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Biology subjects

He, X.

Publications and source records attributed to He, X..

At least 19 recordsLinked to original sources

Acetylation of NDUFA13 at K7 Senses OXPHOS Dysfunction to Disrupt Prohibitin Scaffolds and Drive Cristae Remodelling in Heart Failure

Mitochondrial cristae disintegration is a hallmark of heart failure, yet how oxidative phosphorylation (OXPHOS) dysfunction triggers this structural collapse remains unclear. Here we identify acetylation of the complex I subunit NDUFA13 at lysine 7 (K7) as a metabolic sensor coupling OXPHOS dysfunction to cristae remodelling. OXPHOS inhibition drove NDUFA13-K7 hyperacetylation, inducing conformational compaction and translocation to the intermembrane space. Acetylated NDUFA13 competitively engaged PHB2 through its coiled-coil domain, displacing PHB1 and disassembling prohibitin scaffolds. The acetylation-mimetic NDUFA13-K7Q induced cristae fragmentation in vitro, and cardiomyocyte-specific K7Q knock-in mice showed accelerated mortality and cristae loss under pressure overload, accompanied by impaired TCA cycle flux, defective fatty acid oxidation and selective depletion of polyunsaturated cardiolipins. These findings establish NDUFA13-K7 acetylation as a reversible switch converting bioenergetic failure into pathological cristae remodelling and metabolic reprogramming, highlighting therapeutic potential for targeted deacetylation.

cell biology↗

The human hippocampus retroactively binds experiences across time

Creating memories requires linking events across seconds and minutes, but canonical mechanisms of associative plasticity operate over much shorter timescales. This poses a fundamental problem: how can the brain bind information that arrives at different times? Computational model and animal studies implicate the hippocampus in associating events across time, but when the hippocampus becomes engaged and how it interacts with representations of preceding events are unknown. Here we show, using intracranial recordings in humans forming image-word associations across a ~2-s temporal gap, that hippocampal high-frequency activity predicts later memory when the second event arrives, but not during the initial image or intervening delay. At this moment, neurons tuned to the preceding image reinstate stimulus-specific firing despite its sensory absence. Across trials, hippocampal high-frequency activity was selectively coupled to this reinstatement during successful encoding and increased before spiking of image-tuned neurons. These findings reveal a retroactive mode of hippocampal binding, whereby representations of preceding events are recruited when new information arrives. This mechanism provides a neural solution for binding experiences across the temporal discontinuities that characterize everyday life.

neuroscience↗

RAPPL-Seq Enables Rapid, High-Sensitivity Ribosome Profiling of Scarce Samples

Ribosome profiling is a powerful method for transcriptome-wide monitoring of translation activity in vivo, but its use is limited by lengthy protocols, reliance on specialized equipment and inefficient recovery of ribosome-protected fragments (RPFs). To overcome these challenges, we developed RAPPL-Seq, a magnetic bead-based affinity capture method that enables fast and efficient RPF isolation. RAPPL-Seq achieves high recovery efficiency, short processing time, and robust performance with limited input material. It is broadly applicable across multiple sample types, including cell lines, primary tissues, and human biospecimens. RAPPL-Seq can also be paired with translation inhibitors to characterize inhibitor- and tissue-specific mRNA translation. RAPPL-Seq efficiently captured translatomes of glioblastoma (GBM) and adjacent non-tumor brain tissues from patient biopsies. Together, these findings establish RAPPL-Seq as a straightforward, easily accessible, and highly sensitive approach that extends translatome studies to scarce experimental and clinical samples, opening new avenues for mechanistic insight and therapeutic discovery.

molecular biology↗

A domesticated prophage enzyme links bacterial metabolism, episymbiosis and phage fitness

Prophages are increasingly recognized as drivers of bacterial adaptation, yet how domesticated prophage genes shape microbial symbiosis remains poorly understood. Here, we identify GDPDXhp1, a glycerophosphodiester phosphodiesterase encoded by prophage Xhp1 of Schaalia odontolytica XH001, as a metabolic regulator linking bacterial physiology, episymbiosis and phage fitness. Association with the epibiotic bacterium Nanosynbacter lyticus TM7x strongly induced gdpdXhp1 expression and triggered GDPDXhp1-dependent lipid droplet accumulation in XH001. GDPDXhp1 orchestrated glycerophospholipid remodeling, membrane biophysical properties, respiration and cell-surface glycan composition in XH001, thereby promoting TM7x episymbiosis. Remarkably, GDPDXhp1 was also required for productive Xhp1 infection by remodeling susceptible bacterial host envelope structures to facilitate phage-host interactions. Together, our findings reveal a domesticated prophage enzyme that contributes to bacterial metabolism, interspecies symbiosis and viral reproduction, highlighting prophage as an overlooked source of metabolic innovation in microbial symbiosis.

microbiology↗

In situ identification of pre-hematopoietic stem cells and their niche

Definitive hematopoietic stem cells (HSCs) emerge predominantly along the ventral surface of the dorsal aorta in the embryonic aorta-gonad-mesonephros (AGM) region, yet the spatial organization of the niche underlying this developmental asymmetry remains poorly understood. Defining the native niche of pre-HSCs has also been hindered by their rarity, transient nature and lack of a single definitive marker for their identification in situ. Here, we integrated single-cell and spatial transcriptomics to construct a spatiotemporal atlas of the niche in the AGM. Neural cells and chondrogenic mesenchyme predominated dorsally, whereas N-cadherin mesenchymal stromal cells (N-cad+ MSCs) formed a ventral layer between the aortic endothelium and mesonephric cells. BMP and SHH signaling showed overall dorsal enrichment, whereas WNT, NOTCH and KIT signaling were enriched ventrally, with these cellular and signaling patterns dynamically remodeled across HSC ontogeny. Imaging-based spatial transcriptomics validated the findings and further enabled the identification of individual pre-HSCs in situ and revealed a multicellular niche comprising endothelial cells, N-cad+ MSCs, intra-aortic hematopoietic cluster cells and macrophages. TGF{beta} and NOTCH signals were primarily supplied locally, whereas major sources of BMP and WNT were more distant. Functional perturbation identified niche-derived CD200 as an immune-regulatory signal promoting HSC development, validating a spatially predicted niche interaction. Together, our findings link tissue-scale spatial organization with the local multicellular niche supporting the preferential development of HSCs along the ventral aorta.

developmental biology↗

Host Cell Wall Polysaccharides Modulate Nanosynbacteria Recognition and Attachment

Ultrasmall episymbiotic bacteria grow on the surface of their obligate bacterial hosts. These episymbionts have a restricted host range and likely rely on host-derived receptor molecules to identify and associate with cognate hosts. Nanosynbacteria from the oral microbiome, like Nanosynbacter lyticus, utilize a pair of distinct type 4 pili (T4P) for host surface interactions, T4P-1 and T4P-2. In this study we identify the adhesin components of both T4P, demonstrate that these adhesins localize to the cell wall of the cognate host Actinobacterium Schaalia odontolytica, and demonstrate that each adhesin binds distinct components of the host cell wall. The adhesin from the motility driving T4P-1, termed PilX1, showed a wide range of affinities in microarray screening and bound peptidoglycan directly, but could not bind purified cell wall polysaccharides (CWPS) from host surfaces. Conversely, the adhesin from T4P-2, termed PilX2, bound host-derived CWPS and preferentially bound episymbiont-naive hosts over episymbiont-exposed hosts. Within the bacterial host we identified two CWPS biosynthesis pathways that altered adhesin binding and episymbiont susceptibility. Disruption of a rhamnose-CWPS pathway left hosts susceptible to episymbiont binding and disruption of a fucose remodeling pathway made hosts more resistant to episymbiont binding. Analyzing polysaccharide compositions revealed multiple CWPS, potentially including a rhamnose- and mannose-rich rhamnomannan. Consistent with this finding, exogenously added rhamnose and mannose were able to block binding by N. lyticus, supporting a role for these sugars in episymbiont defense. Our study reveals that Nanosynbacteria rely on the complex sugars that coat their host bacteria for recognition and attachment.

microbiology↗

EnsPlex: Integrative Protein Complex Prediction through Multi-source Complementary Structural Sampling and Topology-Aware Candidate Selection

Protein complex structure prediction depends on both the breadth of candidate coverage and the ability to select accurate models within a limited output budget. Most existing approaches emphasize only one stage. End-to-end models and molecular docking workflows generate candidate structures, whereas quality-assessment models primarily rerank a predefined candidate pool. A complete strategy must account for complementary sampling across generators, differences among scoring scales and the allocation of final candidate quotas. Here, EnsPlex is presented as a multi-source framework that couples structural sampling with candidate selection for protein-protein interaction complex prediction. EnsPlex combines conformation-expanded docking, AlphaFold-Multimer, AlphaFold3 and Boltz-1 to expand the sampled conformational space. Conformation-expanded docking comprises monomer conformational expansion followed by flexible HADDOCK docking. FACET is trained to predict candidate quality using DockQ and its component metrics as supervision. Across 102 antigen-antibody systems, EnsPlex achieved up to a 27.8% relative improvement in target success rate over AlphaFold3 with its built-in ranking under matched output budgets. FACET also improved within-source ranking in the internal candidate pools and in homology-filtered external data. These findings support the utility of EnsPlex for structure prediction in the evaluated antigen-antibody systems.

bioinformatics↗

β-arrestin1 directly engages Gαs to sustain endosomal GPCR signaling

{beta}-arrestins ({beta}arrs) are canonical terminators of G protein-coupled receptor (GPCR) signaling, yet internalized receptors paradoxically sustain {beta}arr-dependent Gs/cAMP signaling from endosomes. How a desensitizing scaffold can instead promote G protein activation has remained unresolved, because the receptor-{beta}arr-G protein assemblies described so far position the two transducers on opposite faces of the receptor, with no contact between them. Using the parathyroid hormone type 1 receptor (PTH1R), we show that {beta}arr1 binds Gs directly, engages its nucleotide-free state, and does so far more strongly than it engages G{beta}{gamma}. A cryo-EM structure of the agonist-bound PTH1R-{beta}arr1-Gs complex at ~3.1 &Aring resolution reveals how: {beta}arr1 anchors to the phosphorylated receptor C-tail, leaving the transmembrane core free for Gs, and reorients to build a direct, three-node interface in which its finger, C- and back loops clamp the Switch I, Switch II and 3/{beta}5 regions of Gs. Disrupting either side of this interface abolishes complex assembly and selectively collapses the internalization-dependent phase of cAMP production, and the same interface governs {beta}arr1-Gs coupling at the glucagon-like peptide-1 receptor. {beta}-arrestin1 therefore acts as a positive regulator that holds Gs in a signaling-competent state, providing a unifying structural basis for sustained endosomal GPCR signaling.

biochemistry↗

A lactate-HIF1-VDR positive feedback loop drives protective SPP1+ macrophage differentiation to inhibit schistosomiasis induced liver fibrosis

Hepatic fibrosis remains a major cause of morbidity and mortality in patients with schistosomiasis without effective therapy. Here we show that vitamin D receptor (VDR) signaling in macrophages is essential for limiting fibrosis, acting through a previously unrecognized metabolic epigenetic circuit. Hepatic macrophages exhibit the highest VDR expression among liver resident cells, and pharmacological VDR activation with paricalcitol selectively expands a protective SPP1+ macrophage subset derived from circulating monocytes. Myeloid specific VDR knockout exacerbates fibrosis and abrogates paricalcitol's hepatoprotective effects, whereas SPP1+ macrophage depletion worsens disease. Mechanistically, VDR activation synergizes with hypoxia and lactate to drive SPP1 expression via glycolytic reprogramming. Moreover, lactate and HIF1alpha cooperatively induce VDR transcription by binding to the Vdr promoter and enhancing histone lactylation, forming a positive feedback loop that amplifies the antifibrotic response. Our findings establish the lactate-HIF1alpha-VDR-SPP1 axis as an endogenous defense mechanism and identify macrophage VDR as a promising therapeutic target for fibrotic liver diseases.

immunology↗

The RNA modification N6-methyladenosine restrains SIRPα to license myeloid efferocytosis in atherosclerosis

Background: Macrophages participate in atherosclerotic plaque development, progression and resolution, integrating lipid and inflammatory cues to balance tissue damage and repair. Despite considerable understanding of macrophage function in atherosclerosis, the underlying regulatory mechanisms governing these responses continue to evolve. Emerging evidence implicates epitranscriptomic gene regulation by the RNA modification N6-methyladenosine (m6A) in control of context specific immune responses, but its role in atherosclerosis is not well-defined. Methods: We integrated m6A quantitative trait loci (m6A QTLs) with Coronary Artery Disease (CAD) GWAS summary statistics. Using Oxford Nanopore direct RNA sequencing and MeRIP-seq, we constructed a high-resolution map of m6A modifications in macrophages. To determine the functional consequences of myeloid m6A loss in-vivo, we generated a myeloid-specific loss-of-function mouse model of m6A (Mettl14f/f LysMCre) and performed atherosclerosis studies using bone marrow transplantation into irradiated Ldlr-/- mice. Atherosclerosis lesions were analyzed using light microscopy and immunofluorescence imaging, with complementary in-vitro mechanistic studies performed in murine bone marrow derived macrophages (BMDMs). Results: m6A quantitative trait loci (m6A-QTLs) were enriched in CAD GWAS loci relevant to immune and lipid function, implicating m6A regulation in human CAD genetics. Using Oxford Nanopore direct RNA sequencing in conjunction with public MeRIP-sequencing data, we generated a single-base resolution, stoichiometric map of m6A modification in macrophages, nominating genes central to macrophage biology in atherosclerosis as high-confidence m6A targets. Myeloid specific deletion of the m6A "writer" complex subunit Mettl14 drove the development of larger, more necrotic and unstable atherosclerotic plaques on an Ldlr-/- background compared with Mettl14f/f controls, establishing an atheroprotective role for myeloid m6A. Loss of myeloid Mettl14 impaired macrophage efferocytosis in atherosclerotic plaques, in a dexamethasone-induced apoptosis in the thymus and in-vitro. Molecularly, m6A methylation of the "don't eat me" antiphagocytic receptor SIRP RNA suppressed its protein expression. Conclusions: These findings identify the myeloid m6A - SIRP axis as a novel homeostatic regulator of efferocytosis and establish epitranscriptomic remodeling as a crucial layer of macrophage regulation in atherosclerosis.

physiology↗

Glutamine-regulated VCP acetylation creates a proteostasis vulnerability in RB1-deficient tumor

RB1 deficiency defines an aggressive tumor state with limited therapeutic options. Osteosarcoma represents a clinically relevant model of RB1 deficient malignancy, as RB1 alterations are among the most frequent genomic events in this tumor type. However, the non cell cycle vulnerabilities created by RB1 loss remain incompletely understood. We therefore used osteosarcoma to identify therapeutic dependencies associated with RB1 deficiency. Through high-throughput compound screening, we identified a selective vulnerability of RB1-deficient tumor cells to VCP inhibition. NMS-873 and other VCP-targeting compounds preferentially suppressed RB1 deficient cells across two-dimensional cultures, three-dimensional spheroids, patient-derived organoids, and in vivo tumor models. Mechanistically, VCP inhibition exacerbated proteostasis stress and activated endoplasmic reticulum stress responses in RB1-deficient cells. We further found that VCP function was regulated by glutamine-dependent acetylation, with lysine 615 serving as a dominant acetylation site that modulated sensitivity to VCP inhibition. Glutamine restriction phenocopied, whereas glutamine supplementation partially rescued, the effects of NMS-873 on VCP acetylation and tumor cell growth. Clinical and transcriptomic analyses further linked VCP expression and protein-folding stress programs to aggressive disease features. Together, these findings identify glutamine regulated VCP acetylation as a metabolic proteostasis dependency in RB1-deficient tumors and nominate VCP inhibition as a therapeutic strategy for this difficult-to-target tumor state.

cell biology↗

Hypomineralized Enamel Alters Trigeminal Sensory Afferent Architecture, Transcriptome, and Dental Injury Responses

At barrier tissues such as the skin and gut, sensory neurons interface with environmental stimuli and coordinate with neighboring epithelial and immune cells to detect tissue perturbations. In teeth, however, the sensory dentin-pulp complex is insulated from the oral environment by highly mineralized enamel. Although dentin-pulp responses to severe injury have been studied in models with direct pulp exposure, it remains unclear whether enamel barrier dysfunction alone alters pulpal and neuronal homeostasis. Using a kallikrein-related peptidase 4 knockout (KLK4 KO) mouse model of enamel hypomineralization, we demonstrated that defective enamel induced structural, molecular, and transcriptional responses in the dental pulp and in the trigeminal system innervating teeth, despite the absence of direct pulp exposure to the oral cavity. Hypomineralized molars exhibited increased reactionary dentin formation accompanied by retraction of sensory afferents from the dentin-pulp junction. Consistent with these structural findings, trigeminal ganglia of KLK4 KO mice displayed upregulation of genes associated with cytoskeletal remodeling and stimulus response pathways. Despite increased bacterial burden and biofilm accumulation on the enamel surface, enamel hypomineralization did not induce substantial innate immune responses in the pulp. In addition, following severe dental pulp injury, teeth with hypomineralized enamel exhibited reduced sensory afferent loss and tissue damage. Together, these findings reveal that enamel integrity functions as a critical regulator of dentin-pulp homeostasis and that barrier dysfunction alone can precondition tissue responses to subsequent injury.

neuroscience↗

OmniSyn unifies target-aware molecular generation and optimization within a synthesis-native LLM framework across the human proteome

Designing target-specific bioactive molecules with actionable synthesis routes for the human proteome holds enormous potential for expanding therapeutic discovery, but remains a challenge. Existing target-aware generative models often depend on protein structures and generate molecules before assessing synthetic feasibility. Here we present OmniSyn, a protein-sequence-conditioned Mixture-of-Experts (MoE) language model that couples a task-conditioned interaction module with a synthesis-action decoder to generate molecules with explicit synthesis traces, thereby unifying de novo ligand generation, synthesizability projection and hit-to-lead (H2L) optimization within synthesis-traceable chemical space. OmniSyn is pre-trained with self-distillation and post-trained with task-specific reinforcement learning (RL) to adapt expert routing and optimize molecular properties across design modes. On unseen protein targets from MolGenBench, a real-world drug-discovery benchmark, OmniSyn achieves state-of-the-art performance across de novo design and H2L optimization, including target-awareness and hit-rediscovery metrics, despite relying only on protein sequences rather than three-dimensional (3D) pocket structures. By embedding synthesis planning into the design process, OmniSyn shifts molecular generation from a generate-then-filter paradigm toward design-with-synthesis paradigm, transforming virtual predictions into experimentally actionable candidates. Independent AiZynthFinder evaluation yielded retrosynthetic success rates of 68.47% for de novo generation and 71.92% for H2L optimization, improving over the strongest baselines by 61.3% and 184.2%, respectively, and supporting the synthetic feasibility of OmniSyn-generated molecules. In synthesizability projection, OmniSyn further converts outputs from external generative models into close analogues with improved retrosynthetic feasibility while preserving molecular similarity. Having established strong benchmark performance and external retrosynthetic feasibility, we next applied OmniSyn at human-proteome scale, spanning more than 21,000 targets. Rapid sequence-conditioned sampling enabled the construction of, to our knowledge, the largest human-proteome-scale generative virtual library, comprising 2.7 billion target-specific molecules, each accompanied by model-derived synthesis traces and target-specific prioritization scores. By enabling scalable target-specific molecular design with synthesis-aware generation across the human proteome, OmniSyn opens new opportunities for exploring previously inaccessible therapeutic targets, including those lacking experimentally resolved structures.

bioinformatics↗

A multi-tissue epigenomic atlas links the sheep non-coding genome to domestication and complex traits

Non-coding regulatory variation drives complex traits, domestication, and evolutionary adaptation, yet the sheep genome lacks high-resolution functional annotation. Here we present SheepEpimap, a multi-tissue regulatory atlas harmonizing 516 CUT&Tag histone modifications, ATAC-seq, and RNA-seq datasets across 43 adult tissues in sheep. We annotated 2.93 million cis-regulatory elements, yielding 557,441 enhancer-gene pairs and 145,407 variants with allele-specific effects. By training a sequence-to-function deep-learning model, we decoded the base-pair syntax of chromatin accessibility, annotated transcription factor motif instances genome-wide, and constructed 12,210 tissue-specific gene regulatory networks (GRNs). Integrating this resource with multi-tissue expression quantitative trait loci, selection sweeps, and genome-wide association studies prioritized non-coding variants driving domestication and complex traits. Finally, cross-species analysis revealed that sequence-conserved, tissue-matched enhancers were significantly enriched in the heritability of complex traits and diseases in humans. In summary, SheepEpimap (https://genome.ucsc.edu/s/mengzhu/SheepEpimap) provides an open-access foundational ecosystem for sheep functional genomics, precision breeding, and comparative biology.

genetics↗

Imaging guided single-cell multiomics unveils shared autoreactive CD4+ T-cell responses in blood, locoregional lymph node and affected tissues of patients with systemic autoimmunity

Systemic autoimmune connective tissue diseases (CTDs) are characterized by anti-nuclear antibodies, shared HLA-associated genetic risk, and frequent disease overlap, suggesting a central role for CD4+ T cells in pathogenesis. However, defining disease-driving CD4+ T-cell responses remains challenging due to their localization within lymphoid and affected tissues and the lack of approaches linking these responses to circulating counterparts. We combined [18F]-labeled thymidine PET/CT-guided tissue sampling, ex vivo antigen stimulation, and single-cell multiomics to characterize CD4+ T-cell responses in blood, PET-avid locoregional lymph nodes (LNs), and disease-affected tissues from patients with the immunologically distinct CTDs systemic sclerosis and Sjogren's disease. PET-avid LNs from both diseases exhibited enhanced adaptive immune activity and contained an expanded population of interferon-stimulated gene (ISG)-expressing TRAIL+ CD4+ T cells. In Sjogren's disease, active LNs and affected tissues harbored diverse effector CD4+ T-cell populations, including follicular and peripheral helper T cells and Th2/Th17 cells. In contrast, systemic sclerosis tissues lacked effector CD4+ T cells, while active LNs were enriched for naive, regulatory, and TRAIL+ ISG CD4+ T cells. Antigen stimulation of peripheral blood mononuclear cells enriched for expanded effector CD4+ T-cell populations that shared activation profiles and clonal relationships with cells in LNs and affected tissues, many representing autoreactive antigen-specific T cells. TRAIL+ CD4+ T cells suppressed effector T-cell differentiation, autoreactive plasma cell generation, and autoantibody production in vitro, identifying a previously unrecognized immunoregulatory population. Together, this workflow enables comprehensive characterization of pathogenic and regulatory CD4+ T-cell responses across CTDs.

immunology↗

PASTRI: Resolving Stage-Specific Cell-State Dynamics from Annotated Cell Lineage Trees

Cellular transitions between phenotypic states are fundamental to development and disease, yet quantitative analysis of their dynamics remains challenging. Here we present PASTRI (Phylogenetic Adjacency-based State Transition Rate Inference), a computational framework that infers transition rates from cell lineages/phylogenies annotated with terminal phenotypic states, such as single-cell transcriptomes. We validate PASTRI using simulated lineages and the Caenorhabditis elegans embryonic lineage. Importantly, by leveraging cell pairs at varying phylogenetic distances, PASTRI accurately resolves stage-specific transition rates, circumventing the issue of developmental changes in dynamics. Applied to three cell phylogeny datasets from our lineage-tracing experiments spanning diverse developmental/disease models and tracing systems, PASTRI uncovers rate-limiting steps in the activation of hepatic stellate cells and the differentiation of primordial lung progenitors, as well as attractor states that support cancer cell proliferation. PASTRI thus opens up a venue for dissecting cell state transition dynamics from annotated cell lineage/phylogeny.

bioinformatics↗

Spatial cholesterol homeostasis gatekeeps T-cell development and activation by orchestrating signaling and fitness

Cholesterol is essential for T-cell immunity, and its spatial distribution is tightly regulated. Although cholesterol is synthesized in the endoplasmic reticulum (ER), it is predominantly transported to the plasma membrane (PM); however, the machinery mediating this anterograde transport in T cells remains unknown. Here, through a functional genetic screen, we identify oxysterol-binding protein (OSBP) as the principal mediator of ER-to-PM cholesterol transport in T cells. OSBP deficiency depletes accessible PM cholesterol while causing cholesterol accumulation in the ER, resulting in impaired T-cell receptor (TCR) signaling and disruption of ER homeostasis. Using stage-specific conditional knockout mice, we demonstrate that OSBP is required at multiple developmental checkpoints in the thymus, including {beta}-selection, positive selection, and post-selection maturation. Loss of OSBP during early thymocyte development causes a near-complete block in T-cell development, resulting in a profound absence of mature peripheral T cells. In mature T cells, activation markedly increases dependence on OSBP-mediated cholesterol transport, with its inhibition causing ER perturbation and extensive cell death. Finally, we show that disease-associated oxysterols disrupt OSBP-mediated cholesterol transport, leading to T-cell dysfunction and providing a mechanistic explanation for impaired T-cell immunity in pathological settings. Together, our findings identify OSBP as a central regulator of intracellular cholesterol transport that couples membrane cholesterol homeostasis to TCR signaling and ER integrity. These results establish the spatial distribution of cholesterol, rather than its abundance alone, as a fundamental metabolic determinant of thymocyte development and peripheral T-cell function.

immunology↗

HMCES DNA-protein cross-links promote template slippage during DNA replication

During replication, nucleolytic processing of apurinic/apyrimidinic (AP) sites in single-stranded (ss)DNA is attenuated by the evolutionarily conserved 5-hydroxymethylcytosine binding, embryonic-specific (HMCES) protein. HMCES forms a covalent thiazolidine linkage with the ring-opened aldehyde form of a ssDNA AP site to stabilize the AP site and suppress the formation of DNA double-strand breaks. The resulting HMCES DNA-protein cross-link (DPC) can then be digested by the SPRTN protease and bypassed through mutagenic translesion synthesis (TLS). Here, we use Xenopus egg extracts and molecular dynamics simulations to investigate how HMCES-DPC formation influences the mutagenicity of AP site bypass. We show that SPRTN processes the HMCES-DPC to a five amino acid peptide adduct prior to TLS. Surprisingly, the mutagenicity of HMCES-DPC bypass is insensitive to the extent of DPC proteolysis and depends only on cross-link formation, which traps the AP site in a more dynamic ring-opened configuration. We further show that the spectrum of mutations produced during bypass of HMCES-adducts strongly depends on the template strand nucleotide immediately 5 of the AP site. Our data support a model in which HMCES-DPC formation increases the conformational flexibility of the DNA template, allowing template slippage and use of the 5 template nucleotide to direct insertion opposite the adducted AP site.

biochemistry↗