Biology subjects

Zheng, N.

Publications and source records attributed to Zheng, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Multiple non-redundant functions underlie the essentiality of lipopolysaccharide in Gram-negative bacteria

Lipopolysaccharide (LPS) is well known for its immunostimulatory activity and its contribution to the permeability barrier of the Gram-negative outer membrane. Yet LPS is essential for growth in nearly all Gram-negative bacteria, even under laboratory conditions where its barrier function should be less critical. LPS contributes to envelope mechanics, membrane biogenesis, and cell division, but whether any of these functions accounts for its essentiality remains unclear. Here, we systematically tested these possibilities by depleting LPS in Escherichia coli and Klebsiella pneumoniae. Osmoprotection largely preserved cell-envelope integrity but not viability, demonstrating that the mechanical contribution of LPS alone cannot explain its essentiality. Genetic perturbations that increase peptidoglycan rigidity or alter phospholipid homeostasis partially improved growth, supporting independent roles for LPS in envelope mechanics and membrane biogenesis. An unbiased suppressor screen further identified activation of the MarA regulon as a potent mechanism for surviving LPS depletion. Protection required transport by the AcrAB-TolC multidrug efflux pump, revealing an unexpected connection between efflux and cell-envelope homeostasis. Thus, LPS essentiality emerges from multiple non-redundant contributions to Gram-negative cell-envelope physiology.

microbiology↗

bioRxiv2026

Integrated immunopeptidomics and transcriptomics reveal a cancer-restricted non-canonical HLA class I ligandome in medulloblastoma

Medulloblastoma is the leading type of malignant brain tumor in children. Despite recent advances in T cell-based immunotherapy, the low mutational burden of medulloblastoma poses a considerable challenge to the discovery of mutation-derived neoantigens. Previous studies of medulloblastoma have investigated tumor-associated antigens and neoantigens derived from aberrant splice junctions. The landscape of non-canonical HLA class I-bound peptides (ncHLAp) remains poorly defined. To broaden the repertoire of targetable antigens in medulloblastoma, we mapped the ncHLAp landscape across the four major molecular subgroups of medulloblastoma (Group 3, Group 4, WNT, and SHH) by integrating immunopeptidomic and transcriptomic analyses. Candidates were further filtered against normal-tissue immunopeptidomic and ribosome profiling datasets to identify cancer-restricted ncHLAp (CR ncHLAp). A total of 2,298 ncHLAp were identified across 21 human medulloblastoma samples, of which 598 were CR ncHLAp. These CR ncHLAp were primarily derived from non-canonical translational sources, including retained introns, out-of-frame open reading frames, and long non-coding RNAs. Some CR ncHLAp were shared across medulloblastoma samples and detected in other cancer types. More than 70% of the CR ncHLAp were predicted to bind at least one patient-matched HLA allotype, and approximately 87% were predicted to have immunogenic potential. Collectively, this study defines a previously undercharacterized component, CR ncHLAp, of the medulloblastoma HLA-I ligandome and suggests that CR ncHLAp may serve as promising candidate targets for T-cell-based immunotherapy in medulloblastoma.

cancer biology↗

bioRxiv2026

Single Amino Acid Disease Mutations Act as Genetic Glues to Drive Neo-substrate Degradation

Disease-associated missense mutations are well known to affect protein turnover by either impairing substrate-ubiquitin ligase interactions or inducing misfolding-triggered protein clearance. Whether such mutations can drive gain-of-function in substrate-selective protein degradation remains under-explored. Here, we develop a tri-color global protein stability system to profile mutational effects across the intrinsically disordered regions (IDRs) of human transcription factors. Unexpectedly, we identify single amino acid substitutions that create functional short linear motif degrons to drive targeted protein degradation independently of protein quality control. These disease variants are exemplified by the gain of an FRY-like degron in GATA2, whose deficiency leads to impaired immune function. Structural analyses reveal that the substituted residue enables neomorphic interaction between the transcription factor and the KLHL15 E3 ligase by complementing their otherwise suboptimal interface. The prevalence of such glue-like mutations beyond transcription factors suggests that quasi-degrons are widespread in protein IDRs and can be functionalized by simple mutations, a finding that may inform the rational discovery of molecular glue degraders.

molecular biology↗

bioRxiv2026

Cyclin D-CDK4/6 couple proliferation to membrane and mitochondrial protein supply through a DeSI1 phospho-switch

Cyclin D-CDK4/6 complexes drive cell-cycle entry through an RB-E2F-dependent transcriptional program, but how they coordinate proliferation with the membrane and organelle protein supply required for growth is unclear. We identify DeSI1 as a cyclin D-CDK4/6 substrate whose phosphorylation at S25 converts a latent homodimer into an active monomeric deubiquitylase that recognizes hydrophobic proteins such as those bearing transmembrane domains and mitochondrial targeting sequences. Phosphorylated DeSI1 extends the lifetime of newly synthesized hydrophobic proteins that support membrane and mitochondrial capacity. Constitutive DeSI1 activation uncouples this proteostatic program from metabolic supply, creating a cytidine-nucleotide supply-demand imbalance associated with impaired CTP-dependent phospholipid homeostasis, cardiolipin depletion, and mitochondrial decompensation - defects that cytidine reverses. In mice, constitutive DeSI1 activation causes progressive cerebellar degeneration with membrane-protein accumulation, respiratory-chain loss, and phospholipid depletion. These findings define a post-translational mechanism coupling cell-cycle entry to membrane and mitochondrial capacity, and reveal the cost of uncoupling this program from its metabolic support.

cell biology↗

bioRxiv2026

Cyclic-thiourea ionizable lipids program multilamellar mRNA-LNPs for sustained expression, frozen-storage stability and potent vaccine immunotherapy

Lipid nanoparticles (LNPs) are the leading delivery vehicles for mRNA therapeutics, yet transient expression and limited formulation stability constrain their broader therapeutic application. Here we report the phenotype-driven discovery of an ionizable lipid (HI-62) bearing a 1,3,5-triazinane-2-thione (cyclic-thiourea) headgroup that reproducibly assemble mRNA into onion-like multilamellar nanoparticles. Cryogenic electron microscopy revealed ordered internal lamellae, while molecular-dynamics simulations supported a cooperative assembly model involving pH-responsive electrostatics, headgroup preorganization and short-range directional interactions. The lead HI-62 formulation maintained greater than 95% mRNA encapsulation across N/P ratios of 3-7, prolonged reporter expression for substantially longer durations than benchmark formulations following intravenous and intramuscular delivery, and retains physicochemical integrity and in vivo potency after repeated freeze-thaw cycles and 18 months of storage at -20. In the tumor models, HI-62 elicits robust cellular responses and achieved significant inhibition of tumor growth. These findings establish cyclic-thiourea headgroup chemistry as a design principle for programming lipid-RNA organization into multilamellar architecture, and provide a design principle for next-generation LNPs platform with durable protein expression and superior stability. The HI-62-based formulation has been utilized in mRNA cancer vaccine program which will enter clinical development in months.

bioengineering↗

bioRxiv2026

Scaffold Affinity Tunes Biomolecular Condensate Function

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

biochemistry↗

bioRxiv2026

Blood-Mediated Opto-Acoustic Stimulation of Brain Cortex at Sub-millimeter Precision

Direct modulation of neural activity with high spatiotemporal precision is a cornerstone in experimental neuroscience. Here, we present a blood-mediated optoacoustic stimulation (BOAS) approach that utilizes blood as an endogenous transducer for brain stimulation. By delivering 532-nm nanosecond pulsed laser to the cortex, we demonstrate that the absorption of hemoglobin generates sufficient acoustic pressure to trigger neuronal activity. By integrating BOAS with calcium imaging in GCaMP6f-expressing mice, localized neuronal responses were observed. Quantitative analysis reveals that BOAS produces responses comparable to natural visual stimulation and is significantly more efficient than the photothermal stimulation. Furthermore, we show that the response is dose-dependent. At high energy doses, BOAS induces cortical spreading depression. Histological evaluation confirmed that the brain maintains tissue integrity even under these stimulation parameters. Together, this work establishes a versatile method for precise brain stimulation as an alternative method for stimulating neuron at cortex.

bioengineering↗

bioRxiv2026

Integrated Interactomics Reveals Novel Protein Associations: The FOXA1-PBX1 Complex as a Case Study

Protein-protein interactions (PPIs) are fundamental to cellular signaling networks, yet many remain undetected due to technical limitations of individual affinity purification approaches. To address this, we systematically mapped the interaction landscapes of six regulatory proteins involved in cell proliferation, immunity, and inflammation, including three transcription factors (TFs) and three kinases. We implemented an integrated proteomics workflow that combined four complementary affinity purification strategies: native immunoprecipitation, two crosslinking-assisted capture methods, and proximity labeling. Combining these approaches revealed distinct yet overlapping interaction profiles, uncovered numerous previously unreported interactors not reliably detected by individual methods, and robustly recovered known interactions while substantially extending PPI networks. Despite method-specific differences at the protein level, functional enrichment analyses showed strong convergence on coherent biological pathways. Biochemical approaches validated most of the previously unreported interactions, including putative weak and transient complexes stabilized by crosslinking. Functional assays revealed a previously unrecognized physical interaction between FOXA1 and PBX1 TFs and demonstrated their cooperative regulation of transcriptional programs and cell fitness in estrogen receptor (ER) positive breast cells. We propose that the FOXA1-PBX1 complex could represent a higher-order regulatory node integrating chromatin accessibility and ER-driven transcriptional output. HIGHLIGHTSO_LIComplementary affinity purification strategies uncover putative weak and transient protein-protein interactions C_LIO_LIFunctional pathway convergence validates biologically coherent interactome expansion C_LIO_LIBiochemical validations confirm unreported interactions C_LIO_LIFunctional validation studies identify a FOXA1-PBX1 pioneer factor complex that regulates estrogen receptor transcriptional programs C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/738938v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@193cc1borg.highwire.dtl.DTLVardef@3d068dorg.highwire.dtl.DTLVardef@791d5corg.highwire.dtl.DTLVardef@1769ce7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Functional Locality-Aligned Learning Reveals Structure-Function Causality in Enzyme Kinetics

Accurate estimation of enzyme kinetic parameters is essential for enzyme engineering and industrial biocatalysis, yet their experimental measurement remains labor-intensive and costly. Although machine learning offers an efficient alternative, existing methods still struggle to generalize to unseen enzymes and substrates. In particular, current three-dimensional (3D) structure-aware approaches rely on whole-enzyme 3D geometric structures and neglect substrate 3D geometry, often yielding limited or even degraded performance. We identify a fundamental limitation underlying these methods: a mismatch between the structural representation learning scale and the functional locality scale, which weakens structure-function causality in enzyme kinetics. To address this issue, we introduce EnzymePlex, a functional locality-aligned framework that aligns inductive biases with the localized structural determinants of enzyme function by prioritizing catalytic pockets, integrating substrate 3D geometry, and modeling nuanced enzyme-substrate interplay under the guidance of pocket-level structural priors. EnzymePlex achieves state-of-the-art performance across multiple benchmarks and substantially improves generalization under stringent out-of-distribution evaluations. Beyond predictive accuracy, EnzymePlex learns mechanistically aligned representations, with attention enriched at catalytic pocket residues and substrate reaction centers despite receiving no explicit super-vision for either. Moreover, when applied to recently reported wet-lab data, EnzymePlex effectively prioritizes high-activity enzyme variants and identifies potent inhibitors, highlighting its potential to accelerate enzyme engineering and drug discovery.

biochemistry↗

bioRxiv2025

Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency

Orthosteric inhibitors block enzyme active sites and prevent substrates from binding. Enhancing their specificity through substrate dependence seems inherently unlikely, as their mechanism hinges on direct competition rather than selective recognition. Here, we show that a molecular glue mechanism unexpectedly imparts substrate-dependent potency to CSN5i-3, an orthosteric inhibitor of the COP9 signalosome (CSN). We first confirm that CSN5i-3 inhibits CSN, which catalyzes NEDD8 deconjugation from the cullin-RING ubiquitin ligases (CRLs), by occupying the active site of its catalytic subunit, CSN5, and directly competing with the iso-peptide bond substrate. Curiously, the orthosteric inhibitor binds free CSN with only micromolar affinity, yet achieves nanomolar potency in blocking its deneddylase activity. Cryo-EM structures of the enzyme-substrate-inhibitor complex reveal that active site-engaged CSN5i-3 occludes the substrate iso-peptide linkage while simultaneously extending an NEDD8-binding exosite of CSN5, acting as a molecular glue to cement the NEDD8-CSN5 interaction. The cooperativity of this tri-molecular CSN5i-3-NEDD8-CSN5 assembly, in turn, sequesters CSN5i-3 at its binding site, conferring high potency to the orthosteric inhibitor despite its low affinity for the free enzyme. Together, our findings highlight the modest affinity requirements of molecule glues for individual target proteins and establish "orthosteric molecular glue inhibitors" as a new class of substrate-dependent enzyme antagonists.

molecular biology↗

bioRxiv2025

UBR-1 enzyme network regulates glutamate homeostasis to affect organismal behavior and developmental viability

Johanson-Blizzard Syndrome (JBS) is an autosomal recessive spectrum disorder associated with the UBR-1 ubiquitin ligase that features developmental delay including motor abnormalities. Here, we demonstrate that C. elegans UBR-1 regulates high-intensity locomotor behavior and developmental viability via both ubiquitin ligase and scaffolding mechanisms. Super-resolution imaging with CRISPR-engineered UBR-1 and genetic results demonstrated that UBR-1 is expressed and functions in the nervous system including in pre-motor interneurons. To decipher mechanisms of UBR-1 function, we deployed CRISPR-based proteomics using C. elegans which identified a cadre of glutamate metabolic enzymes physically associated with UBR-1 including GLN-3, GOT-2.2, GFAT-1 and GDH-1. Similar to UBR-1, all four glutamate enzymes are genetically linked to human developmental and neurological deficits. Proteomics, multi-gene interaction studies, and pharmacological findings indicated that UBR-1, GLN-3 and GOT-2.2 form a signaling axis that regulates glutamate homeostasis. Developmentally, UBR-1 is expressed in embryos and functions with GLN-3 to regulate viability. Overall, our results suggest UBR-1 is an enzyme hub in a GOT-2.2/UBR-1/GLN-3 axis that maintains glutamate homeostasis required for efficient locomotion and organismal viability. Given the prominent role of glutamate within and outside the nervous system, the UBR-1 glutamate homeostatic network we have identified could contribute to JBS etiology.

neuroscience↗

bioRxiv2025

PCMT1 generates the C-terminal cyclic imide degron on CRBN substrates

The E3 ligase substrate adapter cereblon (CRBN), the primary target of clinical agents thalidomide and lenalidomide, recognizes endogenous substrates bearing the C-terminal cyclic imide modification. Although C-terminal cyclic imides can form spontaneously, an enzyme that regulates the formation of these modifications and thereby promotes a biological pathway connecting substrates to CRBN is unknown. Here, we report that protein carboxymethyltransferase (PCMT1) promotes formation of the C-terminal cyclic imide on C-terminal asparagine residues of CRBN substrates. PCMT1 and CRBN co-regulate the levels of metabolic enzymes glutamine synthetase (GLUL) and inorganic pyrophosphatase 1 (PPA1) in vitro, in cells, and in vivo, and this regulation is associated with the proepileptic phenotype of CRBN knockout mouse models. The discovery of an enzyme that regulates CRBN substrates through the C-terminal cyclic imide modification reveals a previously unknown biological pathway that is perturbed by thalidomide derivatives and provides a biochemical basis for the connection between multiple biological processes and CRBN.

biochemistry↗

bioRxiv2025

Characterization of the gut mycobiome in patients with non-alcoholic fatty liver disease and correlations with serum metabolome

BackgroundThe relationship between the gut mycobiome and non-alcoholic fatty liver disease (NAFLD) is largely unexplored. MethodsIn this study, we used a publicly available shotgun metagenomic sequencing dataset of fecal samples to compare the gut fungal communities of 90 NAFLD patients with those of 90 healthy controls. We examined their correlations with gut bacterial communities, host serum metabolite s, and the interactions of key taxa within these networks. ResultsOur results revealed a remarked dysbiosis in the gut mycobiome of NAFLD patients, cha racterized by a significant increase in four fungal taxa, including Pseudopithomyces sp. c174, Muc or sp. c176, Aspergillus sp. c25, and uncultured Ascochyta c213. Multi-omics analysis showed that the gut mycobiome significantly contributes to the host serum metabolome. Pseudopithomyces s p. c174 was notably associated with some beneficial blood metabolites, such as glycoursodeoxycholic acid and alpha-linolenic acid, whereas the NAFLD-enriched pro-inflammatory phenylacetic acid was linked to Aureobasidium sp. c170 and Basipetospora sp. c193. Network analysis revealed significant alterations in the gut microbiome of NAFLD patients, highlighting the potential roles of Alternaria alternata c42, Penicillium sp. c5, and uncultured Malassezia c303. Moreover, predictive models that integrated both bacterial and fungal taxa improved prediction accuracy, emphasizing the crucial role of gut fungi in NAFLD. ImportanceNon-alcoholic fatty liver disease (NAFLD) is a widespread liver condition with limited treatments. While gut bacteria have been extensively studied in NAFLD, the role of gut fungi (the mycobiome) remains poorly understood. This study fills that gap by characterizing gut fungal communities in NAFLD patients and linking these changes to their blood metabolite profiles. We found that NAFLD patients show distinct fungal imbalances, which correlate with key metabolic alterations in the host. These findings expand our understanding of NAFLD by spotlighting gut fungi as important contributors to disease progression. This insight opens new avenues for potential biomarkers and therapies targeting the gut-liver axis. ConclusionsOur findings provide a comprehensive understanding of the link between the gut mycobiome and NAFLD progression, which is essential for guiding future therapeutic research.

microbiology↗

bioRxiv2024

Distinct Perception Mechanisms of BACH1 Quaternary Structure Degrons by Two F-box Proteins under Oxidative Stress

The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.

cancer biology↗

bioRxiv2024

Asymmetric Engagement of Dimeric CRL3KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

molecular biology↗

bioRxiv2024

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1. As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB)2, the most common embryonal brain tumor in children, and pineoblastoma3. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST4. However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

molecular biology↗

bioRxiv2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we developed a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screened multi-billion compound libraries against two unrelated targets, a novel ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. On both targets, we discover hits, including seven novel hits (14% hit rate) to KLHDC2 and four novel hits (44% hit rate) to NaV1.7 with single digit micromolar binding affinities. Screening in both cases was completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

biochemistry↗

bioRxiv2024

LRRK2 G2019S mutation suppresses differentiation of Th9 and Treg cells via JAK/STAT3

The Leucine-rich repeat kinase-2 (LRRK2) G2019S mutation, resulting in aberrantly enhanced kinase activity, is one of the well-recognized genetic risk factors in Parkinsons Disease (PD). Increased LRRK2 activity was also observed in immune cells from PD patients. Emerging results have also unveiled an upsurge in -synuclein (-syn)-specific CD4+ T cell responses in PD patients. Given that LRRK2 mutations in PD are germline mutations, there are unmet meets to explore whether LRRK2 G2019S mutation contributes to the pathogenesis of PD via altering CD4+ T-cell functions. To fill this knowledge gap, we generated a new T cell receptor (TCR) transgenic mouse strain bearing LRRK2 G2019S knock-in mutation, OT-II/LRRK2 (Refer to Mut). As CD4+ T cells from OT-II mice specifically recognize ovalbumin, this new strain enables us to explore the impact of LRRK2 G2019S mutation on T-cell functions in an antigen-specific manner. We found that the abundance and proliferation of major immune subsets in spleen tissue from Mut mice are comparable to wild-type (OT-II, Refer to WT) control. However, when we characterized T cell differentiation in these two strains, T cells derived from Mut mice displayed increased Th2 differentiation (IL-4) and decreased Th9 (IL-9) and Treg (Foxp3+ %) differentiation. LRRK2 G2019S mutation significantly altered the expression levels of master transcription factors (TFs) for T cell differentiation. Specifically, Mut T cells displayed an increase in mRNA expression of Gata3 (TF for Th2), a decrease in expression of Irf4 and Foxp3 (TFs for Th9 and Treg, respectively). Mechanistically, LRRK2 mutation decreased IL-9 production and Treg cell population through the JAK/STAT3 signaling. In conclusion, LRRK2 plays a critical role in regulating T cell differentiation, warranting further studies to evaluate the impacts of altered T cell differentiation led by LRRK2 mutation in dopaminergic neuron damages.

immunology↗