strategies. ImportanceUnderstanding the differential impacts of SARS-CoV-2 variants is crucial for effective public health response and treatment development. This study provides insights into the pathogenesis of the original Wuhan strain and the Omicron variant of SARS-CoV-2, revealing significant differences in host mortality, viral load, and lung pathology. The use of the K18-hACE2 transgenic mouse model enables detailed examination of these differences in a controlled setting. Furthermore, this study highlights the importance of the microbiome in modulating disease severity and host responses to viral infections. By uncovering distinct microbial shifts associated with infection by different SARS-CoV-2 variants, this study suggests potential microbiome-related mechanisms that might be targeted to mitigate disease outcomes.

microbiology↗

bioRxiv2024

RNA sequencing depth guidelines for the study of alternative splicing

A key parameter in the experimental design of RNA-seq projects is the choice of sequencing depth. Considering a limited budget, one needs to find a tradeoff between the number of samples and the sensitivity of the analysis, particularly concerning lowly expressed genes. While previous studies have proposed a lower bound for the comprehensive analysis of differential gene expression, for the analysis of alternative splicing, it has only been proposed for human adipose tissue. However, alternative splicing differs across tissues and conditions. We analyzed publicly available and newly generated deep-sequenced paired-end RNA-seq samples (between 150 and >500 million reads, read length 50-150 bp) from human buffy coat cells and diverse sets of tissues, including gluteal subcutaneous fat, heart, and hypothalamus. Our results show that the sequencing depth typically used in published cohorts is not sufficient to comprehensively capture the landscape of alternative splicing. This motivates the use of deeper sequencing or long-read technologies in future studies. Toward this goal, we offer guidelines for choosing a suitable sequencing depth. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/617406v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1aecd9org.highwire.dtl.DTLVardef@1b3d0fborg.highwire.dtl.DTLVardef@5d15c4org.highwire.dtl.DTLVardef@140035b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Gut microbiota dysbiosis induced by brain tumor modulates the efficacy of immunotherapy

BackgroundAlthough the influence of gut microbiota on various tumors has gained recognition, the intricate mechanisms underlying their effects on brain tumors remain largely unexplored. In this study, our objective was to unveil pivotal gut microbiota that contribute significantly to the anti-tumor immune response against brain tumors. ResultsDuring the progression of brain tumors, our research uncovered a notable shift in the gut microbiome, as revealed through comprehensive 16S rRNA sequencing analysis. This shift coincided with a substantial decrease in the concentration of tryptophan in fecal samples, indicating a potential link between tryptophan levels and microbiome composition. Intriguingly, dietary supplementation with tryptophan significantly mitigated these microbial alterations, suggesting a restorative effect on the gut microbiota composition. This intervention not only reversed the changes observed in the gut microbiome but also markedly improved survival rates, a phenomenon that was determined to be dependent on the gut microbiota. The mechanism underlying this improvement appears to involve an enhancement in T cell circulation, which in turn boosts the efficacy of immunotherapeutic approaches. Upon further investigation into the specific microbial species that were positively influenced by tryptophan supplementation, some candidates were screened. Among several gut microbiota strains restored by tryptophan supplementation, the most significant is Duncaniella dubosii. Through sole colonization into germ-free mice, we found that the presence of Duncaniella dubosii alone was able to replicate the beneficial effects observed with tryptophan supplementation. ConclusionsThis study highlights Duncaniella dubosiis crucial role in linking tryptophan supplementation to positive shifts in the gut microbiome, immune modulation, and enhanced survival during brain tumor progression. It underscores the complex interaction between diet, microbiota, and immune responses, offering novel insights for boosting cancer immunotherapys success.

microbiology↗

bioRxiv2024

Impaired Hippocampal Reactivation Preceding Robust Aβ Deposition in a Model of Alzheimer's Disease

Current therapeutic strategies for Alzheimers disease (AD) target amyloid-beta (A{beta}) fibrils and high molecular weight protofibrils associated with plaques, but molecular cascades associated with AD may drive neural systems failure before A{beta} plaque deposition in AD. Employing hippocampal electrophysiological recordings and dynamic calcium imaging across the sleep-wake cycle in a freely behaving mouse model of AD before A{beta} plaques accumulated, we detected marked impairments of hippocampal systems function: In a spatial behavioral task, phase-amplitude coupling (PAC) of the hippocampal theta and gamma oscillations was impaired and place cell calcium fluctuations were hyper-synchronized with the theta oscillation. These changes were not observed in REM sleep. In subsequent slow wave sleep (SWS), place cell reactivation was reduced. These degraded neural functions underlying memory encoding and consolidation support targeting pathological processes of the pre-plaque phase of AD to treat and prevent hippocampal impairments.

neuroscience↗