Biology subjects

Chen, E.

Publications and source records attributed to Chen, E..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Spatially Distinct Myosin II Architectures Regulate Protrusion Dynamics and Directional Persistence during Immune Cell Migration

Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments. HighlightsO_LIMigrating neutrophils assemble spatially distinct myosin II architectures at the leading edge and rear C_LIO_LIProtrusion dynamics and directional persistence are linked to sustained myosin II engagement C_LIO_LIDirectional migration emerges from the temporal organization of protrusion states rather than their abundance C_LI

cell biology↗

bioRxiv2026

Telomere-to-telomere assembly and haplotype analysis of tetraploid Dendrobium officinale illuminate Orchidaceae polyploid evolution and mycorrhizal symbiosis genes

Dendrobium officinale is a typical epiphytic orchid. We report the telomere-to-telomere (T2T) genome assembly for D. officinale, representing the first T2T reference genome within the Orchidaceae family. The assembly is anchored to 19 chromosomes and contains 38 complete telomeres and 15 characterized centromeres. We further generated haplotype-resolved assemblies of the autotetraploid genome, identifying 12,761 sets of tetra-allelic genes. Based on synonymous substitution analysis, we inferred that the autotetraploidization event occurred approximately 0.86 million years ago. A systematic analysis of the SWEET gene family across the genus Dendrobium revealed that the gene family size is shaped primarily by epiphytic types and environmental factors. In D. officinale from Langshan, eight SWEET genes were specifically expressed in roots, suggesting they may play specialized roles in the root mycorrhizal system, potentially contributing to the D. officinales ability to recruit and maintain fungal partners. Together, these resources provide valuable foundations for studies of orchid evolution, functional genomics, and molecular breeding.

bioinformatics↗

bioRxiv2026

UBE3C retrofits the proteasome to enforce degradation of ultra-stable folds

Neurodegeneration and proteinopathies arise from mutated or misfolded proteins that form ultra-stable, proteotoxic aggregates that evade proteasomal degradation despite ubiquitylation1-8. These conditions are exacerbated by the deubiquitylase USP14 that can prematurely rescue substrates9-11. To counter these obstacles to effective proteolysis, eukaryotes evolved the highly conserved HECT-type E3 ligase UBE3C--dysregulated in neurodegeneration12,13 and overexpressed in cancers14-17--to reprogram the 26S proteasome toward heightened activity via unknown mechanisms18-25. Here we visualized functional dynamics of the human UBE3C-retrofitted proteasome enforcing degradation of a re-engineered superfolder GFP that normally escapes proteolysis, using time-resolved cryo-electron microscopy. A continuum of non-equilibrium conformations, comprising fourteen proteasome conformers orthogonally combined with four UBE3C states or three USP14 states, reveals a cryptic UBE3C-receptor site in the proteasomal lid and captures key intermediates of ubiquitin-chain elongation and branching at four linkage-specific ubiquitin-binding sites. Remarkably, UBE3C creates an extreme shortcut for ubiquitin shuttling that bypasses USP14, promotes USP14 recycling and allosterically strengthens substrate-unfolding forces of the AAA-ATPase unfoldase to ultimately surmount the unfolding energy barrier of ultra-stable proteins. These findings define the complete functional cycle of the UBE3C-reprogrammed proteasome, illuminating how UBE3C simultaneously controls USP14 and the proteasome to enforce clearance of ultra-stable folds, and establish crucial mechanistic foundations for proteolysis-targeting therapeutic discovery.

biochemistry↗

bioRxiv2026

Enhancing Pan-cancer Spatial Transcriptomics atSingle-cell Resolution with stPainter

Subcellular spatial transcriptomics technologies offer unprecedented views of tissue architecture but are fundamentally constrained by sparse gene panels and limited detection sensitivity. Current computational enhancement strategies typically rely on tissue-matched single-cell RNA sequencing (scRNA-seq) references and necessitate computationally intensive retraining for each dataset, impeding their scalability and clinical applicability. Here, we present STPAINTER, a conditional generative model that leverages a massive pretraining pan-cancer scRNA-seq atlas to universally enhance spatial transcriptomics data. Built upon a latent diffusion architecture with stochastic differential equation-guided generation, STPAINTER learns a universal manifold of cellular states to reconstruct genome-wide expression profiles from sparse spatial measurements. Uniquely, our pretraining paradigm enables zero-shot generalization and empowers downstream tasks by providing imputed transcriptomes and informative latent variables to enhance resolution at both the gene and cluster levels. Applied STPAINTER upon 6 spatial transcriptomics datasets of different cancer types, we demonstrate that our model empowers high-fidelity downstream analyses, including fine-grained subpopulation clustering and pathway enrichment. Furthermore, cross-validation with spatially resolved proteomics (CODEX) confirms the biological veracity of the imputed cellular landscapes. STPAINTER provides a robust, scalable framework for decoding complex tumor microenvironments without the need for auxiliary sequencing data.

bioinformatics↗

bioRxiv2026

Volume EM reveals three-dimensional architecture of the desmosome in epithelial cells and tissue models

Desmosomes are a type of cell-cell adhesive junction present in cardiac tissue and epithelial tissues such as the epidermis. These intercellular junctions anchor to the intermediate filament cytoskeleton, providing mechanical integrity to the tissues in which they reside. Our understanding of desmosome architecture has largely been influenced by observations of two-dimensional images obtained through conventional electron microscopy. Here, using focused ion beam scanning electron microscopy, we report the three-dimensional ultrastructure of desmosomes in A431 and S1 human mammary epithelial cells. We also reveal differences in desmosome ultrastructure at homo- and heterotypic junctions of human nasal airway epithelial cells. Quantitative analyses of these volume EM datasets reveal variations in desmosome size, shape, and organization. Importantly, we report the presence of discontinuities or "holes" within the desmosome outer dense plaque, a novel feature that is observed in either one or both halves of a desmosome. This study provides the first comprehensive description of the epithelial desmosome as a three-dimensional structure, and emphasizes the need to investigate the effects of dynamic morphogenetic processes and disease states on desmosome ultrastructure.

cell biology↗

bioRxiv2025

Bioenergetic responses to β-adrenergic stimulation in beige adipocyte depend on actomyosin driven forces

Beige (BeAT) and white adipoctyes (WAT) reside within the same fat depots but differ in their responses to {beta}-adrenergic stimulation. While both activate classical PKA-mediated pathways, only beige adipocytes show induction of UCP1 expression and enhanced mitochondrial respiration, suggesting additional mechanisms that distinguish their responses. Here we uncover a parallel, non-canonical biomechanical pathway specific to beige adipocytes that is essential for thermogenic activation. Mechanistically, {beta}-AR stimulation induces a rapid Myh9-mediated actomyosin contraction and stiffening in beige, but not white, adipocytes that activates focal adhesion kinase (FAK), which we show to be critical for the expression of oxidative and thermogenic genes including UCP1 and PLIN5. These findings reveal a new biomechanical Myh9-FAK signaling arm downstream of b-adrenergic activation that differentiates thermogenic from white adipocytes.

cell biology↗

bioRxiv2025

Expanding the scope of Mycobacterium abscessus reference strains to improve pulmonary disease modeling

Mycobacterium abscessus (Mab) pulmonary disease is an emerging clinical challenge, particularly among individuals with immunosuppression or underlying structural lung conditions. There are currently no FDA-approved therapies for Mab disease. Existing treatment strategies using repurposed drugs are prolonged, complex, and yield low cure rates (30-50%), underscoring the urgent need for more effective therapeutics. Developing new treatments requires preclinical disease models that faithfully replicate human disease, and the choice of Mab strain is a key determinant of model relevance. The commonly used reference strain, ATCC 19977, was isolated from a non-pulmonary source but became the default due to its early availability. To evaluate its relevance for pulmonary disease modeling, we compared ATCC 19977 with 15 clinical Mab isolates derived from lung infections across diverse regions of the United States. In both in vitro assays and a validated mouse lung infection model, ATCC 19977 behavior differed from the clinical isolates for key traits including rapid systemic dissemination, failure to develop robust lung granulomas, and early mortality. In contrast, clinical isolates demonstrated greater pulmonary tropism and reduced dissemination, with several producing robust lung pathology. Based on these findings, we propose a set of pulmonary clinical isolates representing the major Mab subspecies for use in lung infection research. These isolates more accurately recapitulate the pathological features of human Mab lung disease and are expected to enhance the translational value of future mechanistic and therapeutic studies. SUMMARY STATEMENTUsing a mouse model, this study identified Mycobacterium abscessus clinical isolates whose infection profiles more closely resemble human pulmonary disease, establishing them as superior reference strains to ATCC 19977 for future translational and therapeutic research.

microbiology↗

bioRxiv2025

Longitudinal Imaging of the Premalignant Tumor Microenvironment Reveals Transient Myeloid States Predictive of Tumor Fate

The spontaneous regression of cancer lesions illustrates the power of immune surveillance; yet these transient events have largely escaped systematic analysis. Using longitudinal intravital microscopy in a carcinogen-induced model of head and neck cancer, we followed premalignant lesions within the same animals for 24 weeks at single-cell resolution. This strategy uncovered three trajectories: progression, stability, or regression, and enabled direct analysis of immune dynamics underlying each fate. Lesion outcome was determined by the spatial organization of myeloid-derived antigen-presenting cells: regressing lesions were characterized by dense clusters of myeloid-derived cells associated with CXCL9+/CXCL10+ expression and T cell recruitment, whereas progressing lesions displayed a scattered infiltration of these cells. Remarkably, transient myeloid clusters arose prior to any detectable lesion formation and marked regions that would later develop into premalignant lesions. These findings identify spatiotemporal myeloid organization as an early determinant of tumor fate and provide a mechanistic framework for predicting and intercepting cancer at its inception. Summary SentenceEarly myeloid architecture dictates cancer fate: dense CXCL9/CXCL10 clusters with T-cell enrichment accompany regression, whereas sparse infiltration predicts progression. Transient pre-lesional myeloid clusters emerge at future tumor sites, revealing immune organization as an early determinant of malignancy.

cancer biology↗

bioRxiv2025

Regulation of Muscle Stem Cell Dynamics and Quiescence by Netrin-1 Cytoskeletal Signaling

The hallmark property of muscle stem cells (MuSCs) at homeostasis is quiescence. However, MuSCs have cellular protrusions that are heterogeneous, complex, and tipped with filopodia, all signs of motile structures. Such protrusions may serve as sensors of the MuSC niche. Through development of a novel ex vivo live imaging assay for MuSC protrusion dynamics, we report here the identification of a regulatory pathway in this process. The axon guidance cue Netrin-1 promotes MuSC protrusion outgrowth ex vivo in a manner dependent on its receptors Dcc and Neogenin, the small GTPase Rac1, and the actin-branching factor Arp2/3; this pathway is also required for Netrin-dependent axonal growth cone motility. Adult MuSC-specific genetic removal of Netrin-1 receptors, Rac1, and Arp2/3 each result in failure to maintain homeostatic protrusion lengths, spontaneous quiescence exit, and MuSC attrition in uninjured mice. These findings reveal an unanticipated level of morphological dynamism by a quiescent stem cell at homeostasis and link this phenomenon to preservation of the quiescent state.

cell biology↗

bioRxiv2025

Fine-tuned Protein Language Model Identifies Antigen-specific B Cell Receptors from Immune Repertoires

Scalable identification of antigen-specific antibodies from whole immune repertoire V(D)J sequences is a central challenge in biomedical engineering. We show that protein language models (PLMs) fine-tuned on antibody heavy-chain sequences can directly predict antigen specificity from unselected immune repertoires. We assessed our model, Antigen Specificity Predictor (ASPred), against SARS-CoV-2, influenza, and HIV-AIDS antigens, observing comparable predictive performance. In the whole immune repertoire V(D)J sequences of mice immunized with the SARS-CoV-2 spike proteins receptor-binding domain (RBD), ASPred identified antibody sequences specific to RBD. Several candidate sequences were validated, including one as a heavy chain-only nanobody with 20.7 nM dissociation constant. Molecular dynamics simulations supported the predicted interactions at coarse-grained and atomic levels. Benchmarking against Barcode-Enabled Antigen Mapping (BEAM) of B cell receptor sequence data had highly significant overlaps with ASPred predictions, suggesting scalability. The predicted SARS-CoV-2 binders differed substantially from training sequences, demonstrating generalization beyond sequence memorization. Together, we establish that heavy chain antibody sequences encode sufficient information for PLMs to infer specificity, offering a scalable framework for antibody discovery with broad applications.

bioinformatics↗

bioRxiv2025

KDM5-driven transcriptional noise fuels plasticity-led awakening and relapse in paediatric cancer

How drug-tolerant persister (DTP) cells escape quiescence to drive tumour relapse is a central unresolved question in cancer evolution. Here, we identify transcriptional noise (TN), defined as the stochastic variability in gene expression, as a latent property of paediatric cancer cells that becomes a driver of adaptive regrowth after treatment withdrawal. Using functional assays, lineage tracing, single-cell transcriptomics, and multiscale landscape modelling, we show that therapy enriches mesenchymal-like tolerant states in neuroblastoma without clonal selection, while post-treatment awakening is a stochastic process fuelled by noise-enabled plasticity in cell-identity programmes. The histone demethylase KDM5A relocates to noisy cell-state genes during awakening, promoting H3K4me3 removal and chromatin remodelling at these loci. KDM5 inhibition abrogates this process, and suppresses transcriptional noise, halts DTP exit, and prevents tumour recovery in both neuroblastoma and hepatoblastoma models. These results establish DTP as an exploitable evolutionary bottleneck, positioning KDM5-mediated transcriptional noise as an actionable therapeutic target to limit cancer adaptation and relapse GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/682004v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@edba70org.highwire.dtl.DTLVardef@16b1bd9org.highwire.dtl.DTLVardef@f153acorg.highwire.dtl.DTLVardef@1ac5a4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Dual Targeting of MAPK Signaling and Senescence-Associated Survival Pathways Overcomes Vincristine Resistance in Fusion-Negative Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with limited therapeutic options for relapsed or metastatic disease. Fusion-negative (FN) RMS, the predominant subtype, frequently harbors mutations activating the RAS/MAPK pathway, yet the mechanisms underlying chemoresistance remain poorly defined. Here, we generated vincristine-resistant FN RMS cell lines through long-term dose escalation and identified increased MAPK signaling activity, evidenced by elevated phosphorylated ERK levels in vitro and in patient tumors post-chemotherapy. MEK inhibition via trametinib or MEK1 knockdown significantly impaired growth of resistant cells and enhanced vincristine efficacy in zebrafish tumor models, reducing tumor burden and relapse. Mechanistically, trametinib induced cell cycle arrest and senescence--marked by p21 upregulation--in resistant cells. This effect was dependent on suppression of MYC, as MYC disruption promoted senescence while MYC overexpression rescued the phenotype. Senescent cells remained partially viable via BCL-XL-mediated survival, rendering them sensitive to the BCL-XL inhibitor, A-1155463. Combined trametinib and A-1155463 significantly reduced growth of resistant RMS cells. Our findings highlight the MEK/MYC/p21 axis and BCL-XL dependency as key vulnerabilities in therapy-resistant FN RMS and support dual targeting of MAPK and senescence-survival pathways as a rational therapeutic strategy. STATEMENT OF SIGNIFICANCETherapeutic resistance remains a major barrier to improving survival outcomes in fusion-negative rhabdomyosarcoma (FN RMS), the most common pediatric soft tissue sarcoma. This study identifies enhanced MAPK signaling and MYC-driven suppression of p21 as key mechanisms by which FN RMS cells evade vincristine-induced senescence. Moreover, we reveal that senescent-resistant cells rely on BCL-XL for survival, uncovering a new vulnerability that can be exploited therapeutically. Dual targeting of the MEK/MYC/p21 axis and BCL-XL- mediated survival pathways significantly suppresses growth of vincristine-resistant FN RMS cells. These findings provide a compelling rationale for combination therapies that simultaneously disrupt oncogenic signaling and senescence escape mechanisms in treatment-refractory FN RMS.

cancer biology↗

bioRxiv2025

In vivo screening reveals cell-intrinsic mediators of solid tumor resistance to CAR T cell-therapy

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematologic cancers, but has shown limited efficacy in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). The cellular and molecular factors that influence CAR T cell therapy response remain largely unknown. By integrating modular in vivo CRISPR screens with single cell genomics and immunocompetent orthotopic models of PDAC, we uncover oxidative and proteotoxic stress pathways as previously unknown tumor-intrinsic modulators of CAR T cell therapy response. Disruption of stress-regulatory genes, particularly Keap1 and Slc33a1, sensitizes PDAC tumors to CAR T cell killing in vivo. Mechanistically, hyperactivation of the Nrf2 pathway by genetic ablation of the Keap1 tumor suppressor or endogenous engineering of a clinically observed Keap1 mutation enhances tumor susceptibility to CAR T cell therapy. Thus, tumor-intrinsic molecular stress phenotypes accompanying malignant tumor progression can induce unexpected cell state-specific vulnerabilities to cell-based immunotherapies. These findings provide a mechanistic molecular foundation to improve the efficacy of CAR T cell therapy in solid malignancies and to better stratify cancer patients by tumor genotype. Statement of significanceCAR T cell therapy remains an unsolved challenge for pancreatic cancer. Factors that influence CAR-T cell therapy response remain largely unknown, in part due to lack of robust and scalable experimental models. By integrating in vivo genetic screens with single cell genomics in an orthotopic, immunocompetent model of pancreatic cancer, we uncover cell-intrinsic stress states as key regulators of CAR T cell response.

cancer biology↗

bioRxiv2025

Type-I interferon priming signal balances anti-bacterial and anti-tumor trained immunity in alveolar macrophages

Tissue-resident macrophages may be trained to confer enhanced response to heterologous re-stimulation, and thus foster versatile trained immunity (TI) against both infections and tumors. However, key priming signals that contribute to such functional versatility in trained macrophages remain not fully understood. Here we show that influenza A virus (IAV) infection in mice induces lasting transcriptional imprints of acute type-I interferon (IFN-I) signaling in lung-resident alveolar macrophages (AMs) that confer balanced anti-bacterial and anti-tumor TI responses. In both IAV-infected mice and an ex vivo cytokine-mediated training system, IFN-I signaling is found to be critical to developing anti-tumor TI functions, although it limits anti-bacterial TI functions in AMs. Moreover, human AMs carrying transcriptional imprints of IFN-I signaling are associated with anti-tumor immunity and immune activation in lung cancer tissues. Our findings highlight IFN-I as a key priming signal required to developing versatile TI functions in AMs with balanced anti-bacterial and anti-tumor potential.

immunology↗

bioRxiv2025

β-Glucan Modulates Monocyte Plasticity and Differentiation Capacity to Mitigate DSS-Induced Colitis

Trained immunity involves the reprogramming of innate immune cells after an initial exposure, resulting in heightened inflammatory responses to subsequent stimuli and enhanced bactericidal capacity during infection. However, this proinflammatory state could also exacerbate chronic conditions like inflammatory bowel disease (IBD), which is characterized by persistent inflammation and microbial imbalance. It remains unclear how trained immunity influences IBD pathogenesis and whether it can be harnessed therapeutically. In our study, pretreatment with {beta}-glucan reprogrammed bone marrow hematopoietic progenitors and peripheral monocytes, inducing a profound shift in monocyte plasticity and significantly reducing the severity of dextran sulfate sodium (DSS)-induced colitis. Adoptive transfer of bone marrow or peripheral monocytes from {beta}-glucan-trained mice into naive mice conferred robust protection against colitis, demonstrating that this protective effect is transferable. Trained mice also displayed improved clearance of intestinal bacterial infections. Single-cell RNA sequencing revealed an expansion of reparative Cx3cr1 macrophages derived from Ly6Chi monocytes, correlating with accelerated colonic epithelial regeneration. Collectively, these findings reveal how {beta}-glucan-induced trained immunity modulates monocyte differentiation to ameliorate experimental colitis, highlighting the potential of harnessing trained immunity as a therapeutic strategy to recalibrate innate immune responses and restore gut homeostasis in IBD, shedding light for future clinical applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/653036v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1b969c2org.highwire.dtl.DTLVardef@1ce603dorg.highwire.dtl.DTLVardef@10582forg.highwire.dtl.DTLVardef@10d7de6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Self-Motion Perception Influences Postural Sway More than Environmental Motion Perception

Motion of the visual field can alter postural sway and cause illusions of self-motion. The relative perceptual sensitivity of self-motion versus visual field motion induced by virtual reality (VR) stimulation and whether observable sway differs based on perceptual task is unknown. Methods to quantify sway perception while concurrently measuring sway do not exist. We measured head sway and motion perception (self or world) in healthy adults who stood with feet together wearing a VR headset while experiencing adaptive staircases of virtual sinusoidal pitch rotation about the ankle axis. In separate conditions of randomly ordered blocks, subjects were asked to indicate (yes/no) if the room moved (regardless of perceived postural sway) or if their postural sway increased (regardless of perceived room motion). Head sway area was measured by tracking movement of the VR headset. Yes/No responses were fit with psychometric curves to determine points of subjective equality (PSEs) for room motion and postural sway. PSEs were compared between conditions. Effects of motion perception (binary responses) on head sway area before, during, and after visual stimulation were examined. The mean PSE for room motion (0.42 degrees) was significantly lower than for postural sway (2.02 degrees) [t(1,18) = 4.4714, p = 0.00029]. Head sway area was significantly larger during (z = 11.53, p < 0.001) and after (z = 5.09, p < 0.001) visual stimulation only when participants perceived increased postural sway. Nearly 5-fold greater amplitudes of oscillating VR visual stimuli were required to induce perceptions of altered self-versus visual field motion. Observed head sway during visual motion was only linked to perceptual responses when participants focused internally on self-motion, not externally on room motion.

neuroscience↗

bioRxiv2025

Strain-level metagenomic profiling using pangenome graphs with PanTax

Microbes are omnipresent, thriving in a range of habitats, from oceans to soils, and even within our gastrointestinal tracts. They play a vital role in maintaining ecological equilibrium and promoting the health of their hosts. Consequently, understanding the diversity in terms of strains in microbial communities is crucial, as variations between strains can lead to different phenotypic expressions or diverse biological functions. However, current methods for taxonomic classification from metagenomic sequencing data have several limitations, including their reliance solely on species resolution, support for either short or long reads, or their confinement to a given single species. Most notably, most existing strain-level taxonomic classifiers rely on the sequence representation of multiple linear reference genomes, which fails to capture the sequence correlations among these genomes, potentially introducing ambiguity and biases in metagenomic profiling. Here, we present PanTax, a pangenome graph-based taxonomic profiler that overcomes the short-comings of sequence-based approaches, because pangenome graphs possess the capability to depict the full range of genetic variability present across multiple evolutionarily or environmentally related genomes. PanTax provides a comprehensive solution to taxonomic classification for strain resolution, compatibility with both short and long reads, and compatibility with single or multiple species. Extensive benchmarking results demonstrate that PanTax drastically outperforms state-of-the-art approaches, primarily evidenced by its significantly higher precision or recall at strain level, while maintaining comparable or better performance in other aspects across various datasets. PanTax is an open-source user-friendly tool that is publicly available at https://github.com/LuoGroup2023/PanTax.

bioinformatics↗

bioRxiv2025

Segmental Isotope Labelling of the Prion Protein: Identification of a Key Residue for Copper-Mediated Interdomain Structure

The cellular prion protein is composed of two domains: a disordered N-terminal toxic effector domain and a three-helix C-terminal regulatory domain. Copper is thought to form a bridge between these two domains, inhibiting the proteins inherent neurotoxicity. However, the molecular details of how copper interacts with the C-terminal regulatory surface are unclear. To assess the potential role of conserved C-terminal His residues in copper coordination, we applied sortase-mediated ligation to create an expressed, murine prion protein with segmental 15N-labeling of the N-terminal domain. Pulsed EPR methods applied to a 1:1 protein:copper complex revealed both 14N and 15N couplings, consistent with simultaneous coordination of the two proteins domains to the copper center. Mutagenesis studies localized C-terminal copper coordination to His176, present on the second -helix. The cumulative EPR results reveal a copper coordination environment composed of three His residues from the proteins N-terminal domain, along with His176. The feasibility of these findings was tested with AlphaFold 3 simulations. These results further refine the molecular details of the prion proteins autoregulation, emphasizing the critical role of its copper cofactor. Moreover, this interdisciplinary work demonstrates how sortase-mediated ligation combined with pulsed EPR sensitive to distinct nuclear spin systems provides a new strategy for assessing metal ion binding to proteins.

biophysics↗