Biology subjects

Lin, H.

Publications and source records attributed to Lin, H..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Carbon monoxide metabolism in freshwater anaerobic methanotrophic archaea

Anaerobic methanotrophic archaea mitigate methane emissions in anoxic environments as key members of the biological methane filter. Despite their ecological significance, physiology of anaerobic methanotrophs remains poorly understood. Here, we demonstrate that the freshwater methanotroph Candidatus Methanoperedens BLZ2 prefers carbon monoxide (CO) over methane as an electron donor. In the absence of respiratory nitrate, CO oxidation led to acetogenesis and methanogenesis with rates comparable to methane oxidation. The circularized genome of Ca. M. BLZ2 encodes six Ni-dependent carbon monoxide dehydrogenases (CODHs), three of which were highly expressed. Furthermore, we identified a 156-kbp mobile genetic element carrying central metabolic gene clusters, including two additional, highly expressed CODHs. CODH genes were widespread in Methanoperedenaceae and showed diverse evolutionary affiliations, including Methanocomedenaceae anaerobic methanotrophs and bacterial lineages. These findings highlight a novel metabolism and genome plasticity in anaerobic methanotrophs challenging their classification as obligate methanotrophs and their ecological role in anoxic carbon cycling.

microbiology↗

bioRxiv2025

Cutaneous suction-mediated transfection in mice for delivery of DNA-encoded vaccines and proteins

An important step to fulfill the functionalities of DNA vaccines and therapeutics is transfection in vivo to produce the encoded antigens or therapeutic proteins. A cutaneous suction-based method has demonstrated effectiveness in many animal models and has been successfully applied in human clinical trials, but has not been extended to mouse models, where numerous disease models, transgenic strains, and murine-specific reagents exist. The current work establishes and optimizes methods for cutaneous suction-mediated DNA transfection in mice. By adapting a smaller cup diameter and smaller injection volume, the challenges of skin hyperelasticity and decreased skin thickness can be effectively addressed, and vaccinating mice with the GLS-5310 SARS-CoV-2 DNA vaccine yielded high levels of binding antibody and T cell responses. Additionally, suction following injection of a novel pVAX1-based expression vector yielded systemic levels of a SEAP transgene. Thus, suction-mediated delivery of nucleic acid-based therapies and vaccines can be a valuable tool for the study in pre-clinical mouse models.

bioengineering↗

bioRxiv2025

Cholesterol metabolism modulation facilitates CAR-T induced killing of ovarian cancer

Ovarian cancer is one of the most lethal gynecological cancers worldwide and has one of the highest recurrence rates. Recently developed Chimeric Antigen Receptor T (CAR-T) cell therapy has shown potent clinical efficacy against hematological malignancies. However, solid tumors, including ovarian cancer, possess several mechanisms that hinder T cell activity, and metabolic alteration of cancer cells has been shown to contribute to resistance to immune cell attack against solid tumors. Here, we explored the metabolic response of ovarian cancer cells to CAR-T cell attack using label-free high-content hyperspectral stimulated Raman scattering (h2SRS) imaging. Utilizing visible h2SRS imaging with much improved spatial resolution, we found an altered cholesterol metabolism, featured by increased storage of cholesteryl ester in lipid droplets and free cholesterol, in ovarian cancer cells that survived the CAR-T treatment. Administration of Avasimibe, an inhibitor of cholesteryl esterification, further enhanced CAR-T cytotoxicity. Our study shows the promise of implementing metabolic modulation to facilitate CAR-T cell treatment of solid tumors. Significance StatementOvarian cancer is a major global health challenge, mainly due to relapse driven by chemotherapy resistance development. Meanwhile, immunotherapies such as CAR-T have revolutionized treatment for blood cancers, but their application in solid tumors like ovarian cancer is hindered by limited efficacy. This study uses high-content stimulated Raman scattering (SRS) imaging to reveal metabolic adaptations that help ovarian cancer cells survive CAR-T-mediated cytotoxicity. Guided by these insights, we apply metabolic interventions that enhance CAR-T cytotoxicity efficacy. This approach reveals therapeutic vulnerabilities in ovarian cancer and demonstrates an investigative strategy applicable for overcoming resistance in solid tumors.

cell biology↗

bioRxiv2025

Nuclear polysaccharides maintain H3K9me3-heterochromatin and genomic stability

Polysaccharides are known to be synthesized by enzymes in the endoplasmic reticulum and Golgi apparatus and transported through the secretory pathway to the cell surface or extracellular space1, where they mediate essential biological processes2-6. While classical localization and functions of polysaccharides are well established, their presence and potential roles in the nucleus remain unclear. Here, we demonstrate that N-glycans, a type of polysaccharides, are present in the cell nucleus and modify inner nuclear membrane (INM) proteins across diverse cell types--a modification referred to as N-linked glycosylation (N-glycosylation). N-glycosylation is enriched in chromatin regions marked by H3K9me3 and long interspersed nuclear element-1 (LINE-1) retrotransposons. N-glycosylation inhibition and INM protein N-glycosylation site mutation both downregulate H3K9me3 within lamina-associated domains (LADs) and lead to genomic instability. Mechanistically, N-glycosylation regulates the interaction between the histone H3K9 methyltransferase SETDB1 and INM proteins, promotes the association of SETDB1 with the INM, and maintains H3K9me3. Moreover, we reveal that canonical N-glycan biosynthetic machinery in ER contributes to the N-glycosylation of INM proteins. These findings uncover a previously unrecognized nuclear role for polysaccharides, broadening our understanding beyond their traditional subcellular distributions and functional profiles.

molecular biology↗

bioRxiv2025

Deficiency of Mesencephalic Astrocyte-derived Neurotrophic Factor Aggravates Acute Pancreatitis in Mice

Acute pancreatitis (AP) is a complex and potentially severe inflammatory disorder of the pancreas and is one of the most common causes of gastrointestinal hospitalization. Although environmental risk factors such as alcohol and gallstones are well recognized, only a subset of exposed individuals develop AP, suggesting that intrinsic factors, including genetic susceptibility, influence disease onset and progression. Endoplasmic reticulum (ER) stress has emerged as a key mechanism in AP pathogenesis, because ER is essential for protein synthesis, folding, degradation and secretion (proteostasis). Mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER stress-inducible protein highly expressed in the pancreas, plays a critical role in maintaining proteostasis, yet its involvement in AP remains unclear. To investigate the functional role of MANF in AP, we generated pancreas-specific MANF knockout (MANF-KO) mice using the Cre/loxP system and subjected them to moderate experimental AP, that is caerulein- or alcohol-induced AP in mice. In the caerulein model, MANF deficiency exacerbated pancreatic injury in both sexes, as indicated by increased apoptosis (cleaved caspase-3, caspase-12), ER stress markers (eIF2, p-eIF2, GRP78), inflammation (IL-6, TNF), regenerative activity (Ki67), and pancreatic lipase levels. Notably, male MANF-KO mice exhibited enhanced inflammation (HMGB1), macrophage infiltration (CD68), and oxidative stress (DNP, HNE), which were not observed in females. In the alcoholic AP model, both male and female MANF-KO mice showed increased ER stress (p-IRE1, p-eIF2, GRP78), apoptosis, inflammation, macrophage infiltration, regeneration, and lipase levels, whereas elevated HMGB1 expression and oxidative stress again predominantly occurred in male MANF-KO mice. Together, these findings reveal a critical and sex-specific role for MANF in regulating pancreatic stress responses and inflammatory injury, supporting its potential contribution as a genetic factor in AP pathogenesis.

pathology↗

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

Age-Related Alterations in the Expression of Mesencephalic Astrocyte-derived Neurotrophic Factor in the Brain and Their Impact on Neurobehavioral Functions

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic protein localized in the endoplasmic reticulum (ER) and pivotally involved in maintaining ER homeostasis. MANF plays an important role in mitigating neurodegenerative processes. Aging, the primary risk factor for neurodegenerative diseases (NDDs), is associated with significant alterations in ER function. The ER, central to protein synthesis, folding, degradation and secretion (proteostasis), experiences considerable stress in NDDs, which activates the unfolded protein response (UPR). We hypothesized that MANF and UPR is crucial for maintaining proteostasis during aging, but their efficacy declines with age, therefore increasing vulnerability to NDDs. We measured MANF levels in the brain and plasma of 1-, 4-, 11-, and 22-month-old male and female mice. A progressive decline of MANF levels was observed, with the lowest levels detected in 22 months. Reduced MANF expression was found in aged mice across several brain areas, including the cerebral cortex, olfactory bulb, thalamus, hypothalamus, hippocampus, and cerebellum. There was a sex difference in MANF levels in aged mice. Aging also altered the expression of UPR and MANF interacting proteins. Using cerebellar Purkinje cell (PC)-specific MANF deficient mice, we showed that MANF deficiency impaired motor coordination in female, but not male mice. MANF deficiency weakened spatial learning and memory in both male and female mice. Male MANF deficient mice displayed increased sociability, whereas female mice exhibit social withdrawal. Taken together, MANF expression in the brain declined with age and MANF deficiency impacted neurobehaviors in the aging animal in a sex-specific manner.

neuroscience↗

bioRxiv2025

Cell-free DNA fragmentomic characteristics in transposon elements inform molecular regulators and enhance cancer diagnosis

Fragmentomics of plasma cell-free DNA (cfDNA) are emerging diagnostic biomarkers in cancer liquid biopsy, while the molecular regulations of cfDNA fragmentation remain elusive. In this study, we investigated the cfDNA fragmentomics in transposon elements (TEs), a special category of sequences accounting for around half of human genome. We discovered dynamics in fragmentomic features across various types of TEs in human cfDNA, including size, ending patterns and coverage, which were validated in mice and dogs. These dynamics highly correlate with epigenomic features associated with chromatin states, including DNA methylation and histone modification signals, demonstrating fundamental regulatory roles of chromatin state in cfDNA production. Furthermore, fragmentomic features within TEs were significantly altered in cancer samples, offering improved diagnostic performance compared to genome-wide fragmentomic measurements. Additionally, cfDNA coverage in TEs showed frequent imbalances between cancer and control samples in a TE- and cancer type-dependent manner, presenting a promising biomarker for cancer diagnosis. Leveraging artificial intelligence (AI) on cfDNA fragmentomic features within TEs, we developed high-performance models, named TEANA (TE Analysis in cell-free DNA), for cancer diagnosis and tumor-origin prediction. The models were validated across two pan-cancer datasets with more than 1600 samples. Hence, dynamics of cfDNA fragmentomics within TEs, which is highly correlated with cfDNA epigenomics, shed light on the regulatory mechanism of cfDNA biology and highlight translational potential in cancer liquid biopsy.

cancer biology↗

bioRxiv2025

The evolution of high-order genome architecture revealed from 1,000 species

Spatial genome organization plays a crucial regulatory role, but its evolution remains unknown. Leveraging Hi-C data from 1,025 species, we trace the evolutionary trajectories of 3D genome, through two higher-order architectures, global folding (spatial organization of karyotype) and checkerboard (chromatin compartments). Early life forms, including prokaryotes and unicellular eukaryotes, mostly display random configurations. Through the evolution of plants, global folding became the prominent architecture with eudicots and remained dominant. Animals, however, progressively developed stronger checkerboard throughout vertebrate and invertebrate evolution, and even during early embryogenesis, suggesting a conserved gene co-regulation mechanism. In contrast, plants prefer linear gene clusters over checkerboard. Both strategies of gene arrangement reinforce the biological principle structure determines function: divergent evolutionary paths converge on architectural solutions to meet the greater regulatory demands.

evolutionary biology↗

bioRxiv2025

A Novel 'Three-in-one' Mucosal Vaccine Elicits Broad Protection against Three Distinct Clusters of ACE2-using Sarbecoviruseses

With the persistent risk of coronavirus epidemics, the development of universal vaccines is urgently needed and challenging to achieve. In this study, we aimed to design a multivalent immunogen to provide broad protection against diverse ACE2-using sarbecoviruses, which pose a significant threat to global health. Our analysis revealed that the receptor-binding domains (RBDs) of ACE2-using sarbecoviruses segregate into three antigenically distinct clusters, including one cluster that encompasses viruses related to SARS-CoV-2. Based on these findings, we engineered a three-in-one immunogen, designated as 3Rs-NC, which incorporates representative RBDs from all three clusters. 3Rs-NC preserved the natural epitope configuration of each monomeric RBD component, and efficiently elicited high levels of neutralizing antibodies against representative viruses and closely related sarbecoviruses. When administered intranasally with flagellin-based mucosal adjuvant KFD, 3Rs-NC induced robust and durable RBD-specific serum IgG and mucosal IgA responses in mice. Furthermore, KFD-adjuvanted 3Rs-NC conferred sustained protection in both the upper and lower respiratory tracts against SARS-CoV-2 Omicron BA.1 and SARS-like coronavirus WIV1. Additionally, 3Rs-NC immunization protected mice from lethal challenge of SARS-like coronavirus rRsSHC014S, with more efficient protection observed in female mice than male mice. This needle-free three-in-one vaccine represents a promising candidate for a universal mucosal vaccine against ACE2-using sarbecoviruses and could serve as a foundational component of three-in-one vaccine series to form a comprehensive coronavirus vaccine package. HighlightO_LInovel three-in-one immunogen 3Rs-NC induced a high level of neutralizing antibodies against three distinct ACE2-using Sabecovirus clusters. C_LIO_LIadministration of 3Rs-NC plus KFD adjuvant (3Rs-NC+KFDi.n) generated potent and long-lasting RBD-specific serum IgG and mucosal IgA. C_LIO_LI3Rs-NC+KFDi.n provided long-term protection in both upper- and lower-respiratory tracts in mice. C_LIO_LI3Rs-NC+KFDi.n could serve as a foundational component for next-generation coronavirus vaccine strategies. C_LI

immunology↗

bioRxiv2025

EVODEX: A Mechanistic Framework for Extracting, Structuring, and Predicting Enzymatic Reactivity

Accurately modeling enzymatic reactivity is essential for synthesis planning, mass spectrometry interpretation, and biochemical knowledge extraction, but existing reaction datasets are often incomplete or inconsistently annotated. EVODEX introduces two abstractions to address this: partial reactions, which isolate atom-mapped substrate-product transformations from complex biochemical reactions, and Electronic Reaction Operators (EROs), which encode the bonding and orbital environment of atoms undergoing change. From 349,458 curated reactions in EnzymeMap, EVODEX constructs over 186,000 partial reactions and mines a core set of 1,404 EROs, each supported by at least 10 examples. Together, these explain 91 percent of test reactions at the formula level and 62 percent at the mechanistic level. A further filtered synthesis subset of 436 one-to-one operators that exclude ubiquitous metabolites provides a compact and generalizable vocabulary of single-step enzymatic transformations. EVODEX operators are auditable, reusable, and available as part of an open-source Python package with datasets, Jupyter notebooks, and a browsable website.

bioinformatics↗

bioRxiv2025

Chromosome-level genome assembly of the spider mite Tetranychus cinnabarinus

Tetranychus cinnabarinus is an important agricultural pest, primarily due to its rapid development of pesticide resistance. In this study, we constructed a chromosome-level genome of T. cinnabarinus using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled genome spans 86.67 Mb, with an N50 value of 21.77 Mb, and 84.54 Mb of sequences anchored across three chromosomes. The BUSCO completeness of the genome is 92.78%. Through gene prediction, we identified 11,388 protein-coding genes, 98.53% of which are functionally annotated. Furthermore, we systematically identified 304 genes from five major detoxification enzyme systems. This high-quality genome provides a solid foundation for investigating the physiological mechanisms of T. cinnabarinus and for identifying potential targets for biocontrol strategies.

genomics↗

bioRxiv2025

COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale

Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine kinact and KI values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using the BTK inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and novel off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target BTK. We further demonstrate that COOKIE-Pro is compatible with streamlined cysteine activity-based protein profiling (SLC-ABPP) datasets, enabling efficient conversion of competition ratios to meaningful kinetic parameters. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful new tool for optimizing the potency and selectivity of covalent drugs during preclinical development.

biochemistry↗

bioRxiv2025

Spatial Transcriptomics Analysis Uncovers ER stress in MANF-deficient Purkinje Cells Underlying Alcohol-induced Cerebellar Vulnerability in Mice

Cerebellar Purkinje cells (PCs) are among the most vulnerable neurons to alcohol neurotoxicity. Alcohol can induce endoplasmic reticulum (ER) stress and alter the structure and function of PCs. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress inducible protein highly expressed in PCs. It is neuroprotective in various pathological conditions where ER stress is induced. However, it is unknown whether MANF plays a role in protecting PCs from alcohol induced ER stress. In this study, we generated PC-specific MANF knockout (KO) mouse model to test the hypothesis that MANF deficient PCs are more susceptible to binge alcohol exposure induced ER stress and neurodegeneration in the adult brain. We found that PC-specific MANF KO animals show moderate motor function deficit, which was exacerbated by alcohol exposure. Interestingly, female KOs were more sensitive than male KOs to alcohol-induced motor function impairments. In accordance with the behavior changes, alcohol exposure also caused UPR activation, increased intranuclear expression of calcium binding protein Calbindin, and PC degeneration in female but not male MANF KO mice. Spatial transcriptomics and high throughput in situ analyses demonstrated that MANF deficiency altered the transcriptomic landscape in PCs in a sex-specific manner and triggered the expression of genes involved in protein folding and response to ER stress. These results suggests that MANF KO PCs may be predisposed with a higher risk to UPR activation and ER stress in a sex dependent manner, contributing to their vulnerability to alcohol neurotoxicity.

molecular biology↗

bioRxiv2025

Mapping of individual somatosensory representations - comparison of fMRI and TMS

Invasive brain mapping, functional magnetic resonance imaging (fMRI) and navigated transcranial magnetic stimulation (nTMS) results indicate that somatosensory representations vary between individuals. However, it is unknown how well somatosensory representations determined using nTMS and fMRI correspond. Here, we used single-pulse nTMS and fMRI in 17 right-handed subjects to determine the S1 representation of the tip of the right index finger stimulated mechanically with a Braille device. In an nTMS mapping experiment, the S1 site at which tactile sensation was blocked by nTMS, was considered the S1 representation site (S1HS) of the fingertip. The location of S1HS varied up to 36 mm between subjects. In the fMRI experiment, passive and oddball tactile tasks were employed. The location of the somatosensory peak fMRI activation varied up to 39 mm between subjects in the passive condition and up to 84 mm in the oddball condition. Within subjects, the mean distance between the S1HS and the peak fMRI activation was 10 + 2 mm (S.E.M.) in the passive condition and 14 + 3 mm in the oddball task. Taken together, our findings underscore the importance of multimodal approaches to brain mapping in future studies.

neuroscience↗

bioRxiv2025

Structural Basis for Cas9-Directed Spacer Acquisition in Type II-A CRISPR-Cas Systems

CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas92-Csn28-Cas18-Cas24) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

molecular biology↗

bioRxiv2025

Beyond the G-Loop: CRBN Molecular Glues Potently Target VAV1 via a Novel SH3 RT-Loop Degron

This study reports the discovery and characterization of novel CRBN molecular glues that selectively induce the proteasomal degradation of the hematopoietic-specific signaling protein VAV1, a key target in hematological malignancies and autoimmune diseases. Utilizing unbiased global proteomics, we identified phenyl-glutarimide derivatives NGT-201-12, as effective VAV1 degraders, with its C-terminal SH3 domain (SH3-2) being crucial for this interaction. A significant finding is the elucidation of a non-canonical RT-loop degron (RDxS motif, residues 796-799) within VAV1 SH3-2, distinct from previously characterized G-loop degrons. This discovery, supported by advanced computational modeling using the physics- and AI-driven GluePlex workflow and validated by site-directed mutagenesis, highlights versatility of CRBN in recognizing diverse neosubstrate motifs. Furthermore, we demonstrate that applying Free Energy Perturbation (FEP+) calculations to these predicted ternary structures yields cooperativity metrics that correlate with experimental degradation potency, overcoming the limitations of standard molecular docking. This establishes a robust workflow where, once a ternary complex is predicted, even with initial weak binders, FEP+ can be utilized to prospectively rank analogs and optimize molecular glue potency. Additionally, we demonstrate that strategic chemical modifications, particularly conformational restriction via halogen substitution (e.g., NGT-201-18), markedly potentiate VAV1 degradation, a principle supported by density functional theory (DFT) calculations. Comprehensive structure-activity relationship (SAR) studies provided a roadmap for designing next-generation VAV1 degraders. Importantly, dose-response proteomics not only confirmed VAV1 as the primary target but also revealed LIMD1, possessing a canonical G-loop, as an off-target for some analogs, indicating a single molecular glue can engage disparate degron motifs. The identification of the VAV1 RT-loop degron prompted a proteome-wide search, revealing other SH3-containing proteins as potential targets or off-targets. In conclusion, this research unveils a novel non-canonical RT-loop degron in VAV1, demonstrates the utility of conformational restriction in enhancing degrader potency, and underscores the critical role of integrating global proteomics with advanced structural modeling and FEP calculations for understanding degrader potency and selectivity. These findings offer a promising therapeutic strategy for targeting VAV1 and significantly expand the landscape of CRBN neosubstrate recognition and the rational design of molecular glue degraders.

biochemistry↗

bioRxiv2025

Cryo-EM structure of plant urea transporter DUR3 reveals essential role of C-terminal domain in tetramer assembly and insights into proton-coupled transport

Nitrogen pollution, caused by the overuse of nitrogen-fertilizers such as urea in agricultural production, has become a serious problem that threatens both the environment and human health. Improving the nitrogen use efficiency of crops is an important approach to reduce fertilizer use. DUR3, a high-affinity urea transporter, enables plants to acquire urea from natural or agricultural soils with low urea concentrations and has been proposed as a promising target for engineering crops to improve nitrogen-fertilizer use efficiency. Despite extensive studies for more than 20 years, the structure, substrate recognition and transport mechanism of DUR3 have remained unknown. Here we report the cryo-EM structure of maize DUR3 (ZmDUR3) at 3.41 [A] resolution. ZmDUR3 adopts an amino acid-polyamine-organocation (APC) superfamily fold and is structurally distinct from animal facilitative urea transporters. Our structure provides important insights into urea recognition by DUR3 and suggests that a pair of acidic residues is potentially involved in proton coupling during urea transport. Furthermore, our structural and functional studies indicate that DUR3 assembles into a tetramer and tetramer formation is important for its function. Our results pave the way for engineering plant DUR3 protein for applications in sustainable agricultural production.

plant biology↗