Biology subjects

Ma, W.

Publications and source records attributed to Ma, W..

At least 55 records · Page 3Linked to original sources

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

Population density triggers wing dimorphism via NlNompC-mediated insulin signaling in Nilaparvata lugens

The Nilaparvata lugens employs wing dimorphism as a key adaptive strategy to environmental heterogeneity. While hormonal pathways and transcription factors governing nutrient-induced wing plasticity are known, how population density triggers developmental divergence remains unresolved. Here, we mechanistically dissect density-dependent wing morph determination, revealing tactile inter-individual contact as the primary signal. High-density conditions amplify gentle-touch frequency, which is detected by mechanosensitive ion channels NlNompC and NlNMDARs. Genetic silencing or pharmacological inhibition of these channels abolished density-induced long-winged morphogenesis. The conserved role of NlNompC in gentle-touch sensation was validated by rescuing tactile perception deficits in Drosophila nompC mutants. Crucially, NlNompC integrates tactile cues into insulin/insulin-like growth factor signaling (IIS): crowding-driven mechanical stimuli upregulate NlIlp3, while suppressing NlInR2, to activate wing elongation programs. Epistasis experiments confirmed IIS as the key downstream pathway, with co-silencing NlInR2 or NlFoxO rescuing the short-winged phenotype induced by NlNompC knockdown or low population density, silencing NlIlp3 or NlInR1 rescuing the long-winged phenotype induced by high population density. Our work uncovers a mechano-endocrine axis linking tactile perception to developmental plasticity, bridging ecological cues with molecular pathways. These findings redefine density sensing beyond chemical modalities and offer actionable targets for disrupting pest dispersal strategies, advancing sustainable agriculture.

molecular biology↗

bioRxiv2025

Optimal transport modeling uncovers spatial domain dynamics in spatiotemporal transcriptomics studies

Spatiotemporal transcriptomics is an emerging and powerful approach that adds a temporal dimension to spatial transcriptomics, enabling the characterization of dynamic changes in tissue architecture during development or disease progression. Here, we present SpaDOT (Spatial Domain Transition detection), a computational method designed to identify spatial domains and infer their temporal dynamics across time points for spatiotemporal transcriptomics studies. SpaDOT employs a variational autoencoder (VAE) framework to capture a low-dimensional representation of the data, incorporating hidden clustering variables to define distinct spatial domains. In the process, SpaDOT integrates a Gaussian Process prior and graph neighbor information to explicitly model both global spatial continuity and local structural heterogeneity among spatial locations within each time point, while using optimal transport (OT) to derive time-varying embeddings and infer the relationships between spatial domains across time points. Through simulation and real data applications, we demonstrate the superiority of SpaDOT in spatial domain detection, latent space preservation, and domain transition tracking over time. In real data applications, SpaDOT accurately captures dynamic domain transitions, including the disappearance and re-emergence of domains due to technical variation. Notably, SpaDOT uncovers key aspects of valvulogenesis in the developing heart, revealing the splitting of a single valve structure into two distinct functional valves -- the atrioventricular valve and the semilunar valve.

bioinformatics↗

bioRxiv2025

A secreted citrus protease cleaves an outer membrane protein of the Huanglongbing pathogen

Plants secrete a variety of proteases as a defense response during infection by microbial pathogens. However, the relationship between their catalytic activities and antimicrobial functions remains largely unknown. Particularly, few biologically relevant substrates of these proteases have been identified. Huanglongbing (HLB) has been a major threat to the citrus industry worldwide. The HLB-associated bacterium, Candidatus Liberibacter asiaticus (Las), was previously shown to deploy an inhibitor of papain-like cysteine proteases (PLCPs) to promote disease in citrus. In this study, we identified an outer membrane protein (OMP) of Las, LasOMP1, as a substrate of the citrus PLCP CsRD21a. LasOMP1 is one of the most highly expressed genes in Las. CsRD21a cleaves LasOMP1 and produces cleaved peptide products, which could be detected in vitro and in HLB-diseased citrus plants. We found that CsRD21a targets the N-terminal portion of LasOMP1, potentially at an extracellular loop region. Importantly, transgenic sweet orange overexpressing CsRD21a showed reduced Las titers and delayed disease symptoms, highlighting that engineering this protease is a promising strategy to enhance HLB resistance in citrus. Together, our work reveals a pathogen-derived substrate of plant PLCPs and highlights bacterial OMPs as direct targets of plant defense. Significance StatementHuanglongbing (HLB) is the most devastating disease of citrus with no resistance having been identified in commercial cultivars. Previous work implicated papain-like cysteine proteases (PLCPs) as an important hub of defense in citrus; however, their precise role in HLB tolerance remained unclear. Here, we identify and characterize an outer membrane protein (OMP) from the HLB-associated bacterium as a substrate of the citrus PLCP RD21a. We demonstrate a specific cleavage of the bacterial OMP by citrus RD21a, which may impair pathogen growth and/or activate plant immunity. Importantly, overexpression of RD21a enhances HLB tolerance in sweet oranges. This work identifies OMPs as substrates of plant PLCPs and provides insights into protease defense functions.

plant biology↗

bioRxiv2025

Desmoglein-2 deficiency results in cardiac dysfunction by compromising both Z-disc- and intercalated disc-mediated mechanotransduction

Desmoglein-2 (DSG2), a critical component of the cardiac desmosome and located at the cardiomyocyte-cardiomyocyte intercalated disc, is essential for cell-cell adhesion, cardiomyocyte mechanical stability, and electrical coupling between cells. However, its relative contribution in maintaining cardiac function at the sarcomere level remains unclear. Using 4-week-old (adolescent) and 16-week-old (adult) homozygous knock-in Dsg2-mutant (Dsg2mut/mut) mice, we found that loss of DSG2 leads to early onset chamber- and age-dependent cardiac dysfunction driven by Z-disc structural defects and increased myosin detachment rate. Interestingly, Ca{superscript 2}-activated force was markedly reduced in adolescent Dsg2mut/mut permeabilized left ventricular cardiac muscle bundles but preserved in permeabilized isolated cardiomyocytes. This disparity demonstrates that DSG2 is not only crucial for mechanical coupling between cardiomyocytes but also for force transmission within and between sarcomeres, revealing a novel role for DSG2 in maintaining contractile integrity at both the cellular and tissue levels.

biophysics↗

bioRxiv2025

Characterisation of cold-selective lamina I spinal projection neurons

Skin cooling is detected by primary afferents that express the Trpm8 channel, but how this information is conveyed to the brain remains poorly understood. We have previously identified a population of lamina I projection neurons belonging to the anterolateral system (ALS) that receive numerous contacts from Trpm8-expressing primary afferents. Here, using a semi-intact somatosensory preparation, we provide evidence that these cells correspond to the cold-selective ALS neurons identified in previous physiological studies. We also confirm the presence of synapses from Trpm8 afferents onto these cells at the ultrastructural level and with optogenetics. Based on our previous transcriptomic findings, we identify calbindin as a molecular marker, and show that this can be used to target the cold-selective ALS neurons for anterograde tracing studies. We provide evidence that they project to brain regions that have been implicated in thermosensation: the rostralmost part of the lateral parabrachial area, the caudal part of the periaqueductal grey matter, and the posterior triangular and ventral posterolateral nuclei of the thalamus. Our findings provide important insights into the organisation of neuronal circuits that underlie thermoregulation and the perception of cold stimuli applied to the skin.

neuroscience↗

bioRxiv2025

A Tonic Signaling Code Predicts CAR-T Cell Efficacy in Diffuse Midline Glioma

Diffuse midline glioma (DIPG/DMG) is a uniformly fatal pediatric brain tumor with no effective cure. Although CAR T-cell therapy shows promise, clinical outcomes remain inconsistent due to limited persistence and premature exhaustion. Reliable predictive biomarkers are lacking, and proposed exhaustion or stemness markers provide limited utility. Here, we systematically compare multiple CAR-T constructs targeting clinically-relevant antigen B7-H3 and identify antigen-independent CAR activation, or tonic signaling, as a key determinant of therapeutic performance. We find that B7-H3 CAR-T cells with restrained tonic signaling display superior tumor killing, persistence, and resistance to exhaustion, along with reduced CAR membrane clustering, in patient-derived DIPG models. Integrated multi-omics and single-cell profiling further reveal a CAR-T tonic signaling-associated gene signature that outperforms conventional exhaustion or stemness markers in predicting therapeutic efficacy across multiple clinical trials, including DIPG and other tumor types. Together, these findings define a mechanistic and predictive framework to guide CAR design and improve clinical outcomes.

cancer biology↗

bioRxiv2025

Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity

Cross-kingdom RNAs are emerging as critical mediators of interspecies interactions, yet the functions of long RNAs such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms remain largely unexplored. Here, we show that the brown planthopper (Nilaparvata lugens, BPH), a major rice pest, translocates mRNAs and lncRNAs into rice plants, where they migrate systemically from feeding sites to distal tissues. Compared with BPH mRNAs, BPH Salivary gland Cross-kingdom LncRNA (BSCLs) exhibit markedly higher stability in rice. Among them, BSCL1 functions as a virulence factor that promotes BPH feeding and reproduction by suppressing host defense. Mechanistically, BSCL1 associates with the HIRA histone chaperone complex and displaces histone H3.3 from the promoters of transcription factors, including bHLH genes central to jasmonic acid signaling, thereby repressing transcriptional immunity. Our results identify BSCLs as systemic, RNA-based effectors that reprogram host defense at the epigenetic level, revealing a previously unrecognized mode of insect-mediated manipulation of plant immunity and highlighting lncRNAs as cross-kingdom regulators. Significance StatementPlant-herbivore interactions are traditionally viewed as battles over nutrients and defense signaling, mediated largely by proteins and small RNAs. Here, we demonstrate that long non-coding RNAs from brown planthopper saliva are translocated into rice, migrate systemically, and function as epigenetic effectors that suppress key transcription factors in jasmonic acid-mediated defense. Unlike insect mRNAs, these lncRNAs persist in the plant and reprogram immunity by interfering with histone deposition. This discovery uncovers a new class of mobile, RNA-based virulence factors, expands our understanding of cross-kingdom regulation, and suggests innovative strategies for pest control targeting RNA effectors.

plant biology↗

bioRxiv2025

Molecular mechanisms of altered contraction with the β-myosin R403Q mutation in porcine ventricular muscle and a human stem cell-derived cardiomyocyte model.

I.The R403Q mutation in the sarcomere protein beta-myosin heavy chain ({beta}-MHC) is a known genetic cause of hypertrophic cardiomyopathy (HCM), associated with ventricular hypercontractility, impaired relaxation, and cardiac arrhythmias. Despite extensive research, the mutations impact on myosin contractile properties remains unclear, likely due, at least in part, to discrepancies cross different model systems. In this study, we used a multidisciplinary approach to explore mutational effects using two distinct heterozygous R403Q systems: a Yucatan minipig model and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). X-ray diffraction of R403Q minipig ventricular muscle demonstrated reduced order of the thick filament, suggesting destabilization of the inhibited OFF (vs. ON) state of myosin in relaxed muscle, which correlated with elevated force at submaximal calcium. Super-resolution, single-molecule fluorescence microscopy indicated elevated ATPase activity in thick filament zones lacking cMyBP-C. Furthermore, R403Q myofibrils exhibited slower activation and relaxation kinetics, with reduced sensitivity to ADP. Molecular dynamics simulations suggested that altered interactions at the actomyosin interface contribute to these effects, rather than changes at the nucleotide binding pocket, typically associated with ADP release. Human engineered heterozygous R403Q hiPSC-CMs exhibited reduced maximal myofibril force, slowed contractile kinetics, and hypercontraction in engineered heart tissue constructs-consistent with HCM phenotypes observed in the heterozygous porcine model. Our results demonstrate that the R403Q mutation induces early and persistent contractile dysfunction, and that hypercontractility and slower contractile kinetics may result from a combination of an increased population of activated (ON) myosin heads and delayed detachment during cross-bridge cycling, respectively.

biophysics↗

bioRxiv2025

Temporal Clonal Tracing and Functional Perturbation Reveal Niche-Adaptive and Tumor-Intrinsic IFNγ Dependencies Driving Ovarian Cancer Metastasis

Metastasis is an emergent continuum driven by evolving reciprocal adaptations between disseminating tumor cells (DTCs) and specialized niches of different organs. The interplay between intrinsic and niche-driven mechanisms that enable DTCs to survive and home to distant organs in peritoneal ovarian cancer metastasis remains incompletely understood. Here, we present MetTag, a single-cell barcoding and transcriptome profiling approach with single cell clonality barcodes and time-stamped batch identifiers (BC.IDs) to resolve metastasis clonality and temporal dynamics of DTC colonization. Deep sequencing of MetTag barcodes revealed enrichment of early-disseminated clones across metastatic sites, and targeted depletion of pioneer DTCs diminished the outgrowth of subsequent arriving DTCs. Subsequent MetTag-coupled single cell RNA sequencing (scRNA-seq) on ascites and metastasis-bearing omenta revealed a distinct interferon-gamma (IFN{gamma})-centric transcriptional trajectory selectively enriched among pioneer clones. In vivo CRISPR/Cas9 screening of niche-specific signatures demonstrated that the tumor-intrinsic IFN{gamma} response is functionally required for peritoneal metastasis. Knockout of the IFN{gamma} receptor 1 (Ifngr1) in the initial pioneer DTCs significantly reduced total metastatic burden, revealing a critical window of time in which IFN{gamma} signaling shapes the post-seeding metastatic niche (PSMN) and subsequent metastatic evolution. Mechanistically, the tumor-intrinsic IFN{gamma} response induced Poly(ADP-Ribose) polymerase family member 14 (Parp14) and peritoneal macrophages cooperatively shield DTCs from anoikis by promoting pro-survival signaling. Our study defines the temporal clonal architecture of peritoneal niches and reveals a "first come, first served" adaptation principle, where pioneer colonizer fitness determines the success of subsequent colonizers.

cancer biology↗

bioRxiv2025

Differential transcriptomics and metabolomics analyses of the skin of coarse and fine Liaoning cashmere goats

Secondary follicles in cashmere goats determine cashmere yield and quality. The present study investigated the relationship between the regulatory factors and cashmere fiber diameter through transcriptomics and metabolomics methods. Diameters of the fibers of 400 (18 months old) female Liaoning cashmere goats were measured and categorized into coarse and fine hair types (200 goats per category). Six goats were selected as representatives of each group for further analysis. Fiber diameters were significantly different between groups (P < 0.05). Transcriptomic analysis revealed 211 differentially expressed genes between groups, including FGF18, KRT36, KRT79, AWAT2, and MOGAT1, which were enriched in the PPAR and glycerol-lipid metabolism signaling pathways that regulate hair follicle growth and sebum secretion. Metabolomics analysis revealed 50 differentially expressed metabolites in the skin, which regulate cellular ion channels and participate in neurological channels by enriching ABC transporter protein pathways, amino acid biosynthesis and metabolism, and arachidonic acid metabolism. The findings of the present study could facilitate improvement of cashmere quality in cashmere goats.

genetics↗

bioRxiv2025

Efficient spatial gene expression profiling using split-probe ligation and rolling circle amplification

Spatial transcriptomics has transformed our understanding of gene regulation by enabling high-resolution mapping of RNA molecules within their native cellular and tissue environments. This is typically accomplished by capturing or imaging RNA in situ, thereby preserving spatial context. Here, we introduce an in situ RNA imaging method based on split-probe ligation and rolling circle amplification (RCA) for profiling spatial gene expression. In this approach, split-probes hybridize to adjacent regions of a target RNA fragment and are then enzymatically ligated to form circular DNA templates, which are subsequently amplified via RCA to boost signal. We demonstrate that this method enables robust in situ RNA detection and genotyping in both tissue sections and whole-mount tissue samples. By coupling this technique with in situ sequencing, we mapped the spatial expression patterns of 82 genes in the kidneys of healthy and diabetic male and female mice. This analysis revealed distinct localization of Aqp4 in proximal tubules and principal cells of the collecting ducts, and uncovered sex-specific transcriptomic alterations in diabetic kidneys with spatial resolution. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/668044v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@c8efaborg.highwire.dtl.DTLVardef@1eef010org.highwire.dtl.DTLVardef@1d2bbd7org.highwire.dtl.DTLVardef@2a482b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

From Dataset Curation to Unified Evaluation: Revisiting Structure Prediction Benchmarks with PXMeter

Recent advances in deep learning have significantly improved the accuracy of structure prediction for biomolecular complexes; however, robust evaluation of these models remains a major challenge. We introduce PXMeter, an open-source toolkit that support consistent and reproducible evaluation of diverse predictive models across a broad spectrum of biological complex structures. PXMeter provides a unified and reproducible benchmarking framework, offering valuable insights to support the ongoing improvement of structure prediction methods. We also present a high-quality benchmark dataset curated from recently deposited structures in the Protein Data Bank (PDB). These entries are manually reviewed to exclude non-biological interactions, ensuring reliable evaluation. Using these resources, we conducted a comprehensive benchmark of several structure prediction models, namely Chai-1, Boltz-1, and Protenix. Our benchmarking results demonstrate the advancements achieved by deep learning models, while also identifying ongoing challenges--especially in modeling protein-protein and protein-RNA interactions. Project Pagehttps://github.com/bytedance/PXMeter

bioinformatics↗

bioRxiv2025

A conserved small RNA-generating gene cluster undergoes sequence diversification and contributes to plant immunity

Small RNA-mediated gene silencing contributes to plant immunity. The secondary small interfering RNA (siRNA) pathway promotes defense by silencing target genes in invading fungal and oomycete pathogens. Many secondary siRNAs derive from transcripts potentially encoding pentatricopeptide repeat (PPR) proteins. Here, we report that siRNA production is an ancient function of an evolutionarily conserved clade of PPR genes that undergo extensive within-species diversification. In Arabidopsis thaliana, siRNA-source PPRs are physically clustered in one locus on Chromosome 1. These sequences are diversified through gene duplication followed by sequence diversification as well as accumulation of high-impact variations including pseudogenization. This diversity leads to the accumulation of a diverse PPR-siRNA pool, consistent with an engagement in a co-evolutionary arms race with the pathogens. This study defines siRNA-producing PPRs as a family of defense genes and highlights the potential of PPR-siRNA-based engineering for enhancing broad-spectrum disease resistance.

plant biology↗

bioRxiv2025

Multi-omics characterization of the skin microbiota reveals the anti-aging roles of Stenotrophomonas maltophilia

Shifts in the skin microbiome have shown a close link to chronological age. However, the contribution of skin microbiome in skin aging phenotypes remains unclear. To explore this, we performed phenotypic, metabolomic, metagenomic, and functional analyses on a cohort with divergent skin aging phenotypes. Genome-scale metabolic models (GEMs) integrated with metabolomic analysis revealed that Stenotrophomonas maltophilia, enriched in the younger group (categorized by AI-predicted age and skin elasticity), utilizes the glutathione cycle to maintain redox homeostasis. Cellular experiments showed its metabolites enhanced GSH synthesis and alleviated oxidative stress-induced skin aging by upregulating key genes in fibroblasts, including GCLM, PGD, SOD2, and NQO1. Additionally, GEMs highlighted its potential anti-aging roles in regulating host metabolic pathways involving betaine, lysolecithin, and porphyrin. In parallel, Acinetobacter guillouiae was found to influence host melanin metabolism by degrading dopamine (DA) and 3-methoxytyramine (3-MT), offering potential therapeutic strategies for mitigating pigmentation. Our findings highlight the dynamic interplay between skin microbiota and the host in aging, offering new insights for designing targeted anti-aging interventions.

microbiology↗

bioRxiv2025

Pathogen effector forms a hexameric phosphatase holoenzyme with host core enzyme to promote disease

Pathogen effectors play critical roles in pathogenesis by directly manipulating host cellular processes. The Ser/Thr protein phosphatase 2A (PP2A) is commonly targeted by pathogens. Effectors from devastating plant pathogen Phytophthora hijack the PP2A core enzyme in plant hosts, altering the host phosphoproteome. In this study, we present a series of cryo-electron microscopy structures of the Phytophthora effector PSR2 in complex with the host PP2A core enzyme to form a functional holoenzyme. The PSR2-PP2A complex adopts a unique hexameric architecture, driven by PSR2-mediated dimerization of two heterotrimers. This hexamer exhibits greater stability and features a more exposed catalytic pocket compared to the canonical trimeric form of PP2A, likely enhancing its virulence activity. Mutational analyses underscore the importance of this hexameric structure for PSR2s virulence function. These findings provide mechanistic insights into pathogen-mediated manipulation of a key host phosphatase and offer targets for developing disease resistance strategies.

biophysics↗

bioRxiv2025

Annotating the X-ray diffraction pattern of vertebrate striated muscle

Low-angle X-ray diffraction is a powerful technique for analyzing the molecular structure of the myofilaments of striated muscle in situ. It has contributed greatly to our understanding of the relaxed, 430-[A]-repeating organization of myosin heads in thick filaments in skeletal and cardiac muscle. Using X-ray diffraction, changes in filament structure can be detected on the [A] length scale and millisecond time scale, leading to models that are the foundation of our understanding of the structural basis of contraction. As with all X-ray fiber diffraction studies, interpretation requires modeling, which has previously been based on low-resolution knowledge of thick filament structure and is complicated by the contributions of multiple filament components to most X-ray reflections. Here, we use an atomic model of the human cardiac thick filament C-zone, derived from cryo-EM, to compute objectively the contributions of myosin heads, tails, titin, and cMyBP-C to the diffraction pattern, by including/excluding these components in the calculations. Our results support some previous interpretations but contradict others. We confirm that the myosin heads are responsible for most of the intensity on the myosin layer-lines, including the M3 meridional. Contrary to expectation, we find that myosin tails contribute little to the pattern, including the M6 meridional; this reflection arises mainly from heads and other components. The M11 layer line (39 [A] spacing) arises mostly from the curved and kinked structure of titin, which allows eleven [~]42-[A]-long domains to fit into the 430 [A] repeat. The M11 spacing can be used as a measure of strain in the myosin filament backbone as there is negligible head contribution. These insights should aid future understanding of the X-ray pattern of intact muscle in different conditions such as contraction and drug treatment. Significance statementX-ray diffraction is widely used to study the structure of striated muscle, revealing the molecular organization of the thick and thin filaments in situ. Changes in the X-ray pattern during contraction provide insights into contractile mechanisms on the [A] length scale and millisecond timescale. Interpretation of X-ray patterns is based on modeling, which is complicated by contributions of multiple filament components to different reflections and the lack of a reliable thick filament model. Here, we use a cryo-EM-based atomic model of the thick filament to compute contributions of different filament components to the diffraction pattern, by including/excluding these components in the calculations. The insights gained will aid interpretation of the X-ray pattern in relaxation and contraction and following drug treatment.

biophysics↗

bioRxiv2025

Constitutive activation and allosteric mechanisms underlying Gi/Gq signaling bias in SUCNR1 revealed by AlphaFold-based modeling and enhanced sampling simulations

Succinate Receptor 1 (SUCNR1) is a key metabolite sensing GPCR activated by high local succinate concentrations through Gi and Gq signaling pathways. By combining AlphaFold-based modeling, Gaussian accelerated molecular dynamics simulations (72s in total), receptor mutagenesis, and signaling assays, we here investigate the spontaneous and G-protein binding-associated activation mechanism of SUCNR1. Our molecular simulations predict that, in the absence of G protein or ligand, human SUCNR1 (hSUCNR1), in contrast to murine SUCNR1 (mSUCNR1), adopts canonical active- like conformations in its intracellular domains and undergoes critical conformational changes in its extracellular domain. This finding is consistent with our signaling-assay results showing that hSUCNR1, unlike mSUCNR1, displays a high degree of constitutive signaling. When complexed with either Gi or Gq, both h- and mSUCNR1 adopt highly energetically favorable conformations in the extracellular domain, characterized by the unlocking of extracellular loop 2b (ECL2b) and an expanded ligand entry pathway. Interestingly, the simulations reveal that helix 5 of Gq binds less firmly to the TM3/6 cleft of mSUCNR1, resulting in an unstable extracellular domain of the Gq- mSUCNR1 complex, which however can be stabilized by agonist binding. This result is supported by our BRET assays, which show that, in contrast to hSUCNR1, mSUCNR1 fails to recruit mini-Gq to the cell surface in the absence of an agonist. Furthermore, the signaling effects of key receptor residues at G-protein binding site and within ECL2b, as predicted by our simulations, were confirmed by mutagenesis assays. Overall, our integrative approach demonstrates that hSUCNR1 can spontaneously adopt all key conformations associated with receptor activation, and that G protein binding further primes the extracellular receptor domain for agonist binding, which in turn stabilizes the active complex.

biophysics↗