Biology subjects

Hu, J.

Publications and source records attributed to Hu, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Pericyte contact alters endothelial cell metabolism by promoting exchange of lactate through SLC16A3

Intimate crosstalk between endothelial cells and pericytes is fundamental for vascular development and stability, but the metabolic dimension of this interaction remains poorly defined. Using a filter-based co-culture system that mimics the shared basement membrane of the microcirculation, we performed an integrated multi-omics analysis to investigate how direct contact reprograms both cell types. We found that initial contact does not induce immediate quiescence but rather triggers a transient, low-level activation of an endothelial-to-mesenchymal transition (EndMT)-like state in endothelial cells, characterized by specific upregulation of genes involved in extracellular matrix production (e.g., collagen isoforms) and PDGFR signaling, without a full loss of endothelial identity. Concomitantly, pericytes shifted endothelial cell metabolism, increasing glycolysis and elevating intracellular pyruvate and lactate. Proteomic analysis of the contact interface revealed enrichment of solute carriers, most notably the lactate transporter SLC16A3. Functional studies demonstrated that pericytes act as a glycolytic partner, actively shuttling lactate to endothelial cells via SLC16A3. This lactate did not fuel the TCA cycle but instead served as a signaling metabolite, driving widespread alterations in the endothelial acetylome and lactylome, particularly affecting proteins involved in glycolytic metabolism. In vitro, exogenous lactate potentiated cytokine-induced EndMT and upregulated lactylation-associated genes. The physiological relevance of this lactate shuttle was confirmed in vivo, where endothelial-specific deletion of Slc16a3 in mice impaired postnatal retinal angiogenesis, leading to reduced vessel density and diminished endothelial-pericyte overlap without affecting endothelial cell proliferation. Our findings establish that vessel maturation is orchestrated by a metabolically gated phase of plasticity initiated upon first contact, wherein a targeted EndMT-like program and a pericyte-driven lactate signaling axis are integrated to coordinate vascular network assembly.

physiology↗

bioRxiv2025

An in situ generated CAR-M with IFNg and negative dominance Sirpa isoform augments hepatocellular carcinoma immunotherapy

Background and AimsChimeric antigen receptor (CAR) T cells have shown strong efficacy in hematological cancers but limited success in solid tumors. Macrophages, with their natural ability to infiltrate tumors, modulate immunity, and phagocytose cancer cells, offer a promising alternative when engineered with CARs. While studies have demonstrated the feasibility and anti-tumor activity of CAR macrophages (CAR-M), enhancing their persistence and phagocytic capacity remains a key challenge. Methods and ResultsBuilding on first-generation CD3{zeta}-based CAR-M, we developed a novel CAR targeting human GPC3, incorporating IFN-{gamma} and the extracellular domain of SIRP (SIRPECD) to improve CAR-M persistence and block the CD47-SIRP immune checkpoint. Following delivery via lipid nanoparticle-encapsulated mRNA (LNP-mRNA), the self-secreted IFN-{gamma} sustained M1 polarization through phospho-STAT1 activation. Meanwhile, the ectopically expressed SIRPECD competitively bound to CD47 on tumor cells, thereby blocking the endogenous SIRP-SHP2 interaction in a dominant-negative manner. This design enhanced pro-inflammatory activity and anti-tumor efficacy compared to CD3{zeta}-only CAR-M. Single-cell RNA sequencing and cellular analysis showed that in situ programmed CAR-M reprogrammed the tumor microenvironment toward inflammation in a murine HCC model. Moreover, CAR-M derived from human peripheral blood mononuclear cells (PBMCs) effectively phagocytosed human HCC organoids while sparing healthy tissues, indicating clinical potential. ConclusionsCollectively, our work presents a novel CAR design that enhances phagocytic function and sustains anti-tumor activity, offering a promising strategy for human solid tumor immunotherapy.

cancer biology↗

bioRxiv2025

Soluble epoxide hydrolase upregulation in Alzheimer's disease promotes blood-brain barrier dysfunction

Recent advances in anti-amyloid therapies for Alzheimers disease have been promising, but they have also highlighted critical challenges, including increased vascular complications, such as amyloid-related imaging abnormalities. Emerging evidence suggests that the soluble epoxide hydrolase may be a promising therapeutic target due to the involvement of sEH-derived diols in inflammation, oxidative stress, and vascular destabilization. APPPS1 mice were crossed with an inducible soluble epoxide hydrolase knock-out mouse line. The knock-out was induced before onset of amyloid deposition, and then the mice were analyzed using histological, molecular, and RNA sequencing techniques. Here, we identify astrocytic soluble epoxide hydrolase as a key mediator of vascular instability in Alzheimers disease. Targeted astrocyte-specific deletion of soluble epoxide hydrolase in APPPS1 mice dramatically mitigated vascular changes, reducing the vascular amyloid burden by 67.95% and preserving VE-cadherin architecture. Importantly, vasomotion was markedly impaired in the Alzheimers disease model and was preserved in soluble epoxide hydrolase-deficient animals. Transcriptomic profiling of vasculature in APPPS1xsEH{Delta}AC mice revealed upregulated expression of genes critical for neurovascular protection. These findings identify soluble epoxide hydrolase as a central regulator of neurovascular dysfunction and underscore its therapeutic potential in increasing vascular stability in Alzheimers disease.

neuroscience↗

bioRxiv2025

Context-aware simulation enables systematic optimization of long-read mapping parameters

The performance of long-read mapping is critical yet highly sensitive to parameter choices. We present CycSim, a context-aware simulator that models sequence-context-dependent errors from empirical sequencing data, coupled with a Bayesian optimization framework for systematic parameter tuning. CycSim more accurately reproduces real error profiles than existing simulators, enabling reliable simulation-based optimization. The framework identified parameter configurations that achieved 2.78-fold faster mapping for data from the newly developed Cyclone platform, and consistently improved both mapping efficiency (8.14-32.65% faster) and structural variant calling accuracy (0.75-1.70% higher F1) across ONT, HiFi, and Cyclone datasets, providing a robust and generalizable foundation for analysis-goal-driven parameter refinement.

bioinformatics↗

bioRxiv2025

Deep Mediation Analysis for Multimodal Genotype-Imaging Associations with Disease Phenotypes

Both genotype and imaging data carry entangled information about the phenotypes. Associations between genotypes and phenotypes can be manifested or absent on biomedical images. While there are abundant multimodal association studies integrating genotype and imaging data, few of them disentangle their direct and indirect effects in associations. We propose GIF (Genotype-Image-Phenotype), a novel modeling framework to predict phenotypes by genotype and imaging data with a mediation structure of these three sets of variables. GIF constitutes a backbone of mediation path genotype[->]image[->]phenotype, and direct genotype[->]phenotype and image[->]phenotype paths capturing the additional direct associations not explained by the mediation model. To implement the mediation model with a convolutional neural network (CNN), we constructed a CNN to predict genotypes with images and then employed the joint embedding vectors of the CNN to predict phenotypes. The direct links were sequentially augmented to the mediation model to further improve prediction accuracy. We validated GIF on three datasets: (1) a synthetic polygon dataset where the presence or absence of a polygon specie indicates a genotype and selected combinations of multiple polygon species indicate phenotypes, (2) the PASCAL VOL dataset of object detection and action recognition, where the presence or absence of an object class indicates a genotype and the presence or absence of an action class indicates a phenotype, (3) the ADNI dataset of Alzheimers disease diagnosis comprising the genotype and imaging data of subjects with three cognitive states. GIF prediction outcomes on the ADNI data indicate the associations between genotypes and phenotypes are primarily mediated by image features, and images provide additional information about phenotypes which is not attributed to genotype variations.

bioinformatics↗

bioRxiv2025

Phylogenomics of Rhinogobius gobies reveal northern-southern divergence and trait evolution in China

The goby genus Rhinogobius represents a prominent radiation of freshwater fishes in East Asia, with China serving as its evolutionary hotspot harboring over 39 endemic species. Despite their ecological dominance in riverine ecosystems, phylogenetic relationships among mainland Chinese Rhinogobius species remain poorly resolved due to limited sampling and morphological conservatism, particularly in meristic traits traditionally used for classification. To address these knowledge gaps, we employed a nuclear gene enrichment approach to target 4,434 single-copy nuclear loci across 26 species spanning all major river basins in eastern and southern China. Our phylogenomic analyses based on sequence of 2,137 cleaned-up loci (477,822 bp) recovered a robust topology dividing Rhinogobius into three primary lineages: an early-diverging of R. similis sister to two major clades, the northern vs. southern clades exhibiting strong geographic structuring. Fossil-calibrated molecular dating placed the crown age of Rhinogobius at [~]9.33 million years ago (Ma) during the late Miocene, with a pivotal north-south divergence at [~]5.96 Ma coinciding with major paleo-drainage reorganizations in East Asia. Ancestral state reconstruction revealed that the southern clade uniquely evolved unstable first dorsal fin formula and near-universal presence of predorsal scales. By integrating the most extensive geographic and taxonomic sampling to date with phylogenomic data, this study establishes: (1) a revised evolutionary timeline for Rhinogobius diversification linked to East Asian geomorphological history, (2) diagnostic morphological characters for major clades, and (3) a robust phylogenetic framework to guide future taxonomic revisions and conservation efforts for this ecologically significant group. HighlightsO_LIPhylogenomics resolves north/south split in Chinese Rhinogobius gobies. C_LIO_LIFossil-dated 9.3 MYA origin of Rhinogobius and rapid divergence at 5.96 MYA. C_LIO_LISouthern lineages exhibit more predorsal scales and unstable first dorsal fin formula. C_LIO_LIFirst genomic backbone for species revision and adaptation study. C_LI

zoology↗

bioRxiv2025

NSUN2-dependent 5-methylcytosine Modification Regulates Influenza A virus Gene Expression and Genomic Packaging

Emerging evidence indicates that methyltransferase NSUN2 catalyzes 5-methylcytosine (m5C) modifications on various viral RNAs and plays important roles in viral biology. However, the regulatory roles of NSUN2 in influenza A virus (IAV) replication have not been elucidated. Here, we revealed that NSUN2 negatively regulated the viral RNA transcription and protein production by knocking out and over-expressing NSUN2. By m5C MeRIP-seq and RNA-BisSeq, NSUN2-dependent m5C sites were identified on both the plus and minus viral RNA strands. In NSUN2-KO cells, the m5C modification on vRNAs was reduced, resulting in the production of a large number of deficient interfering particles (DIPs) which had lower vRNA content, imbalanced genome fragments, abnormal morphology and reduced pathogenicity. Mutation of m5C sites at the 5 and 3 ends of PB2 vRNA interfered with the selective packaging of the 8 vRNA segments into virus particles, resulting in the formation of a variety of abnormally packaged virus particles. PB2-vRNA mutants also had reduced replication ability and pathogenicity in mice. Overall, these data demonstrate that the m5C residues catalyzed by NSUN2 are required on vRNAs for the proper assembly of infectious viral particles, suggesting the depletion of m5C modification as a potential strategy that can be utilized to attenuate IAV strains. Significance StatementInfluenza A virus (IAV) infections pose a significant threat to global public health by causing substantial morbidity and mortality. The segmented nature of the IAV genome requires precise regulation of the genomic assembly to produce infectious progeny virus particles. In this study, we demonstrate that m5C modification is crucial for the proper assembly of infectious viral particles. The lowered level of m5C modifications on vRNAs leads to the generation of defective interfering particles with reduced replication capacity and pathogenicity. Our data shed new light on the selective packaging mechanism of IAV segmented genome and highlight a potential new strategy for attenuating IAV strains by targeting the m5C modification machinery.

microbiology↗

bioRxiv2025

Ferroptosis governs lymphatic vessel growth and regression

Whether, when, and how lymphatic vessels undergo cell death remains poorly understood. Here we identify ferroptosis as a physiological, cell-intrinsic regulator of the lymphatic endothelial cell survival during development and following injury, in stark contrast to the resilient organotypic blood endothelial cells. The lymphatic susceptibility to ferroptosis stems from tampered cystine/ hydropersulfide metabolism, alongside reduced glutathione availability triggered by an SH3RF3 E3 ligase mediated GPX4 degradation, and enhanced integration of polyunsaturated fatty acid enriched membrane phospholipids. Inducing ferroptosis genetically or pharmacologically elevated lymphatic lipid peroxidation, halted embryonic lymphangiogenesis and prevented post-injury lymphatic overgrowth while simultaneously shaped immune responses. Conversely, ferroptosis inhibition through saturated fatty acid supplementation led to pathological lymphatic hyperplasia. Targeting lymphatic ferroptotic mechanisms holds promise against pathological lymphatic growth in response to injury.

cell biology↗

bioRxiv2025

InfluenceNet: Encoding Motif Influence for Interpretable Modeling of Cis-Regulatory Syntax

Cis-regulatory elements shape gene expression by recruiting transcription factors (TFs) to DNA motifs, yet how motifs cooperate or compete across distances remains poorly understood. Existing deep learning models predict TF binding with high accuracy but rely on computationally intensive post-hoc analyses to infer regulatory syntax, limiting interpretability and scalability. Here we present InfluenceNet, a transparent convolutional architecture that directly encodes motif influence-a position- and motif-specific profile quantifying how one motif modulates binding at another. Trained on ChIP-nexus data for pluripotency TFs, InfluenceNet achieves performance comparable to state-of-the-art models while providing immediate, global interpretability. The learned influence profiles recapitulate known principles of motif syntax, including Oct4-Sox2 pioneering activity and Nanogs striking 10.5-bp helical periodicity, and reveal a cooperative Nanog-Nanog grammar supported by de novo motif discovery. Integrating these results with structural and molecular-dynamics modeling, we propose that Nanog forms chain-like oligomers on DNA through asymmetric contacts between WRD (tryptophan repeat domain) and DBD (DNA-binding domain) that maintain helical phase alignment. Together, these findings establish InfluenceNet as a general framework for accurate prediction and mechanistic interpretation of cis-regulatory grammar, bridging deep learning and biophysical insight.

bioinformatics↗

bioRxiv2025

Targeting Troponin I Phosphorylation for Precision Treatment of Diastolic Dysfunction

BackgroundDefective cardiac relaxation (diastolic dysfunction) is prevalent in heart failure, particularly in the context of diabetes, obesity, hypertension, and ageing, and is associated with increased mortality. Yet there is no available treatment that directly targets this abnormal relaxation. Phosphorylation of troponin I by cAMP-dependent protein kinase A (PKA) promotes relaxation, but global PKA activation has widespread, undesirable effects. Here we investigated whether selectively enhancing troponin I phosphorylation could improve relaxation without engaging the broader PKA signalling network. MethodsTo resolve cAMP signalling at subcellular scales, we combined real-time cAMP measurements with FRET-based genetically encoded reporters targeted to defined nanodomains, alongside biochemical and genetic approaches. Protein-protein interaction surfaces were mapped using peptide-array technology, which guided the design of a disruptor peptide to displace specific interactions. The peptides effects on cardiomyocyte function were assessed in vitro using rodent and human cardiomyocytes with biochemical assays, real-time imaging, and work-loop analysis. In vivo effects were evaluated through echocardiography and haemodynamic measurements. ResultsWe identified a previously unrecognized regulatory mechanism in which the cAMP-hydrolyzing enzyme PDE4D9 interacts with troponin I and restricts local cAMP levels within a nanometre-scale domain, thereby selectively controlling troponin I phosphorylation. We found that PDE4D9-troponin I association is markedly increased in cardiac disease in both rodents and humans. A peptide that displaces PDE4D9 from troponin I in a mouse model of heart failure selectively enhances troponin I phosphorylation, accelerates cardiomyocyte relaxation, and prevents diastolic dysfunction, without compromising systolic contractility. ConclusionsThese findings reveal a highly specific regulatory nanodomain governing troponin I phosphorylation and points to a first-in-class, mechanism-based therapeutic strategy with the potential to address a major unmet contributor to heart failure. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe identify a previously unrecognized, nanometre-scale regulatory mechanism in which PDE4D9 binds troponin I to locally suppress cAMP, limit troponin I phosphorylation and attenuate relaxation. C_LIO_LIWe demonstrate that this mechanism becomes maladaptive in disease: PDE4D9-troponin I interaction is markedly upregulated in failing hearts in both rodents and humans, which can mechanistically explain the impaired cardiac relaxation. C_LIO_LIWe show that this interaction is druggable: a rationally designed peptide can selectively displace PDE4D9, restore localized cAMP-PKA signalling, and normalize myocardial relaxation without global cAMP perturbation. C_LIO_LIDisplacement of PDE4D9 from TPNI improves relaxation with no impact on the calcium transient amplitude and without adversely affecting systolic output and cardiac reserve. C_LI What Are the Clinical Implications?O_LIDiastolic dysfunction, a major cause of heart failure with no targeted treatments, may be amenable to therapies that modulate cAMP signalling with subcellular precision rather than global pathway modulation. C_LIO_LISelective disruption of PDE4D9-troponin I binding offers a mechanism-based approach with potential to restore diastolic function while minimizing off-target effects typical of GPCR-directed therapies. C_LIO_LIBy enhancing relaxation while preserving systolic function and cardiac reserve, this approach may overcome limitations of myofilament-targeted therapies currently under evaluation for diastolic dysfunction that exhibit negative inotropic effects. C_LIO_LIMore broadly, this work establishes a platform for targeting localized signalling domains, a concept that may extend beyond cardiovascular disease to other pathological conditions in which dysregulated cAMP contributes to dysfunction. C_LI

cell biology↗

bioRxiv2025

Accelerated and High-Accuracy Variant Calling on Oxford Nanopore Technologies Sequencing Data with the Sentieon DNAscope LongRead and Hybrid Pipelines

Oxford Nanopore Technologies (ONT) has emerged as a clinically meaningful long-read sequencing platform, enabled by recent improvements in read accuracy and throughput. However, variant calling on ONT data remains challenging due to platform-specific error profiles, particularly indels in homopolymer regions. Here, we introduce description and comprehensive benchmarks of the Sentieon DNAscope LongRead and DNAscope Hybrid pipelines on ONT whole-genome datasets, demonstrating high accuracy and substantially reduced computational requirements across SNP, Indel, and Structural Variant detection. Benchmarked against multiple truth sets including GIAB v4.2.1, CMRG, and T2T-Q100, DNAscope LongRead reduced SNP errors by [~]50% relative to Clair3 across five reference samples and showed consistently higher accuracy in all non-long-homopolymer stratifications. Integration of ONT and Illumina short reads via DNAscope Hybrid further improved variant detection: with low depth ONT + Illumina datasets, the pipeline achieved F1 scores of 0.9992 (SNPs) and 0.9979 (Indels), outperforming alternative pipelines. In challenging benchmarks such as T2T-Q100 and CMRG genes, DS-Hybrid reduced SNP and Indel errors significantly, compared to next-best methods. For structural variants, DNAscope pipelines outperformed alternative (e.g. Sniffles2), highlighting the advantages of haplotype-resolved SV detection method. Despite its accuracy, the Sentieon software suite remains computationally efficient: complete ONT long read FASTQ-to-VCF analysis completed within [~]190 minutes on a 120-vCPU Azure instance, at costs less than $5 USD. Overall, the DNAscope LongRead and Hybrid pipelines deliver fast, accurate, and scalable germline variant calling for ONT datasets, providing a practical solution for comprehensive whole-genome analysis in both research and clinical genomics.

bioinformatics↗

bioRxiv2025

FuFiHLA: A tool for Full-Field HLA typing from long read data

0.MotivationAllele typing for Human Leukocyte Antigen (HLA) genes has many important clinical applications. Popular short-read typing can only accurately distinguish alleles at the peptide level, which potentially limit our understanding of the effect of variants in non-coding region. Recently, a few methods were declared to distinguish full-field HLA alleles from de-novo assemblies, which motivates us to develop an accurate HLA typing method directly from long reads. ResultsWe developed FuFiHLA, a lightweight open-source software, to type HLA alleles. Currently it supports typing alleles of six HLA genes (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, and HLA-DQB1) from long reads. Evaluation using 47 PacBio HiFi samples from HPRC shows that FuFiHLA achieves 99.57% accuracy in the full field allele typing and QV as 50.1 for consensus allele sequence construction. Additional testing on four Nanopore R10 reads demonstrates slightly reduced accuracy in the fourth field. Availability and implementationFuFiHLA is available on GitHub (https://github.com/jingqing-hu/FuFiHLA) under MIT License.

bioinformatics↗

bioRxiv2025

Lymphatics unload VEGFR3 in response to the endothelial stress upon VEGFR2 insufficiency

Vascular endothelial growth factor receptor 2 (VEGFR2) is a key target for regulating the endothelial cell lineage and angiogenesis. It is also expressed by lymphatic endothelial cells (LEC) while its participation in the process of lymphangiogenesis remains inadequately characterized. We show in this study that VEGFR2 is highly expressed in dermal initial lymphatic vessels and valves. The induced deletion of pan-endothelial Vegfr2 at the neonatal stage produced a potent suppression of dermal lymphatic growth, including the thinner lymphatic diameter, the decrease of LEC number and lymphatic valves. Mechanistically, the VEGFR2 insufficiency led to a dramatic decrease of lymphatic VEGFR3, a key regulator mediating signals for lymphatic growth and remodeling. The RNA sequencing analysis revealed that GO terms enriched for the downregulated genes included biological processes related to the EC development while pathways related to the hematopoiesis and immune responses were upregulated in the skin of Vegfr2 mutants compared with the littermate controls. This was further confirmed by the RNA-seq analysis of dermal tissues 48 hours after the endothelial Vegfr2 deletion. Consistently, an inhibitory effect on the dermal lymphatic growth was also observed by targeting Vegfr2 in PROX1+ cells, manifesting a similarly altered transcriptome signature. The alteration of lymphatic gene expression was further validated by the siRNA mediated knockdown of Vegfr2 in primary lymphatic endothelial cells, showing a transcriptional trend of LEC to hematopoietic transition. Findings from this study imply that VEGFR2 is required for the maintenance of endothelial identity and its insufficiency may trigger the alteration of lymphatic transcriptional programs to diminish the VEGFR3-mediated lymphangiogenesis.

developmental biology↗

bioRxiv2025

Rad54 separation of function mutation highlights unique roles during homologous recombination

Homologous recombination (HR) is a DNA repair pathway that utilizes a template-based approach to repair double-strand breaks within the genome. Template utilization requires the exchange of individual DNA strands, which members of the RecA family of recombinases facilitate. Rad51 is a primary strand exchange factor in eukaryotes. During regular mitotic DNA repair, Rad51 is aided by the DNA translocase Rad54, which acts as a motor to remodel the template DNA and stabilize primary-strand exchange intermediates. The regulation of this activity remains incompletely understood. Here, we have identified a conserved site within the C-terminal region of Rad54. The mutation of this site creates a functional separation at early strand-exchange intermediates in vivo. Using this mutant protein, we identify a novel intermediate essential for stabilizing displacement loop (D-loop) structures. This precedes the removal of Rad51 and DNA extension. Based on our experiments, we hypothesize that this Rad54 mutant cannot stabilize Rad51-mediated strand-exchange intermediates because it cannot topologically isolate dsDNA regions. Identifying a mutant that disrupts this intermediate before Rad51 removal unifies existing models of Rad54-mediated D-loop formation and extension.

biochemistry↗

bioRxiv2025

Beyond P-values: A Multi-Metric Framework for Robust Feature Selection and Predictive Modeling

High-dimensional biomedical datasets routinely contain sparse signals embedded among vast, correlated features, making variable selection central to building models that generalize. Although significance-based selection is widely used across modalities (e.g., imaging, EHR, multi-omics), statistical significance does not guarantee predictive utility, and vice versa. Yet few methods unify inferential and predictive evidence within a single selection framework. We introduce MIXER (Multi-metric Integration for eXplanatory and prEdictive Ranking), a domain-agnostic approach that integrates multiple selection metrics into one consensus model via adaptive weighting that quantifies each criterions contribution. Through simulation studies, we demonstrate that different selection metrics identified markedly different feature sets whose over-laps depended on the underlying feature distributions and signal strength. Applied to Alzhemiers disease in UK Biobank, MIXER outperformed every individual criterion, including statistical significance, and generalized to an external disease-specific cohort, Alzheimers Disease Sequencing Project, yielding higher discrimination and stronger risk stratification. The MIXER framwork is also modular and readily extends to other selection criteria and data modalities, providing a practical route to more accurate, interpretable, and transportable predictive models.

bioinformatics↗

bioRxiv2025

Evolutionarily Conserved and Divergent Mechanisms of Dual Ca2+ Sensors in Synaptic Vesicle Exocytosis

Neurotransmitter release at the C. elegans neuromuscular junction is governed by a dual Ca{superscript 2} sensor system composed of SNT-1 and SNT-3, which are functional analogs of synaptotagmin-1 and -7 (Syt1/Syt7) in mammalian central synapses. In this study, we investigated how SNT-1 and SNT-3 mediate fast and slow neurotransmitter release through their potential interactions with the SNARE complex and their polybasic motifs. AlphaFold 3 models of SNT-1-SNARE and SNT-3-SNARE complexes accurately recapitulated the canonical Syt1 C2B-SNARE primary interface (Zhou et al., 2015, Nature) and precisely identified conserved binding residues within the C2B domains, as well as in SNAP-25 and Syntaxin, highlighting the evolutionary conservation of this interaction. Electrophysiological analyses using targeted mutagenesis demonstrated that both SNT-1 and SNT-3 require C2B-SNARE interactions and polybasic motifs within their C2 domains to drive evoked fast and slow neurotransmitter release. Notably, SNT-1 and SNT-3 exhibited differential dependence on distinct regions of the C2B-SNARE interface and their respective polybasic motifs, suggesting that Ca{superscript 2}-triggered fast and slow release operate via distinct mechanistic strategies. Furthermore, we found that SNT-1 mediates spontaneous neurotransmitter release through multiple pathways, involving not only the primary C2B-SNARE interface but also additional putative SNARE-binding interactions. Together, our findings uncover both conserved and divergent mechanisms for synaptic exocytosis regulated by the dual Ca2+ sensors in C. elegans.

neuroscience↗

bioRxiv2025

Computational design of pH-sensitive binders

pH gradients are central to physiology, from vesicle acidification to the acidic tumor microenvironment. While therapeutics have been developed to exploit these pH changes to modulate activity across different physiological environments, current approaches for generating pH-dependent binders, such as combinatorial histidine scanning and display-based selections, are largely empirical and often labor-intensive. Here we describe two complementary principles and associated computational methods for designing pH-dependent binders: (i) introducing histidine residues adjacent to positively charged residues at binder-target interfaces to induce electrostatic repulsion and weaken binding at low pH, and (ii) introducing buried histidine-containing charged hydrogen-bonding networks in the binder core such that the protein is destabilized under acidic conditions. Using these methods, we designed binders that dissociate at acidic pH against ephrin type-A receptor 2, tumor necrosis factor receptor 2, interleukin-6, proprotein convertase subtilisin/kexin type 9, and the interleukin-2 mimic Neo2. Fusions of the designs to pH-independent binders of lysosomal trafficking receptors function as catalytic degraders, inducing target degradation at substoichiometric levels. Our methods should be broadly useful for designing pH-sensitive protein therapeutics.

biochemistry↗

bioRxiv2025

In Vivo Mutagenesis of a Ketosynthase Domain Uncovers Productivity and Specificity Control in Modular Polyketide Synthases

Ketosynthase domains govern chain transfer and substrate selectivity in modular polyketide synthases (PKS), yet their functional tunability in native contexts remains poorly understood. We performed phylogenetically guided mutagenesis of the KS5 domain from the Streptomyces cinnamonensis monensin PKS and evaluated 72 variants in vivo across wild-type and reductive-loop-null backgrounds. This revealed discrete active-site motifs that control productivity, redox-state specificity, and extender-unit selection, functions traditionally ascribed to other PKS domains. AlphaFold3 structural mapping linked these motifs to substrate-tunnel and catalytic-core features, providing a mechanistic basis for the observed phenotypes. Our findings demonstrate that KS domains can be rationally re-tuned to overcome productivity bottlenecks and alter specificity in intact PKSs, offering a route to improved yields and expanded chemical diversity in engineered polyketides.

biochemistry↗