Biology subjects

Yang, Y.

Publications and source records attributed to Yang, Y..

At least 289 records · Page 16Linked to original sources

bioRxiv2025

Artemisinin-Conjugated Phototheranostic Probe for Golgi-Targeted H2O2 Detection and Side Effect Attenuation

Understanding the mechanism by which Artemisinin conjugated with phototheranostic probes for reducing side effects (AC-Pr-RSE) will guide endeavor to improve the therapeutic outcomes. Herein, the first report describes the application of a cytotoxic artemisinin derivative and N-methyliminodiacetic acid (MIDA) boronates conjugated with a naphthalimide fluorophore for the detection of H2O2 in the Golgi apparatus. A comparative analysis of the localization signals from the fluorescent artemisinin derivative and organelle-specific dyes uncovered that the Golgi apparatus functions as the primary site of its accumulation, which could efficiently enhance the intracellular reactive oxygen species (ROS) level and induce cell apoptosis. This work highlights the potential of AC-Pr-RSE study, which enables us to optimize the design of theranostic probes to improve their biological activities during oxidative stress.

biochemistry↗

bioRxiv2025

FCHo2, instead of talin, enables inside-out activation of integrin αvβ5 in curved adhesions

Inside-out activation of integrins is crucial for transducing mechanical forces through the extracellular ligand-integrin-talin-F-actin axis. Extensive studies have shown that talin is the essential player in this process by binding to the intracellular tail of {beta} integrins. Here, we show that, while talin binding is essential for inside-out integrin activation in focal adhesions, it is dispensable in curved adhesions - a distinct adhesion architecture that is exclusively mediated by integrin v{beta}5 and selectively formed at curved membranes. Instead, a curvature-sensing protein FCHo2 binds to the HDRRE motif in the cytoplasmic tail of integrin {beta}5 (ITG{beta}5) and inside-out activates integrin v{beta}5 in curved adhesions. Intriguingly, FCHo2 does not bind to a similar motif in the homologous integrin {beta}3 tail. Through truncations and mutations, we identified a pivotal tryptophan (W) in the {beta}3 tail, which is conserved in all homologous integrin {beta} isoforms except {beta}5, where it is replaced by a tyrosine (Y766). This tyrosine substitution is crucial for integrin {beta}5s unique capability in forming curved adhesions. A Y766W mutation abolishes integrin {beta}5s capacity to form curved adhesions, without affecting its ability to form focal adhesions. Furthermore, our studies suggest that the phosphorylation state of Y766 regulates whether integrin v{beta}5 forms curved adhesions or focal adhesions, providing a cellular mechanism governing different adhesion types. Overall, our work unveils distinct molecular interactions and regulatory mechanisms between curved adhesions and focal adhesions, and establishes a molecular basis for the formation of curved adhesions by integrin v{beta}5. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/691229v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@eb4addorg.highwire.dtl.DTLVardef@1802a01org.highwire.dtl.DTLVardef@145e805org.highwire.dtl.DTLVardef@83d641_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

An Lrs14 family protein functions as a nucleoid-associated protein regulating cell cycle progression in Sulfolobales

Archaea of the order Sulfolobales possess eukaryotic-like cell cycle. The chromatin organization in these archaea is supposed to rely on nucleoid-associated proteins (NAPs). Genomes of Sulfolobales species encode multiple members of the Lrs14 protein family, which have been postulated to function as NAPs. Here, we show that one of the Lrs14 family protein, named Sul14a, is a potential key player in dynamic chromatin organization in Saccharolobus islandicus. Sul14a is transcribed in a cell cycle-dependent manner and appears to be essential for cell viability. The protein exhibits DNA-binding activity preferring AT-rich sequences and can bridge DNA in vitro. ChIP-seq analysis revealed that Sul14a is enriched at AT-rich regions of the chromatin. Overexpression of Sul14a leads to cell cycle arrest at G2 phase. High-throughput chromosome conformation capture (Hi-C) and transcriptomic analyses indicate that Sul14a overexpression results in chromatin contact reduction and global transcriptional changes. In addition, we observed that the chromatin contact pattern changes markedly between 1h and 5h after release from G2 synchronization, corresponding to the lowest and highest levels of sul14a transcription, respectively, during the cell cycle. Our results establish that Sul14a is an NAP intimately associated with chromatin dynamics and global transcriptional changes during the cell cycle in Sulfolobales.

microbiology↗

bioRxiv2025

PKU tags, novel genetically encoded shape tags for cell labeling in light and electron microscopy

Distinguishing among neuronal cell types is crucial for deciphering complex neural networks and brain functions. However, the current repertoire of cell-labeling tools compatible with light microscopy (LM) and/or electron microscopy (EM) is limited compared to the vast number of cell types in the brain. Here, we introduce PKU (polymer king-size unit) tags, genetically encoded "shape tags" that leverage the polymerization of self-assembling proteins, spectrally distinct fluorescent proteins and, optionally, a nuclear targeting sequence to generate a series of multi-shaped (spherical or filamentous), multi-colored (blue, green, red, near-infrared) and multi-localized (cytosolic or nuclear) tags. By co-expressing multiple PKU tags within the same cell, a combinatorial strategy further expands the repertoire, which can theoretically yield hundreds of unique labeling patterns. Expressing PKU tags in vivo provides multi-cell-type labeling and neuronal circuit tracing, without altering the animals behavior or transcriptomic profiles. Moreover, when fused to the peroxidase APEX2, PKU tags maintain their shape-specific features, providing shape "barcoding" using EM. Thus, PKU tags represent a versatile and efficient toolkit for studying connectomics using both LM and EM.

neuroscience↗

bioRxiv2025

Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

Dendritic cells (DCs) release extracellular vesicles (DEVs) that amplify antigen presentation while incorporating patient-derived, rapidly evolving antigens. By enriching peptide-MHC and co-stimulatory ligands orders-of-magnitude above donor-cell levels, DEVs emerge as potent vesicle-vaccines, although efficacy remains limited by unclear mechanisms. We show that DCs repurpose viral components to generate endogenous virus-like vesicles (VLVs) that preferentially carry peptide-MHC and high-density co-stimulatory ligands, intensifying and extending antigen presentation. Upon antigen exposure, Arc partners with endogenous envelope proteins to assemble VLVs that directly engage T cells and trigger intrinsic adjuvanticity via viral mimicry. Arc-/- DEVs failed to prime antigen-specific T cell responses, whereas Arc overexpression with its 5'-UTR stem-loop shifted DEVs toward VLVs that trafficked to lymphoid organs, drove rapid CD4-assisted priming and durable CD8-biased memory, suppressed melanoma, and prolonged survival. These reveal a viral-mimicry mechanism enabling long-range immune activation and support Arc+ VLVs as an antigen-agnostic vaccine for cancer immunotherapy. HighlightsO_LIArc+ VLVs intensify and extend DC antigen presentation in vivo C_LIO_LIArc+ VLVs directly engage T cells and trigger viral-mimic adjuvanticity C_LIO_LIArc-/- DEVs fail to prime antigen-specific T cell responses C_LIO_LIEngineered Arc+ VLVs drive durable CD8-biased memory and tumor control C_LIO_LIArc+ VLV vaccination provides long-range and cross-tumor protection in vivo C_LI

bioengineering↗

bioRxiv2025

Bacteriophage genome-wide transposon mutagenesis

Bacteriophages have genomes that span a wide size range, are densely packed with coding sequences, and frequently encode genes of unknown function. Classical forward genetics has defined essential genes for phage replication in a few model systems but remains laborious and non-scalable. Unbiased functional genomics approaches are therefore needed for phages, particularly for large lytic phages. Here, we develop a phage transposon sequencing (TnSeq) platform that uses the mariner transposase to insert an anti-CRISPR selectable marker into phage genomes. CRISPR-Cas13a-based enrichment of transposed phages followed by pooled sequencing identifies both fitness-conferring and dispensable genes. Using the Pseudomonas aeruginosa-infecting nucleus-forming jumbo phage {Phi}KZ (280,334 bp; 371 predicted genes) as a model, we show that [~]110 genes are fitness-conferring via phage TnSeq. These include conserved essential genes involved in phage nucleus formation, protein trafficking, transcription, DNA replication, and virion assembly. We also isolate hundreds of individual phages with insertions in non-essential genes and reveal conditionally essential genes that are specifically required in clinical isolates, at environmental temperature, or in the presence of a defensive nuclease. Phage TnSeq is a facile, scalable technology that can define essential phage genes and generate knockouts in all non-essential genes in a single experiment, enabling conditional genetic screens in phages and providing a broadly applicable resource for phage functional genomics.

microbiology↗

bioRxiv2025

Uncovering structural determinants of peptide recognition by public and private T-cell receptors

Public T cell receptors (TCRs) recurrently emerge across individuals in response to common pathogens, yet the structural and biophysical basis distinguishing public from private clonotypes remains incompletely defined. Here, we combine epitope mapping, single-cell TCR sequencing, and single-particle cryo-electron microscopy to dissect CD8+ T cell responses to the immunodominant SARS-CoV-2 ORF3a(207-215) epitope presented by HLA-A*01:01. Among responding clonotypes, we identify a shared public TCR (TCRpub) and an individual-specific private TCR (TCRpriv) that use nearly identical TRBV5-1 {beta} chains but distinct chains. Both clonotypes exhibit comparable micromolar affinity and functional avidity, yet their structures reveal different antigen-recognition modes. We determined cryo-EM structures of the TCRpub and TCRpriv in complex with ORF3a(207-215)/ HLA-A*01:01 at [~]3 [A] resolution. Despite targeting the same epitope, the two receptors engaged the peptide-MHC complex with distinct CDR-loop orientations and contact footprints: TCRpub engages the peptide through a peptide-centric AGDL CDR3{beta} motif and focuses interactions on the MHC 2-helix, whereas TCRpriv distributes contacts across both MHC -helices via a canonical CDR3{beta} configuration. These findings illustrate how near-identical {beta} chains can yield divergent recognition strategies to recognise the same pMHC ligand through alternative -chain pairing. More broadly, this work establishes cryo-EM as a robust approach for resolving physiological-affinity TCR/pMHC complexes, providing mechanistic insight into how public TCRs emerge and persist in antiviral immunity.

immunology↗

bioRxiv2025

Single-Cell Multi-Omics Dissects Transcript Isoform and Immune Repertoire Dynamics in Human Immunosenescence

Immunosenescence, a major hallmark of systemic aging, refers to the progressive functional decline of the immune system. This decline not only compromises host defense and immunological memory but also fuels chronic inflammation and tissue degeneration (collectively known as inflammaging). While single-cell RNA sequencing (scRNA-seq) has revealed transcriptomic alterations in immune aging, analyses restricted to transcript abundance fail to capture deeper regulatory layers, such as transcript isoform diversity and the remodeling of immune receptor repertoires. To address this, we present the human peripheral immune single-cell multi-omics atlas that integrates gene expression, transcript isoforms diversity, and immune receptor repertoires. By combining single-cell full-length transcriptome sequencing (scCycloneSEQ), short-read scRNA-seq, and single-cell immune receptor sequencing (scTCR/BCR-seq), we systematically profiled peripheral blood mononuclear cells (PBMCs) from healthy young and elderly donors. Our analyses uncovered extensive age-related remodeling of immune cell composition, functional states, and TCR/BCR diversity. Notably, we identified in CD4 effector memory T cells exhibited widespread differential isoform usage (DIU), 3'UTR length variation, and a marked reshaping of cytotoxic T lymphocyte (CTL) clonotypes--all closely associated with aging-related inflammation and cellular senescence. This multi-omics atlas delineates key molecular features of immunosenescence and provides a high-resolution resource for deciphering the regulatory architecture underlying immune aging.

immunology↗

bioRxiv2025

Neural and Behavioral Responses to Social Norm Violations Across Trust and Fairness Contexts

Trust and fairness sustain cooperation, but social neuroscience has largely studied violations of these social norms in isolation. We asked whether neural sensitivity to betrayal carries forward into the evaluation of unfairness in a separate social exchange. In 132 adults who completed fMRI during the Trust Game and Ultimatum Game, betrayal and unfairness both recruited overlapping voxels within left anterior insula. Yet, individual differences in regional activation were uncorrelated across tasks, arguing against a simple shared-response account. Instead, cross-task structure emerged in connectivity: stronger anterior insula responses to social versus nonsocial betrayal predicted weaker anterior insula-ventromedial prefrontal coupling during social versus nonsocial fairness evaluation. These findings suggest that betrayal and unfairness are linked less by identical regional responses than by how salience signals engage valuation circuitry across contexts. By bridging two canonical social exchange tasks within the same individuals, this study reveals a network-level architecture for generalizing sensitivity to interpersonal norm violations.

neuroscience↗

bioRxiv2025

Remodeling of KRAS interactome induced by clinically relevant RAS inhibitors reveals convergent responses and a KRAS-mediated regulation of directional cell migration

KRAS is commonly mutated in lung, colorectal, and pancreatic cancers. Small molecule inhibitors targeting KRAS with distinct mechanisms-of-action and variable specificities have entered the clinic, but a comprehensive view of their effect on the RAS signaling network has not been reported. Here, we describe the impact of RAS inhibition on the KRAS protein interactome using Proximity-dependent Biotinylation Identification. Two inhibitors were used: panRAS-ON, which forms a ternary complex between cyclophilin and RAS in the GTP bound state; and panKRAS-off, which binds specifically to the KRAS switch II pocket. RAS inhibitors were found to significantly alter 16.5% of proteins in proximity to KRAS. Despite their distinct mechanisms-of-action, the KRAS inhibitors induced highly correlated changes in the KRAS interactomes. Among proteins in close proximity to KRAS, Afadin was found to be highly regulated by RAS inhibition. AFDN has been characterized as a key regulator of cell motility, invasion, and metastasis. Analysis of AFDN phosphorylation revealed that AKT only partially modulates p-Ser1718, while inhibition of KRAS is sufficient to abolish EGF-mediated AFDN phosphorylation. Knockdown of AFDN in a KRAS-driven non-small cell lung cancer model abolished chemotaxis in a transwell migration assay and disrupted directional movement in an EGF-driven wound healing model. These results suggest that KRAS is a central node in regulating growth factor induced cell migration, and that KRAS inhibition plays a broader role than MAPK-mediated cell proliferation and survival.

cancer biology↗

bioRxiv2025

Deep learning reveals a microprotein atlas in maize and uncovers novel regulators of seed amino acid metabolism

Microproteins represent a vast and functionally important class of genes that remain largely unexplored in plant genomes. Here, we developed DeepMp, a deep learning framework that integrated multi-omics evidence and built the most comprehensive plant microprotein atlas to date, identifying 18,338 high-confidence candidates in maize. The majority of these appear to have originated de novo from regions previously annotated as noncoding, and they show hallmarks of rapid, lineage-specific evolution and pronounced tissue specificity. These novel microproteins were found integrated into core regulatory networks, particularly in organ development and nutrient storage. Focusing on the maize kernel, population-scale analyses linked microprotein expression to natural variation in amino acid content. We functionally validated three grain-filling-specific candidates originating from noncoding regions by CRISPR-Cas9 knockouts, which confirmed their roles as precise modulators of arginine, aspartate, and methionine levels, without pleiotropic effects on kernel morphology. Our study provides a foundational resource and analytical framework, establishes microproteins as a new and functionally important coding layer in maize, and uncovers a previously untapped source of targets for crop improvement.

genomics↗

bioRxiv2025

Autolamellasomes: An Autophagy-Dependent Parallel Pathway for Bulk ER Degradation and Lysosome Biogenesis

Lysosomes containing multilamellar membrane whorls are a hallmark of cellular aging and storage disorders, yet the biogenesis of these structures has remained elusive for decades. Here, we identify a distinct form of endoplasmic reticulum (ER) remodeling, termed autolamellasomes, which mediates bulk ER degradation under chronic mTOR inhibition. Unlike canonical ER-phagy, autolamellasomes are concentric ER stacks that form via an autophagy-dependent but receptor-independent mechanism. Using Cryo-ET, CLEM, and a reconstituted cell-free system, we demonstrate that these structures arise from the cytosolic compaction of fragmented ER membranes, driven by the core autophagy machinery. We find that autolamellasomes accumulate in senescent cells and fibroblasts from patients with Hutchinson-Gilford progeria syndrome, linking sustained mTOR suppression to lysosomal membrane homeostasis. Our results resolve the origin of intralysosomal whorls and define a conserved pathway that couples nutrient sensing to membrane turnover and cellular aging.

cell biology↗

bioRxiv2025

Single-cell transcriptomics reveals spaceflight-induced accelerated aging and impaired recovery in the murine bone marrow

Spaceflight induces physiological changes that resemble accelerated aging, however, how age influences bone marrow response at the cellular level remains poorly understood. Here, we perform single-cell transcriptomic profiling of murine femur and humerus bone marrow from young (12-week-old) and old (29-week-old) mice that underwent a 32-day spaceflight mission followed by a 24-day Earth recovery, with age-matched ground controls. Our analysis reveals that, compared with young cohorts, old mice exhibit persistent dysregulation after spaceflight, most prominently in erythroid and B cell lineages. In erythroid cells, old flight mice show pronounced aging signatures, characterized by impaired maturation, inhibited mitophagy, and increased oxidative stress. In B cells, old flight mice show dysregulation associated with failure of the AP-1 stress-response pathway and complete collapse of the intercellular CXCL signaling network. Our findings dissect the age-dependent effects of spaceflight on the bone marrow hematopoietic and immune system at single-cell resolution, and demonstrate that spaceflight imposes a disproportionate burden on the aged hematopoietic system and blunts post-flight recovery. These insights provide candidate pathways and biomarkers for health monitoring and countermeasures in long-duration missions.

bioinformatics↗

bioRxiv2025

Cross-task, explainable, and real-time decoding of human emotion states by integrating grey and white matter intracranial neural activity

Decoding human emotion states from intracranial neural activity is key in developing brain-computer interfaces and new therapies for affective disorders. However, real-world application of decoding requires high performance that integrates neural activity from both grey and white matter, stable generalization across different contexts, sufficient neural encoding explainability, and robust real-time implementation, all of which remain elusive. Here, we simultaneously recorded intracranial electroencephalogram (iEEG) and abundant self-rated valence and arousal scores across two emotion-eliciting tasks in eighteen subjects, forming the largest intracranial neural dataset for emotion decoding. We then developed personalized decoding models within a hybrid deep learning framework involving both self-supervised and supervised components. The models achieved high-performance decoding of continuous valence/arousal states, doubling the R-squared performance of prior EEG/iEEG decoding. Critically, the models significantly improved performance by integrating grey and white matter signals and demonstrated cross-task generalization. The models further revealed shared and preferred mesolimbic-thalamo-cortical subnetworks encoding valence and arousal, showing neurophysiological explainability. Finally, the models realized robust real-time decoding in four new subjects. Our results have implications for advancing emotion decoding neurotechnology toward deployable affective brain-computer interfaces and closed-loop therapeutic systems for affective disorders.

neuroscience↗

bioRxiv2025

Predicting individual incubation of opioid craving by whole-brain functional connectivity

A high risk of relapse triggered by craving during abstinence remains a main challenge in opioid addiction treatment. Multiple brain regions have been implicated in opioid craving, but the brain-wide neural mechanisms underlying this process remain poorly understood. Using resting-state fMRI and connectome-based predictive modeling, we identified a whole-brain connectome that predicted the time-dependent increases (incubation) in oxycodone craving in individual rats after voluntary abstinence induced by exposure to an electric barrier. Incubation of oxycodone craving was operationally defined as the increase in non-reinforced lever pressing during relapse tests from early (day 1) to late (day 15) abstinence (incubation score). We found that changes in whole-brain functional connectivity during abstinence, but not during oxycodone self-administration, predicted the incubation score. Greater decreases in functional connectivity were associated with higher incubation scores. The predictive connectome involved complex interactions across multiple brain systems, including frontal-striatal, frontal-insula, insula-striatal, and hippocampal and sensorimotor circuits. To test causality of the predictive connectome, we examined the effect of pharmacological inactivation of dorsomedial striatum (DMS), which significantly decreased oxycodone seeking after electric barrier-induced abstinence. DMS inactivation increased connectivity strength within the predictive connectome, supporting a causal role of this connectome in incubation of oxycodone craving. The predictive connectome did not predict food-reward seeking after electric barrier-induced abstinence, indicating specificity to oxycodone craving. Our findings identify a brain-wide connectome marker that predicts individual differences in the incubation of opioid craving and provide potential targets for developing personalized interventions and monitoring therapeutic outcomes in opioid addiction treatment. Significance statementRelapse driven by craving remains a central challenge in treating opioid addiction. Using resting-state fMRI and connectome-based predictive modeling, we identified a whole-brain connectivity pattern that predicted incubation of oxycodone craving after voluntary abstinence in rats. Connectivity changes during abstinence predicted the magnitude of incubation, measured by increased oxycodone seeking from early to late abstinence. The predictive connectome involved interactions among cortical, basal ganglia, insular, hippocampal and sensorimotor systems. Pharmacological inactivation of the dorsomedial striatum modulated connectivity strength within this network, demonstrating a causal role of the connectome in incubation of craving. These findings identify a brain-wide marker of incubation of opioid craving and provide a translational framework for developing targeted interventions and monitoring therapeutic outcomes in opioid addiction treatment.

neuroscience↗

bioRxiv2025

Development and Genome-level Microevolution of Oral Microbiome during Surface Colonization

The oral microbiome is essential to human health, yet its de novo ecological succession and microevolutionary dynamics remain poorly understood due to the absence of tractable in vivo models. Dental implants provide a unique opportunity to investigate these processes by establishing a defined time-zero for initial bacterial attachment. In this cohort study, we performed shotgun metagenomics on 95 subgingival plaque samples from 19 participants, including peri-implant sites at weeks 1-4 after crown placement and adjacent teeth as controls. Peri-implant and adjacent periodontal microbiomes exhibited distinct taxonomic and functional profiles. Even the same taxa had different functional potential across the two sites. These findings indicated that oral microbiome development is a niche-specific process with selective colonization rather than passive microbial translocation from adjacent sites. Longitudinal analysis identified three microbial community modules driving the succession of oral microbiome, including pioneer colonizers, constitutive species, and late commensals. Each module followed distinct temporal abundance patterns and played unique ecological roles throughout the succession process. Strain-level resolution revealed divergent microevolutionary trajectories: pioneer colonizers and late commensals exhibited higher cumulative mutation rates and greater strain heterogeneity overtime, with enrichment of nonsynonymous single nucleotide variants in genes related to virulence and metabolism, whereas constitutive species remained evolutionarily stable by contrast. Our study reveals that the development, succession, and microevolution of the oral microbiome is structured, niche-dependent, and modulated by inter-species facilitation and selective genomic adaptation. These findings advance the understanding of oral microbiome ecology and provide a conceptual foundation for manipulating microbial succession in health and disease.

microbiology↗

bioRxiv2025

Derivation, Characterizations, and Applications of Rabbit Haploid Embryonic Stem Cells

Haploid embryonic stem cells (haESCs), which contain a single set of chromosomes, provide powerful systems for investigating gene function and creating gene-edited animal models. haESCs have been derived from several mammalian species including human, mouse and rats, yet not from rabbits. Here, we report the derivation of parthenogenetic haESCs of rabbit (rbPhESCs) using an optimized culture medium. These cells maintain a stable haploid karyotype during long-term culture and own pluripotency features comparable to diploid embryonic stem cells derived from rabbit fertilized embryos. By integrating gene trapping cassette into ROSA26 locus, rbPhESCs offer an ideal platform for high-throughput functional genomic screening, as shown in haESCs from other mammals. Consistent with their parthenogenetic origin, rbPhESCs durably preserve maternal-specific imprinting patterns even after extended culture in serum-containing conditions, providing a valuable platform for generating disease models deficient in maternally expressed imprinted genes. Therefore, our findings expand the repertoire of mammalian haESCs and establish rbPhESCs as a valuable platform for genetic studies and biotechnological applications in rabbits.

cell biology↗

bioRxiv2025

GNL3 SUMOylation is essential for DNA double-strand break repair by homologous recombination

DNA double-strand break (DSB) repair via homologous recombination (HR) is critical for maintaining genomic integrity and requires proper DNA end resection to generate single-stranded DNA (ssDNA) overhangs. However, the mechanisms governing this critical step in cells remains poorly understood. Here, we report that GNL3, a nucleolar GTP-binding protein, plays a key role in HR repair via regulating DNA end resection dependently on its SUMOylation. Ectopic expression of wild-type, but not the SUMO-defective K196R mutant, GNL3 completely abolished DNA damage response induced by knockdown of endogenous GNL3. GNL3 interacts with the BLM-DNA2 helicase-nuclease complex and is critical for DNA end resection and subsequent loading of RPA and RAD51. This interaction requires SUMOylation and SUMO-interacting motifs (SIMs) in both proteins. We further demonstrate that USP36, a nucleolar deubiquitinating enzyme, functions as a novel SUMO ligase for GNL3, while the SUMO protease SENP3 deSUMOylates GNL3. Notably, several breast cancer-derived GNL3 variants that disrupt its SUMOylation or SIM fail to interact with the BLM-DNA2 complex. Knockdown of GNL3 sensitizes HR-proficient breast cancer cells to etoposide or Olaparib treatment. Together, our results reveal that GNL3 SUMOylation is crucial for HR repair and suggest that targeting GNL3 SUMOylation may induce HR deficiency, thereby sensitizing breast cancers to DNA damage-inducing agents.

cell biology↗