Biology subjects

Wang, S.

Publications and source records attributed to Wang, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

TRAPPC8 Is an Endogenous Brake on UFMylation That Suppresses Tauopathy

UFMylation, a ubiquitin-like protein modification, drives tau spread through the brain, but what keeps this process in check has remained unclear. We show that TRAPPC8 acts as a natural brake on UFMylation, binding directly to the E1 enzyme UBA5 to dampen pathway activity. In Alzheimer's disease brain tissue, TRAPPC8 is reduced while UFMylation is elevated, suggesting this brake fails as disease progresses. Sustaining UFMylation in human iPSC-derived neurons increased tau aggregation and spread while disrupting lysosomal function and lipid balance. Restoring TRAPPC8-UBA5 binding reversed these defects through the lysosomal protein CLN8, which restored lysosomal function and reduced tau pathology. Notably, expressing just the UBA5-binding region of TRAPPC8 was enough to suppress tau pathology in vivo, marking it as a promising therapeutic target.

neuroscience↗

bioRxiv2026

DYNAMIC CONFORMATIONS IN THE ACTIVATION OF PINK1 KINASE

Under mitochondrial stress, the mitochondrial kinase PINK1 is activated to phosphorylate ubiquitin, which in turn recruits the E3 ligase Parkin, thereby initiating clearance of damaged mitochondria. Dysregulation of the PINK1-Parkin signaling pathway is associated with early-onset autosomal recessive Parkinson's disease. However, the mechanisms governing PINK1 conformational dynamics and activation in response to mitochondrial stress remain poorly understood. Here, we report that Tribolium castaneum PINK1 (TcPINK1) forms specific symmetric dimers via interactions between the kinase N-lobe and the regulatory C-terminal domain. These dimers undergo dynamic conformational transitions across distinct states, including an autoinhibited state, an inactive intermediate state, a primed-activation state and an active state. Moreover, TcPINK1 undergoes lateral trans-phosphorylation through dimer-dimer interactions. Notably, symmetric dimer formation is essential for Parkin recruitment during mitophagy. These findings provide a framework for understanding the conformational plasticity and allosteric regulation of PINK1 in mitophagy.

biophysics↗

bioRxiv2026

Regulation of the human voltage-gated proton channel by membrane sterols

Cholesterol is a key component of eukaryotic cell membranes, promoting membrane stability and modulating the function of many membrane proteins, including ion channels. In our previous work using purified human voltage-gated proton channel proteins, we showed that cholesterol inhibits the hHv1 channel by altering the conformational dynamics of its S4 segment, the key element that senses membrane voltage to control proton permeation. In the present work, we examined the effects of cholesterol analogs and potential sites in the hHv1 channel mediating cholesterol inhibition using site-directed mutagenesis and docking simulations. Our results showed that desmosterol, the immediate precursor of cholesterol, markedly attenuates cholesterol inhibition. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we showed that desmosterol attenuates cholesterol inhibition by promoting the intermediate and open state conformations of the S4 segment. Moreover, we identified multiple residues in the hHv1 channel that are critical for cholesterol inhibition, including Y141A in the S2 segment, which reduces cholesterol inhibition by nearly 3-fold. Our smFRET results showed that the Y141A mutation promotes the intermediate conformation in the S4 segment, which underlies the attenuation of cholesterol inhibition. Consistently, docking simulations also revealed multiple residues spanning the transmembrane domain, rather than clustered within a single localized pocket. Our work identified the key molecular determinant in the hHv1 channel that mediates cholesterol inhibition and also provided a mechanism linking the conversion between demosterol and cholesterol by DHCR24 to pH homeostasis in many cells, such as phagocytes, cardiomyocytes, neurons and microglial cells.

biophysics↗

bioRxiv2026

Topology-Based Query Framework for Longitudinal Omics Trajectories

1 Abstract 1.1 Background Many longitudinal omics studies contain only a small number of repeated measurements collected before, during, or after an intervention. Existing approaches, including mixed-effects models and generalized additive models, estimate temporal effects but do not generally provide a discrete representation of trajectory topology that can be queried directly across experimental groups. 1.2 Methods We developed LongOmicsTraj, an open-source R package for topology-based representation and querying of short longitudinal omics trajectories. The framework encodes the direction of change between adjacent visits as up, down, or flat, with the ordered sequence defining an Ordinal Trajectory State (OTS). LongOmicsTraj operates downstream of trajectory estimation and can therefore be applied to empirical summaries or model-derived visit-level estimates, including those from linear mixed-effects models, generalized additive models, and polynomial regression, following a maSigPro-style time-course formulation [1]. OTS labels provide a common representation for topology-based querying, cross-group comparison, and evaluation of higherlevel representations such as trajectory clusters. We evaluated the framework using controlled simulations and bronchial biopsy transcriptomic data from the GLUCOLD corticosteroid intervention study (GEO accession GSE36221), measured at baseline, 6 months, and 30 months. The biological analysis compared continued inhaled corticosteroid (ICS) treatment, ICS withdrawal after 6 months, and placebo. 1.3 Results In simulations, LongOmicsTraj recovered predefined stable, monotonic, transient, rebound, and oscillatory trajectories with high accuracy when longitudinal signal was sufficiently clear, with performance declining under high-noise conditions and depending partly on the upstream estimator. In GLUCOLD, comparator-aware topology queries reduced 20,358 measured transcripts to 168 genes showing a corticosteroid response that was maintained during continued treatment, reversed following withdrawal, and was not reproduced under placebo. The selected genes included established corticosteroid-response genes and were enriched for immune-cell migration, chemotaxis, cell adhesion, and extracellular-matrix organisation. Topology-aware evaluation of FlexMix trajectory clusters additionally revealed substantial within-cluster temporal heterogeneity, with topology purities of approximately 46% to 60%. 1.4 Conclusions LongOmicsTraj provides a compact, directly queryable representation of temporal direction and order in short longitudinal omics studies. It complements existing longitudinal estimation and clustering methods by making trajectory structure explicit, enabling structured cross-group queries and quantification of temporal heterogeneity within trajectory clusters.

bioinformatics↗

bioRxiv2026

Patterns and environmental drivers of amphibian alpha and beta diversity in the Huangshan Mountains, southern Anhui, China

Understanding the spatial patterns of biodiversity and their environmental determinants is fundamental to ecology and conservation, particularly in mountainous transitional zones where species assemblages can shift rapidly. Amphibians, as highly sensitive organisms, are excellent indicators of environmental change, yet quantitative assessments of their diversity in the Huangshan Mountains-a biodiversity hotspot at the junction of the Palaearctic and Oriental realms in southern Anhui, China-remain limited. We conducted systematic line-transect surveys across 200 grids (5 km x 5 km) during the spring and autumn of 2023 and 2024, recording a total of 10,342 individuals belonging to 23 species, 20 genera, 9 families, and 2 orders. Three species (Fejervarya multistriata, Microhyla fissipes, and Bufo gargarizans) were identified as dominant, and two nationally protected species (Hoplobatrachus chinensis and Andrias davidianus) were detected. Inter-annual alpha diversity did not differ significantly, but pronounced seasonal variation was observed, with spring supporting higher Shannon-Wiener and Pielou evenness than autumn in both years. Using generalized additive models and model averaging, we found that annual precipitation and the normalized difference vegetation index (NDVI) were consistently the strongest positive predictors of Shannon-Wiener diversity, Simpson dominance, and species richness, while species richness also declined significantly with increasing human footprint. Total beta diversity (Soerensen dissimilarity) was very high (0.981) and overwhelmingly driven by species turnover (97.86%) rather than nestedness. Partial Mantel tests and distance-based redundancy analysis further revealed that environmental distances-particularly annual precipitation-significantly shaped overall beta diversity and its turnover component after accounting for geographic distance. These results highlight the predominant role of climatic and vegetation gradients in structuring amphibian assemblages in this subtropical mountainous region, providing baseline data to inform local conservation strategies.

ecology↗

bioRxiv2026

SPARKLE: evidence-constrained correction of local RNA leakage in high-resolution spatial transcriptomics

High-resolution sequencing-based spatial transcriptomics, including Stereo-seq and Visium HD, aggregates dense capture units into cell-resolved expression matrices. During tissue processing and permeabilization, RNA released from source cells can spread to neighbouring capture locations, reducing cell-type specificity and biasing downstream analyses. Here we developed SPARKLE (Spatial Ambient RNA Kernel-based Leakage Estimator), a cell-level correction method that uses capture locations outside cell-segmentation masks as within-sample spatial evidence of leakage. SPARKLE fits sparse spatial kernels to out-of-mask observations to estimate a sample-level propagation scale and gene-specific leakage coefficients. It corrects only genes supported by out-of-mask goodness of fit and uses expression-dependent conservative shrinkage to protect highly expressing source cells. In ten simulated scenarios, SPARKLE achieved the highest cell-wise concordance in eight and the lowest RMSE in nine. In axolotl brain, mouse brain and human ovarian cancer, SPARKLE removed ectopic marker signal from neighbouring cells while retaining source-cell expression, improved agreement with independent single-cell and single-nucleus references, and recovered an inferred fibroblast-to-tumor COL1A2-SDC4 communication route that was obscured by ectopic COL1A2 expression. Conclusions remained stable across plausible spatial scales and background-bin sizes. Runtime scaled near-linearly with tissue-window area and was further accelerated on GPU. SPARKLE is therefore a reference-free, fast and scalable method for correcting local RNA leakage from evidence contained within each sample, improving the reliability of cell-type localization, tissue-compartment identification and cell-cell communication inference.

bioinformatics↗

bioRxiv2026

WHaloForce enables chemigenetic imaging of molecular tension in living cells and animals

Piconewton forces borne by individual proteins within complex assemblies underlie cell adhesion, migration, and tissue morphogenesis, yet remain difficult to image in living systems. Here, we introduce WHaloForce, a chemigenetic HaloTag-based tension sensor that converts force-dependent relief of tryptophan-mediated dye quenching into a fluorescence lifetime change. Optical tweezers revealed a switch-like unquenching transition near 5 pN, and the sensor responded reversibly to force changes in cells. WHaloForce enabled quantitative tension imaging of diverse force-bearing proteins (vinculin, E-cadherin, -catenin, and laminin) in mammalian cells, mouse tissue, and C. elegans. Bright synthetic dyes made tension measurements possible at endogenous expression levels. In C. elegans, vinculin and laminin showed opposite tension patterns between tissues, revealing distinct force-transmission routes through adhesions and the extracellular matrix. During ovulation, laminin tension accumulated over repeated stretch-relaxation cycles, scaling with cumulative loading history. WHaloForce thus offers a modular platform for imaging spatiotemporal tension patterns in living systems.

cell biology↗

bioRxiv2026

GWAS and multi-omics uncover the SLC6A3 locus for canine stranger-directed social anxiety and implicate NRIP3 in dopaminergic network regulation

The social anxiety disorder (SAD) is one of the most common mental health disorders, often developing in adolescence. It can severely impair educational achievement, career progression, and social functioning. Certain individuals within the Chinese Kunming dog (CKD) population display spontaneous, SAD-like behaviors towards strangers, offering a valuable model for genetic investigation. Using whole-genome sequencing data, we conducted a genome-wide association study on this SAD-like behavior in 100 CKDs, identifying several genes previously linked to human psychiatric disorders. Notably, SLC6A3 reached genome-wide significance, whereas CADPS2 exceeded only the suggestive threshold and is therefore considered a network-supported candidate locus. By integrating weighted gene co-expression network analysis with public RNA-seq data from the VTA of 38 mice (after quality control), we further showed that Slc6a3 and Cadps2 are part of a co-expression network module associated with the dopamine system. These findings suggest that this specific module may play a functional role in the stranger-directed anxiety-related behaviours in CKDs. Furthermore, we identified Nrip3 as a key candidate transcriptional coregulator in this specific module. Subsequent virus-mediated overexpression and behavioural experiments showed that Nrip3 regulates gene expression within this network and induces a spectrum of behavioural alterations in mice, including social avoidance, anhedonia, and increased locomotor activity. Collectively, this study highlights the involvement of dopamine system genes in canine social anxiety and suggests Nrip3 as an upstream regulator of Slc6a3 associated with anxiety. The identification of conserved anxiety-related signatures between humans and dogs further supports the dog as a valuable translational model for psychiatric research.

genetics↗

bioRxiv2026

Vaccine imprinting drives increased SARS-CoV-2 variant infection in children

Virus exposure history, particularly first exposure, is believed to shape vaccine efficacy and infection susceptibility; however, evidence for mechanistic links between immune responses in individuals and epidemiological outcome in populations is scarce. Recent co-circulation of SARS-CoV-2 variants XFG and BA.3.2 has revealed a striking enrichment in BA.3.2 cases among children. By combining epidemiological modeling, serology and monoclonal antibody analysis in children and adults, we show the dependence of effective variant-specific antibodies on vaccination history which may explain birth-year influence on differential susceptibility to these co-circulating variants. Ancestral cross-reactive site I antibodies frequently neutralize BA.3.2, but not XFG. By contrast, Omicron type-specific site I/III and III antibodies frequently neutralize XFG but not BA.3.2, revealing a tradeoff in the ability to neutralize these two co-circulating strains. These findings mechanistically link immune history, variant neutralization, antibody repertoire and variant infection risk, and suggest that vaccination regimens in children should prioritize neutralization breadth.

immunology↗

bioRxiv2026

Plasma membrane PI4P recruits SNAP47 to mediate SNARE-dependent AMPAR exocytosis during synaptic potentiation

The synaptic delivery of the AMPA-type glutamate receptors (AMPARs) is crucial for longterm potentiation (LTP) of excitatory synapses, yet the mechanisms underlying neuronal activity-dependent AMPAR exocytosis at the plasma membrane (PM) remain unclear. We previously demonstrated that the PM synthesis of the phosphoinositide PI4P is enhanced upon LTP induction and that PM PI4P, not PI(4,5)P2, is required for activity-induced AMPAR exocytic trafficking. Here, we show that AMPARs are exocytosed at PI4P-enriched dendritic PM microdomains in potentiated hippocampal neurons. The Q-SNARE SNAP47 binds PI4P via its pleckstrin homology (PH)-like domain. This interaction recruits SNAP47 to the PM, promoting the exocytic fusion of AMPAR vesicles through the SNAP47-Syntaxin-3-VAMP2 SNARE complex. In the hippocampus, the SNAP47-PI4P interaction is necessary for both LTP and long-term memory. Our findings reveal a mechanistic role for PI4P in mediating activity-dependent, SNARE-driven fusion of AMPAR exocytic vesicles with the PM.

neuroscience↗

bioRxiv2026

High prevalence of KPC-3 in carbapenem-resistant Pseudomonas aeruginosa across multiple clonal lineages in China

Background The epidemiology of Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa is rapidly evolving in China. While blaKPC-2 remains the predominant KPC variant in P. aeruginosa, blaKPC-3 has rarely been documented in this pathogen. This study investigated the molecular epidemiology, resistance and virulence characteristics, and plasmid features of blaKPC-3-producing CRPA isolates collected from a tertiary hospital in eastern China. Methods A total of 65 non-duplicate CRPA isolates collected in 2023 were subjected to whole-genome sequencing. Antimicrobial susceptibility testing, phylogenetic analysis, plasmid characterization, conjugation experiments, and virulence assays were performed. Results Among the 65 CRPA isolates, 37 (56.9%) carried blaKPC-3. These blaKPC-3-positive isolates belonged to four sequence types (STs), including ST1076 (62.2%), ST463 (21.6%), ST646 (10.8%), and ST3393 (5.4%). To our knowledge, this is the first report of blaKPC-3 in P. aeruginosa ST463, ST646 and ST3393. All isolates exhibited extensive drug resistance, and 51.8% were resistant to ceftazidime-avibactam. Phylogenetic analysis indicated that blaKPC-3 dissemination was driven by both clonal expansion and horizontal transmission. Comparative genomic analysis identified three kinds of blaKPC-3 -carrying plasmid. A transferable IncP-2 megaplasmid was widely distributed among ST1076, ST646, and ST3393 isolates, whereas non-transferable IncP-10 plasmids were primarily restricted to ST463. The genetic environments and plasmid backbones of blaKPC-3 were highly conserved and closely related to those of blaKPC-2 and its variants, suggesting evolution from pre-existing blaKPC-2-associated plasmids. Virulence analysis demonstrated marked heterogeneity across lineages. ST463 isolates co-harbored exoU and exoS, exhibited enhanced biofilm formation and pyocyanin production, and caused significantly higher mortality in the G. mellonella infection model, indicating a hypervirulent phenotype. Conclusions The blaKPC-3 is becoming an increasingly important determinant of carbapenem resistance in P. aeruginosa in China. The IncP-2 megaplasmid and IncP-10 plasmid derived blaKPC-3 spread across multiple lineages. Continuous genomic surveillance and enhanced infection control measures are urgently needed to prevent its further prevalence in clinical settings.

microbiology↗

bioRxiv2026

Sustained Volumetric Compression Induces Cell Jamming and Primes Breast Cancer Cells for Enhanced Post-Compression Migration and Invasion

During tumor growth and progression, cancer cells are exposed to sustained physical confinement and volumetric compression that can alter cell volume, cytoskeletal organization, mechanotransduction, and invasive behavior. However, whether breast cancer cells retain a compression-induced mechanical memory after release from sustained volumetric compression, and how this memory influences subsequent migration and invasion, remains poorly understood. Here, by controlling cell volume using PEG - mediated volumetric compression, we investigated the compression and post-compression recovery responses of MCF-7 breast cancer cells. Cells were compressed for four days, followed by four days of recovery after PEG removal, and analyzed using daily morphological tracking, single-cell time-lapse imaging, F-actin and YAP staining, wound healing assays, and 3D spheroid invasion assays. We show that sustained volumetric compression shifts MCF-7 cells into a compact, jammed-like, low-motility state characterized by reduced morphodynamic remodeling, suppressed collective migration, and limited spheroid invasion. In contrast, post-compression recovery induces a distinct mechanobiological state marked by increased cell area and perimeter, altered single-cell trajectories, heterogeneous F-actin remodeling, enhanced YAP nuclear localization in enlarged recovered cells, accelerated wound closure, and increased spheroid invasion and cell dissemination. These findings suggest that prior volumetric compression can prime breast cancer cells for enhanced migration and invasion after stress release, supporting post-compression recovery as a form of mechanical memory that may contribute to tumor dissemination.

bioengineering↗

bioRxiv2026

Myeloid-targeted RNA nanotherapeutics rewire cholesterol metabolism to unleash anti-tumor immunity in glioblastoma

Tumor-associated myeloid cells (TAMCs) dominate the glioblastoma (GBM) microenvironment and suppress anti-tumor immunity. Here, we identify cholesterol efflux via ABCA1 as a targetable metabolic checkpoint controlling TAMC immunosuppression in GBM. Reprogramming TAMC cholesterol metabolism using TAMC-targeting lipid nanoparticle encapsulating ABCA1 siRNA (ABCA1 LNP) converts TAMCs into potent antigen-presenting cells with enhanced pro-inflammatory activity and antigen-presenting capacity, thereby inducing T cell activation, expansion, and tumor infiltration. Mechanistically, ABCA1 blockade induces cholesterol accumulation in TAMC membranes, promoting lipid raft formation and enhancing MHC-I-mediated antigen presentation. In multiple preclinical GBM models, ABCA1 LNP treatment dramatically induces T cell priming, extends animal survival, and overcomes GBM resistance to radiotherapy and immune checkpoint therapy. This efficacy was well-maintained in stem-like and recurrent GBM models, GBM patient specimens, and a renal cell carcinoma model. Altogether, our work identifies cholesterol efflux as a targetable metabolic vulnerability in TAMCs to overcome therapy resistance in myeloid-rich, immunologically "cold" tumors.

immunology↗

bioRxiv2026

Evolution-inspired multi-objective Bayesian optimization for protein engineering

Protein engineering requires efficient navigation of vast sequence spaces under limited evaluation budgets, especially when multiple properties must be optimized simultaneously. We developed Evolution-inspired Multi-Objective Bayesian Optimization (EvoMOBO), an active-learning framework that integrates path-dependent sequence generation, global competition among generated variants, and explicit multi-objective optimization. Benchmarking against state-of-the-art methods on complete steroid receptor DNA-binding domain and ParD3 antitoxin landscapes demonstrated robust target-region enrichment, Pareto-front advancement, and sequence diversity across two- and three-objective tasks. In the DBD landscape, simulation-derived geometric descriptors served as labels for both initialization and iterative updating, enriching variants with favorable measured activities without experimental labels. Building on this validation, we applied EvoMOBO to two enzyme-engineering tasks using simulation-derived mechanistic descriptors, with experiments reserved for final validation. For an old yellow enzyme (GkOYE), 16 of 26 tested variants outperformed the wild type, and the best increased non-native oxidative dehydrogenation conversion from 17.5% to 95%. For a formate oxidase (AoFOx), EvoMOBO identified aggregation-resistant variants, two of which nearly doubled diethyl phthalate degradation in a photoenzymatic cascade. Together, these results establish EvoMOBO as a modular framework for multi-objective protein engineering using experimental or mechanism-derived labels.

bioengineering↗

bioRxiv2026

The S1B-Targeting PEDV Neutralizing Antibody C62 Prematurely Activates S-trimer and Mimics Receptor Function

Porcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in piglets, yet the mechanistic basis of antibody-mediated neutralization remains poorly understood. Here, we determined the structure of PEDV HNXX-strain spike domain B (S1B) simultaneously bound by C62, a neutralizing porcine monoclonal antibody against PEDV G2 strains, and N34, a non-neutralizing porcine PEDV antibody. The structure reveals that C62 targets a conserved, cryptic epitope that is accessible only when S1B adopts an "up" conformation. Functionally, we showed that C62 has substantially stronger activity than N34 in triggering S-trimer disassembly and inducing the formation of proteinase K-resistant, post-fusion-like S2 structures. Despite the weaker triggering activity of N34, both C62 and N34 can function as artificial receptors. Notably, although the C62 epitope is conserved across both G1 and G2 strains, C62 exhibits G2-strain-biased neutralizing activity. We further showed that differences in cell-surface membrane fusion activity among PEDV spikes correlate with distinct viral entry pathways and are jointly determined by the S1A and S1B sequences. Together, our findings identify a strain-specific vulnerable site on the PEDV S-trimer and provide insight into how cell-surface membrane fusion activity may influence viral entry pathway selection and antibody neutralization efficacy.

microbiology↗

bioRxiv2026

Catch-bond engineering surpasses high-affinity maturation for T cell receptor therapies against solid tumors

The sensitivity of T cell receptors (TCRs) has traditionally been attributed to their affinity, a principle that has long underpinned both T cell biology and TCR engineering. Currently, high-affinity maturation remains the predominant strategy employed to enhance TCR sensitivity; however, this approach has been associated with severe off-target toxicities in clinical settings. In this study, using the only FDA-approved TCR-T therapy as a clinical reference, we demonstrate that force-induced catch bonds, rather than static affinity, primarily determine TCR sensitivity and facilitate the development of more effective TCR engineering strategies. Among polar and charged amino acids, histidine was determined to be the most effective residue for pinpointing engineering hotspots. TCRs engineered to form catch bonds exhibited superior performance compared to the FDA-approved TCR subjected to high-affinity maturation, improving the efficacy of TCR-T cell therapies against solid tumors without eliciting off-target toxicity or alloreactivity. Mechanistically, a correlation was observed between TCR sensitivity and the strength of catch bonds, whereas no such relationship was found with affinity. The duration of T cell-tumor cell interactions, immunological synapse formation, and the intensity of subsequent signaling are governed by catch bonds rather than by affinity. Structural and computational investigations have demonstrated that force-induced reconfiguration of the ligand-receptor interface, along with the formation of a novel hydrogen bond network, augments the specificity of interactions between the TCR and antigenic peptides. Furthermore, bispecific T cell engagers utilizing the TCR scaffold also formed catch bonds with tumor antigens, indicating intrinsic mechanosensory properties of the extracellular domains of TCRs and suggesting a novel therapeutic modality based on catch-bond-engineered TCRs.

immunology↗

bioRxiv2026

Gram-scale one-pot enzymatic synthesis of CDP-ribitol by a designed bifunctional fusion enzyme

D-Ribitol-5-phosphate (Rbo5P) plays vital roles in bacterial and mammalian development. In mammals, Rbo5P is an essential component of the O-mannosyl glycan on -dystroglycan, and its defective biosynthesis causes a group of dystroglycanopathies. Supplementation with cytidine diphosphate ribitol (CDP-ribitol, CDP-Rbo) and its analogues has shown therapeutic potential for certain dystroglycanopathies. However, facile and scalable strategies to prepare CDP-Rbo remain unavailable. Here, we report a practical one-pot enzymatic strategy for gram-scale CDP-Rbo production. Starting from inexpensive ribitol, Rbo5P is first generated by L-ribulokinase (AraB) and then converted to CDP-Rbo by CDP-ribitol pyrophosphorylase (TarI) in 90% yield. To streamline the process, AraB and TarI were fused into a single bifunctional biocatalyst, AraB-TarI, which converts ribitol directly into CDP-Rbo on a gram-scale in a one-step reaction. Furthermore, a nucleotide recycling strategy was developed to lower the cost and increase the atom economy from 47% to 75%. This work provides a green and scalable route to CDP-Rbo and a reliable material supply for developing therapeutics against dystroglycanopathies.

biochemistry↗

bioRxiv2026

Molecular principles for graded activation of dopamine D1 receptor

G protein-coupled receptors (GPCRs) signal across a continuum of activation states, yet how ligands encode distinct signaling efficacies remains poorly understood. Here, we define the molecular mechanism underlying graded activation of the dopamine D1 receptor (D1R), a major therapeutic target for neuropsychiatric disorders. Functional analyses reveal that two widely used pharmacological tools, LE300 and SCH23390, possess intrinsic efficacy as an inverse agonist and a weak partial agonist, respectively, rather than the efficacy-silent neutral antagonists. Structural, molecular dynamics and mutagenesis analyses capture previously unrecognized inactive and intermediate receptor activation states that bridge known active conformations, and reveal that ligand efficacy is encoded through the progressive engagement of a conserved activation pathway centered on the W6.48 toggle switch. Guided by this mechanism, a single chemical modification markedly increases the agonist efficacy of SCH23390. Comparison with dopamine D2 receptor structures further reveals a conserved mechanism of inverse agonism despite distinct subtype-specific recognition. Together, these findings establish a structural framework for graded agonism at D1R and provide general principles for the rational design of efficacy-tuned therapeutics at dopamine receptors and related GPCRs.

biochemistry↗