Biology subjects

Lee, J. D.

Publications and source records attributed to Lee, J. D..

17 recordsLinked to original sources

bioRxiv2026

Tumor suppressor-like mutations mark a dosage-constrained dependency in leukemia

Cancer genes are classically categorized as oncogenes or tumor suppressors based on whether gain or loss of function promotes malignancy. Here we show that oncogene-imposed dosage constraints can produce mutations that mimic tumor suppressor loss yet mark essential genes. In acute promyelocytic leukemia (APL), the transcription factor EBF3 gene harbors recurrent heterozygous loss-of-function mutations, a tumor suppressor signature, and is essential for APL survival. We resolve this paradox by showing that while PML::RARA, the fusion oncoprotein defining APL, drives EBF3 expression, excess EBF3 suppresses proliferation. Patient mutations reduce EBF3 activity through heterozygous inactivation, optimizing its expression within a corridor bounded by fitness constraints at both extremes. PML::RARA activates EBF3 via a p300-dependent intronic enhancer, and p300 inhibition exploits this constraint by suppressing EBF3 below the viability threshold. Our findings reveal that oncogenes can impose dosage constraints that drive adaptive mutations conventionally misclassified as tumor suppressor loss, with implications for interpreting cancer genomes.

cancer biology↗

bioRxiv2025

Sex-specific regulation of social play in juvenile rats by oxytocin neurons in the hypothalamus and oxytocin signaling in the nucleus accumbens

Social play is a rewarding behavior shown across juvenile mammalian species and is important for the development of social competency throughout the lifespan. The neuropeptide oxytocin (OXT) regulates various social behaviors and is being used in clinical trials to improve social competency. However, the role of OXT in juvenile social play is largely unknown. To address this gap, we determined the involvement of hypothalamic OXT-producing neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), PVNOXT projections to the nucleus accumbens (NAc), and OXT signaling within the NAc, in the regulation of social play in juvenile male and female rats. We found that neither chemogenetic stimulation of SONOXT cell bodies nor PVNOXT projections to the NAc altered the expression of juvenile social play but instead increased social investigation. However, chemogenetic stimulation of PVNOXT cell bodies as well as intra-NAc infusion of OXT decreased social play in males without an effect in females. Lastly, social play duration correlated negatively with the proportion of activated NAcOXTR neurons, an effect driven by males. Together, these findings suggest that distinct OXT neuronal populations modulate different forms of social behavior and that PVNOXT neurons and NAc-OXT signaling sex-specifically modulate social play behavior.

neuroscience↗

bioRxiv2025

Endothelial CLK2 as a therapeutic target for acute radiation syndrome

Damage to the vascular endothelium is a major contributor to acute radiation injury in multiple organs that underlies acute radiation syndrome (ARS), yet there are no FDA-approved radiation countermeasures targeting endothelial cells. Use of kinome-scale CRISPR screens performed in cultured human vascular endothelial cells isolated from different organs identified CLK2 as a potential radioprotective target. Pharmacological inhibition of CLK2 using TG003 and Cirtuvivint protected these endothelial cells against radiation injury and reversed its effects across the transcriptome and phospho-proteome. Human Organ Chip models of human intestine and lung that contain organ-specific epithelium and microvascular endothelium faithfully replicated clinical features of ARS when exposed to radiation, which were prevented when treated with CLK2 inhibitors. Thus, CLK2 inhibitors may represent a new class of radiation countermeasure drugs that can protect multiple organs against radiation-induced toxicities in patients with ARS.

molecular biology↗

bioRxiv2025

Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors

Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on {beta}arr binding and activation using cryo-EM structures. Interestingly, we observe that {beta}arrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.

biochemistry↗

bioRxiv2024

G protein Inactivation as a Mechanism for Addiction Treatment

The endogenous dynorphin/kappa opioid receptor (KOR) system in the brain mediates the dysphoric effects of stress, and KOR antagonists may have therapeutic potential for the treatment of drug addiction, depression, and psychosis. One class of KOR antagonists, the long-acting norBNI-like antagonists, have been suggested to act by causing KOR inactivation through a cJun-kinase mechanism rather than by competitive inhibition. In this study, we screened for other opioid ligands that might produce norBNI-like KOR inactivation and found that nalfurafine (a G-biased KOR agonist) and nalmefene (a KOR partial agonist) also produce long-lasting KOR inactivation. Neither nalfurafine nor nalmefene are completely selective KOR ligands, but KOR inactivation was observed at doses 10-100 fold lower than necessary for mu opioid receptor actions. Daily microdosing with nalfurafine or nalmefene blocked KORs responsible for antinociceptive effects, blocked KORs mediating stress-induced aversion, and mitigated the aversion during acute and protracted withdrawal in fentanyl-dependent mice. Both nalfurafine and nalmefene have long histories of safety and use in humans and could potentially be repurposed for the treatment of dynorphin-mediated stress disorders.

neuroscience↗

bioRxiv2024

Sodium Benzoate Promotes Fat Accumulation and Aging via the SKN-1/Nrf2 Signaling Pathway: Evidence from the Caenorhabditis elegans Model

Sodium benzoate (SB) is widely used in food products, cosmetics, and medical solutions due to its antimicrobial properties. While it is generally considered safe and has potential neuroprotective benefits, SB has also been linked to adverse effects, including hepatic oxidative stress and inflammation. However, the potential effects of SB on obesity and aging remain poorly understood. In this study, we investigated the effects of SB on fat accumulation and aging using the nematode Caenorhabditis elegans (C. elegans) as a model system. Wild-type worms were exposed to various SB concentrations (0%, 0.0004%, 0.0008%, 0.004%, and 0.1%) and 0.016% glucose as a positive control for 72 hours in liquid or on NGM agar plates. Fat accumulation was assessed through the Oil Red O staining, which revealed that SB induced more fat accumulation compared to vehicle control, even at low concentrations, including the dosage of 0.0004%. Lifespan analysis also demonstrated that SB significantly accelerated aging in wild-type worms in a dose-dependent manner. Further investigations found that SKN-1 (an Nrf2 homolog) is necessary for SB-induced fat accumulation and aging. Moreover, SB inhibited the nuclear localization of SKN-1 under oxidative stress conditions. These findings suggest that SB may induce fat accumulation and aging by inhibiting the oxidative stress-mediated SKN-1 signaling pathway.

pharmacology and toxicology↗

bioRxiv2024

Far-red and sensitive sensor for monitoring real-time H2O2 dynamics with subcellular resolution and inmulti-parametric imaging applications.

H2O2 is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded H2O2 Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF635, oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular H2O2 dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular H2O2 diffusion at subcellular resolution. Lastly, we used oROS-HT with the green fluorescent calcium indicator Fluo-4 to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.

bioengineering↗

bioRxiv2024

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROSs capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A{beta}-putriscine-MAOB axis, highlighting the sensors relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

bioengineering↗

bioRxiv2023

Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.

microbiology↗

bioRxiv2023

Machine Learning Ensemble Directed Engineering of Genetically Encoded Fluorescent Calcium Indicators.

Real-time monitoring of biological activity can be achieved through the use of genetically encoded fluorescent indicators (GEFIs). GEFIs are protein-based sensing tools whose biophysical characteristics can be engineered to meet experimental needs. However, GEFIs are inherently complex proteins with multiple dynamic states, rendering optimization one of the most challenging problems in protein engineering. Most GEFIs are engineered through trial-and-error mutagenesis, which is time and resource-intensive and often relies on empirical knowledge for each GEFI. We applied an alternative approach using machine learning to efficiently predict the outcomes of sensor mutagenesis by analyzing established libraries that link sensor sequences to functions. Using the GCaMP calcium indicator as a scaffold, we developed an ensemble of three regression models trained on experimentally derived GCaMP mutation libraries. We used the trained ensemble to perform an in silico functional screen on a library of 1423 novel, untested GCaMP variants. The mutations were predicted to significantly alter the fluorescent response, and off-rate kinetics were advanced for verification in vitro. We found that the ensembles predictions of novel variants biophysical characteristics closely replicated what we observed of the variants in vitro. As a result, we identified the novel ensemble-derived GCaMP (eGCaMP) variants, eGCaMP and eGCaMP+, that achieve both faster kinetics and larger fluorescent responses upon stimulation than previously published fast variants. Furthermore, we identified a combinatorial mutation with extraordinary dynamic range, eGCaMP2+, that outperforms the tested 6th, 7th, and 8th generation GCaMPs. These findings demonstrate the value of machine learning as a tool to facilitate the efficient prescreening of mutants for functional characteristics. By leveraging the learning capabilities of our ensemble, we were able to accelerate the identification of promising mutations and reduce the experimental burden associated with screening an entire library. Machine learning tools such as this have the potential to complement emerging high-throughput screening methodologies that generate massive datasets, which can be tedious to analyze manually. Overall, these findings have significant implications for developing new GEFIs and other protein-based tools, demonstrating the power of machine learning as an asset in protein engineering.

bioengineering↗

bioRxiv2022

Opto-MASS: a high-throughput engineering platform for genetically encoded fluorescentsensors enabling all optical in vivo detection of monoamines and neuropeptides

Fluorescent sensor proteins are instrumental for detecting biological signals in vivo with high temporal accuracy and cell-type specificity. However, engineering sensors with physiological ligand sensitivity and selectivity is difficult because they need to be optimized through individual mutagenesis in vitro to assess their performance. The vast mutational landscape proteins constitute an obstacle that slows down sensor development. This is particularly true for sensors that require mammalian host systems to be screened. Here, we developed a novel high-throughput engineering platform that functionally tests thousands of variants simultaneously in mammalian cells and thus allows the screening of large variant numbers. We showcase the capabilities of our platform, called Optogenetic Microwell Array Screening System (Opto-MASS), by engineering novel monoamine and neuropeptide in vivo capable sensors with distinct physiological roles at high-throughput.

bioengineering↗

bioRxiv2022

SARS-CoV-2 triggers complement activation through interactions with heparan sulfate

The complement system has been heavily implicated in severe COVID-19 with clinical studies revealing widespread gene induction, deposition, and activation. However, the mechanism by which complement is activated in this disease remains incompletely understood. Herein we examined the relationship between SARS-CoV-2 and complement by inoculating the virus in lepirudin-anticoagulated human blood. This caused progressive C5a production after 30 minutes and 24 hours, which was blocked entirely by inhibitors for factor B, C3, C5, and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface deposition of complement and interactions with heparan sulfate. Additional functional analysis revealed that complement-dependent granulocyte and monocyte activation was delayed. Indeed, C5aR1 internalisation and CD11b upregulation on these cells only occurred after 24 hours. Thus, SARS-CoV-2 is a non-canonical complement activator that triggers the alternative pathway through interactions with heparan sulfate.

immunology↗

bioRxiv2020

The 'C3aR antagonist' SB290157 is a partial C5aR2 agonist

Innate immune complement activation generates the C3 and C5 protein cleavage products C3a and C5a, defined classically as anaphylatoxins. C3a activates C3a receptors (C3aR), while C5a activates two receptors (C5aR1 and C5aR2) to exert their immunomodulatory activities. The non-peptide compound, SB290157, was originally reported in 2001 as the first C3aR antagonist. In 2005, the first report on non-selective nature of SB290157 was published, where the compound exerted clear agonistic, not antagonistic, activity in variety of cells. Other studies also documented non-selective activities of this drug in vivo. These findings severely hamper data interpretation regarding C3aR when using this compound. Unfortunately, given the dearth of C3aR inhibitors, SB290157 still remains widely used to explore C3aR biology (>70 publications to date). Given these issues, in the present study we aimed to further explore SB290157s pharmacological selectivity by screening the drug against three human anaphylatoxin receptors, C3aR, C5aR1 and C5aR2, using transfected cells. We first confirmed that SB290157 acts as a potent agonist at human C3aR. We also identified that SB290157 exerts partial agonist activity at C5aR2 by mediating {beta}-arrestin recruitment at higher compound doses. Notably, SB290157s activity at C5aR2 was more potent and efficacious than the current lead C5aR2 agonist P32. Notwithstanding this, SB290157 showed inhibitory effect on C3a-mediated signalling in primary human macrophages. Our results therefore provide even more caution against using SB290157 as a research tool to explore C3aR function. Given the reported immunomodulatory and anti-inflammatory activities of C3aR and C5aR2 agonism, any function observed with SB290157 could be due to these off target activities.

pharmacology and toxicology↗

bioRxiv2020

Glucose clearance and uptake is increased in the SOD1G93A mouse model of amyotrophic lateral sclerosis in an insulin-independent mechanism

Metabolic disturbances are associated with the progression of the neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), however the molecular events that drive energy imbalances in ALS are not completely understood. In this study we aimed to elucidate deficits in energy homeostasis in the SOD1G93A mouse model of ALS. We identified that SOD1G93A mice at mid-symptomatic disease stage have increased oxygen consumption and faster exogenous glucose uptake, despite presenting with normal insulin tolerance. Fasting glucose homeostasis was also disturbed, along with increased liver glycogen stores, despite elevated circulating glucagon, suggesting that glucagon signalling is impaired. Metabolic gene expression profiling of livers indicated that glucose cannot be utilised efficiently in SOD1G93A mice. Overall, we demonstrate that glucose homeostasis and uptake are altered in SOD1G93A mice, which is linked to an increase in insulin-independent glucose uptake and a disturbance in glucagon sensitivity, suggesting glucagon secretion and signalling could be potential therapeutic targets for ALS.

physiology↗

bioRxiv2020

Absence of receptor for advanced glycation end product (RAGE) reduces inflammation and extends survival in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signaling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE, and compared them to SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signaling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by [~]3 weeks, and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS, and highlights RAGE as a potential immune therapeutic target for ALS.

neuroscience↗

bioRxiv2020

Hematopoietic p53 loss cell-extrinsically defines an immune infiltrated microenvironment in leukemia and pre-leukemia

TP53 is the most frequently mutated gene in human cancers. In Acute Myeloid Leukemia (AML) and Clonal Hematopoiesis of Indeterminate Potential (CHIP), it is one of several recurrent genetic alterations. Despite multiple recent therapeutic advances for AML, TP53 mutated AML is associated with resistance to currently approved therapies and thus, a very poor prognosis. Emerging evidence suggests that mutations in TP53 may be a predictor of positive response to immunotherapy. To model cell - extrinsic consequences of hematopoietic p53 loss, we generated bone marrow chimeric mice bearing p53-/- and congenic wild type cells. Following reconstitution, we observed increased levels of wild type CD8+ and CD4+ T cells in mice transplanted with p53-/- hematopoietic cells compared to controls. In addition, we observed a change in the frequency of T cell subsets in p53-/- chimeras including an increase in Tregs. To determine if these alterations were mirrored in the leukemic setting, we next generated p53-/-;nRasG12D leukemia. While the bone marrow of p53-/-;nRasG12D leukemia showed the presence of both T and B lymphocytes, MLL-AF9 showed a near complete absence of lymphocytes, akin to immune-infiltrated and immune-desert phenotypes seen in solid tumors. These data clearly demonstrate a causal cell-extrinsic effect of hematopoietic p53 loss on the immune system, both in the context of leukemia and preleukemic states. Modeling AML genetics in murine models serves as a powerful tool to define the association between genetic drivers and immune subtypes of AML towards precise patient stratification critical for the application of emerging targeted and immune therapies. Statement of SignificanceTP53 mutations are frequent in both AML and CHIP, and are associated with both resistance to therapy as well as very poor prognosis. We provide evidence to investigate the immunotherapy as a treatment option for this subgroup of AML.

cancer biology↗

bioRxiv2020

A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models

The blood brain barrier (BBB) and blood spinal cord barrier (BSCB) are highly specialised structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In the present study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntingtons disease and amyotrophic lateral sclerosis, while biphasic disruption occurred in a mouse model of Alzheimers disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods, that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.

neuroscience↗