Biology subjects

Zhang, S.

Publications and source records attributed to Zhang, S..

At least 307 records · Page 17Linked to original sources

bioRxiv2025

HPInet: Interpretable prediction of Host-Pathogen protein-protein Interactions using a transformer-based neural network

Gram-negative bacteria utilize a series of secretion systems (T1SS-T10SS) to deliver secreted effector proteins (T1SE-T10SE) into host cells, leading to infections and diseases. Understanding the interactions between these effector proteins and host proteins is crucial for unraveling the pathogenic mechanisms of bacterial pathogens. Despite advancements in sequencing technologies that have significantly enhanced our knowledge of effector protein diversity and structure, the molecular mechanisms underlying their interactions with human host proteins remain unclear. Experimental approaches to validate these interactions are labor-intensive, time-consuming, and insufficient to comprehensively map the extensive effector-host protein interaction (PPI) network. Additionally, specialized computational tools for predicting these interactions remain scarce. To address this gap, we developed HPInet, a transformer-based deep learning model for predicting interactions between bacterial effector proteins and human host proteins. HPInet introduces convolutional layers and global response normalization (GRN) into the feed-forward network (FFN) of the transformer architecture, enhancing the extraction of local sequence features. Compared to existing state-of-the-art methods, HPInet significantly improves prediction performance, increasing accuracy from 0.502 to 0.891 and boosting sensitivity by 35.3%. Furthermore, by leveraging the models attention mechanism, HPInet identifies critical residues at interaction sites, demonstrating its capability to capture local structural features of PPI sites solely from sequence information. To facilitate its application in research, HPInet has been implemented as a freely accessible web server available at https://bis.zju.edu.cn/hpinet. This platform provides a powerful and interpretable tool for studying effector-host protein interactions, offering valuable insights into bacterial pathogenesis and potential therapeutic targets.

bioinformatics↗

bioRxiv2025

The hidden predictors of human haematopoietic clonal fate

Human haematopoietic stem and progenitor cells (HSPCs) exhibit heterogeneous lineage output, but the molecular programs underlying clonal fate remain poorly defined. To address this, we developed a human haematopoietic organoid supporting differentiation into 15 lineages and used it to track barcoded HSPC clones over time. By integrating single-cell transcriptomes, surface phenotypes, and clonal fate, we applied machine learning to identify clonal fate modules - gene and marker signatures predictive of lineage commitment. This approach uncovered hidden transcriptional and surface correlates of multipotency, including CD200, which marked a subset of HSCs with broad output capacity, which we leveraged to increase manufactured type 1 dendritic cell purity for immunotherapy applications. Our study provides a framework for decoding clonal fate decisions in human HSPCs and identifies molecular features that distinguish truly multipotent clones, advancing strategies for stem cell purification and therapeutic engineering.

cell biology↗

bioRxiv2025

Cortical α-synuclein pathology induces cell autonomous neuronal hypoactivity and compensatory circuit changes in a model of early Lewy Body Dementia

Posterior cortical impairments in visuospatial/perceptual function are common non-motor symptoms of the Lewy Body Dementias (LBD) and are relatively specific for LBDs compared to other dementias. Across populations, cognitive impairments correlate with the presence of -synuclein (-syn) pathology in limbic and neocortical brain regions. However, the specific role that -syn pathology plays in driving cortical circuit dysfunction and cognitive impairment remains controversial. We hypothesized that inducing -syn pathology in primary visual cortex (V1) in mice would impair neuronal activity and encoding of visual information, leading to visuoperceptual impairments. To test this, we injected -syn pre-formed fibrils (PFF) into V1, causing the formation of sparse Lewy-like pathology. Using longitudinal in vivo two-photon (2P) calcium imaging over 6 months, we recorded visually evoked activity of pyramidal cells in layer 2/3 (L2/3) and quantified -syn pathology using C05-05, a fluorescent ligand that binds aggregated -syn. Measuring population activity, we found a greater percentage of neurons in PFF-injected mice were responsive to visual stimuli with lower direction selectivity compared to controls at 4-5 months post-injection (MPI). Neurons with somatic Lewy-like inclusions showed reduced activity compared to neighboring neurons without inclusions. Conversely, neurons without somatic inclusions showed increased activity, positively correlated with the local burden of -syn pathology. Using a coherent motion discrimination task, we found no impairments in visuoperceptual ability in PFF-injected mice. Our results demonstrate that -syn pathology leads to reductions in neuronal activity in cells with somatic inclusions and reciprocal changes in local population activity but does not impair visuoperceptual function. Reflecting the early stages of neocortical -syn pathology, our model provides a framework for future studies to better understand the heterogeneity of cognitive symptoms and -syn pathology across patients.

neuroscience↗

bioRxiv2025

MNP33 is a novel mitochondrial protein that reprograms cellular bioenergetics and lipid metabolism

Genomic regions previously annotated as non-coding are now recognized sources of functional microproteins, termed the "dark proteome", though their identification and characterization remain challenging. Addressing this, we developed a novel proteomic strategy combining lipid droplet (LD) enrichment with mass spectrometry against the database of sORF-derived proteins. This approach yielded the first reported LD-resident microprotein, LDANP1, and subsequently revealed other candidates from the same screen, such as MNP33, were localized to mitochondria. Here, we provide the first in-depth functional characterization of MNP33, a novel 28-amino acid protein encoded by an sORF within the Cdhr4 gene locus. We confirm MNP33 localizes to the inner mitochondrial membrane and demonstrate its interaction with the adenine nucleotide translocase 2 (ANT2). Functionally, MNP33 expression remodels mitochondrial bioenergetics, increasing basal respiration and proton leak while paradoxically elevating membrane potential, partly through modulating ANT2 activity linked to enhanced glycolysis. Furthermore, MNP33 shifts cellular lipid metabolism, favoring cholesteryl ester storage over triacylglycerols via stabilization of ACAT1, and is associated with closer ER-mitochondria contacts. MNP33 also induces ROS production and autophagy while inhibiting cell proliferation without increasing apoptosis. This study establishes MNP33 as a novel mitochondrial regulator emerging from the dark proteome, providing a mechanistic link between non-coding genetic elements, ANT2 function, mitochondrial bioenergetics, cellular metabolism, and stress responses. Our work highlights functional importances of the dark proteins and validates organelle-focused proteomics for discovering key cellular regulators.

cell biology↗

bioRxiv2025

Inhibitor fluorination pattern tunes chemically induced protein dimerization

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite Trypanosoma brucei through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.

biophysics↗

bioRxiv2025

Development of ultrasound molecular contrast agents by site-specific conjugation of gas vesicles to antibodies

Ultrasound technology is a powerful tool for medical imaging and diagnosis especially when used in conjunction with a contrast agent to target proteins of interest. However, current ultrasound contrast agents are lacking the combination of specificity, stability, and size optimization. In this study we introduce and confirm the feasibility of using nanometer sized gas vesicles (GV) chemically click conjugated to antibodies (mAb) to make the first site-specific mAb-GV conjugates as a durable cancer cell targeting ultrasound molecular contrast agents. Protein expression of human epidermal growth factor receptor 2 (HER2) and programmed death-ligand 1 (PD-L1) were tested along with the antibody targeting efficiency using cancer cell lines and primary cells isolated from tumor bearing mice. The mAb conjugation was optimized to a site-specific method using the mAb glycans and tested with the addition of a clinically used mAb to target trophoblast cell surface antigen 2 (Trop-2). The developed contrast agents utilize the stability of GVs and the specificity of antibodies to label cancer biomarkers in various types of tumors for ultrasound imaging.

bioengineering↗

bioRxiv2025

Hippocampal input-driven plasticity of prefrontal interneurons reveals a circuit basis for impaired spatial working memory.

Long-range projections from ventral hippocampus (vHPC) to medial prefrontal cortex (mPFC) support cognitive functions including spatial working memory (SWM). vHPC input targets multiple prefrontal interneuron classes, yet how hippocampal input recruits these populations in vivo, whether this recruitment is plastic, and how such plasticity influences cognition remain unknown. Here, we combined optical stimulation of mouse vHPC inputs with calcium recordings from discrete mPFC interneuron populations, revealing persistent activity-induced reweighting of hippocampal recruitment across prefrontal inhibitory microcircuits. Ex vivo electrophysiology and computational modeling implicated weakened monosynaptic hippocampal drive onto vasoactive intestinal polypeptide (VIP)-expressing interneurons in this circuit reconfiguration. Prior vHPC input stimulation and the schizophrenia-associated Df(16)A+/- mutation, which also yielded reduced hippocampal input onto VIP interneurons, produced convergent alterations in task-related VIP interneuron activity associated with poorer SWM task learning. These findings implicate hippocampal input to VIP interneurons as a key locus of plasticity capable of reconfiguring inhibitory microcircuit activity linked to impaired working memory task learning. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/665987v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@e26cb0org.highwire.dtl.DTLVardef@14d9d6borg.highwire.dtl.DTLVardef@9a0fc2org.highwire.dtl.DTLVardef@80afd1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

neuroscience↗

bioRxiv2025

Corticosterone drives behavioral inflexibility via plasticity-related gene expression in the dorsal striatum

Behavioral flexibility allows organisms to modify actions based on new information, such as shifts in reward value or availability, and is promoted by the dorsomedial striatum (DMS). In contrast, behavioral inflexibility provides efficiency and automaticity in familiar contexts, and is promoted by the dorsolateral striatum (DLS). Importantly, chronic elevation of the primary stress hormone, corticosterone (CORT) in rodents or cortisol in humans, impairs behavioral flexibility through dendritic atrophy in the DMS, and promotes inflexible behavioral response strategies through dendritic outgrowth in the DLS. However, understanding of the molecular mechanisms that underlie the structural changes promoting behavioral inflexibility is lacking. We used a food-motivated operant task in male and female mice to define synaptic plasticity gene regulation supporting a decreased DMS activity and increased DLS activity in the shift to inflexible behavior with CORT. We discovered that CORT-accelerated loss of behavioral flexibility is accompanied by decreased DMS- and increased DLS-specific synaptic plasticity gene expression, and that distinct genes are either differentially expressed or spliced in the transition to inflexible behavior. Splicing analysis suggests that repressed activity in the DMS during the transition to inflexible behavior reflects both reduced expression and increased degradation of plasticity-related mRNA transcripts. Finally, given the ability of CORT to influence histone acetylation, we defined CORT-mediated H3K9ac enrichment profiles associated with synaptic plasticity gene regulation stratified by sex and striatal subregion. This study is the first to define CORT-driven epigenetic regulation in the DMS and DLS during the transition from flexible to inflexible behavior in male and female mice.

neuroscience↗

bioRxiv2025

Gene-Corrected Basal Cells Restore CFTR In Vitro; Transplants Regenerate Epithelium in a Preclinical Sinus Model

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene, leading to epithelial dysfunction and progressive lung disease. Although CFTR modulators have transformed care, [~]10% of people with CF remain without effective therapy. Durable, mutation-agnostic approaches are urgently needed. MethodWe used a lentiviral (LV) vector to deliver wild-type CFTR to airway basal cells derived from 13 paediatric CF participants with a range of genotypes. Transduced cells were assessed for transgene expression, epithelial differentiation, and CFTR function using air-liquid interface (ALI) cultures. Separately, to evaluate regenerative capacity in vivo, LVGFP-transduced rabbit airway basal cells were transplanted into the denuded nasal septum of healthy New Zealand white rabbits using a biocompatible scaffold. ResultsTransduced basal cells retained multilineage differentiation capacity, forming well-organized, pseudostratified epithelium with intact barrier function and ciliary activity. CFTR channel activity was restored to levels comparable to or exceeding those achieved with elexacaftor/tezacaftor/ivacaftor (ETI), including in individuals with nonsense mutations. Combined CFTR transduction plus ETI treatment showed additive benefit. In vivo, transplanted rabbit basal cells engrafted and differentiated to regenerate a mucociliary epithelium, with improved nasal potential difference and mucociliary clearance compared to scaffold-only controls. ConclusionOur study demonstrates that LV-mediated CFTR gene addition restores CFTR function in vitro across genotypes and supports epithelial regeneration in a clinically relevant animal airway model. This two-part platform offers a scalable path toward cell therapies for all people with CF and may have broader applications in upper airway epithelial repair.

cell biology↗

bioRxiv2025

CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is a fusion-driven pediatric cancer with poor survival and limited therapeutic options. To uncover novel vulnerabilities, we employed complex-based analysis of the DepMap functional genomic data, identifying CDK8 as a dependency in aRMS. Both CDK8 knockout and pharmacologic inhibition impaired tumor cell growth and induced myogenic differentiation in vitro and in vivo. Compared to genetic loss, CDK8 inhibition induced more dynamic transcriptional changes. With a genome-scale CRISPR-Cas9 drug modifier screen, we determined that the maximal anti-tumor activity of the CDK8 inhibitor requires the presence of the Mediator kinase module and transcriptional cooperation with the SAGA complex. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. These findings suggest a distinct gain-of-function mechanism of the CDK8 inhibitor and establish a strong rationale for CDK8 inhibition as a differentiation-inducing therapeutic strategy in aRMS. STATEMENT OF SIGNIFICANCEWe provide a framework for uncovering therapeutic targets by network-based analysis of functional genomic screens. We identify CDK8 as a druggable target in aRMS and determine that CDK8 inhibition drives myogenic differentiation and impairs tumor progression via a collaborative mechanism involving the Mediator kinase module, SAGA complex, and SIX4.

cancer biology↗

bioRxiv2025

Development and Characterization of a Tunable PDMS Substrate Model for Investigating Elastic Properties and Mechanical Stretching in Intervertebral Disc Mechanobiology

BackgroundAberrant mechanical loading and altered extracellular matrix (ECM) composition favor catabolic cell responses, contributing to intervertebral disc (IVD) degeneration and ultimately impairing the integrity of the annulus fibrosus (AF). This highlights the need for new in vitro models to investigate the interplay of mechanical loading and cell-substrate interactions. Therefore, this study introduces a tunable stretching chamber platform to simultaneously study both factors in AF degeneration. MethodsTunable PDMS substrates were fabricated by adjusting ratios of Sylgard 184 and Sylgard 527, enabling molding into stretching chambers or well plates. Substrates underwent mechanical, optical and chemical characterization. Bovine AF cells were seeded onto the substrates and cultured under static conditions or subjected to cyclic strain (8% strain at 1 Hz). Substrate biocompatibility and cell morphology were assessed over 72 h in static cultures. Dynamic responses were assessed by cell viability and alignment. Digital Image Correlation (DIC) was employed to assess surface strain in custom-designed and commercial (STREX) stretching chambers at strains between 8 to 20%. ResultsPDMS formulations resulted in a stiffness (E modulus) range of 8.72 - 238.00 kPa, demonstrating distinct viscoelastic profiles. All substrate formulations supported AF cell adhesion and viability. DIC analysis revealed non-uniform axial and transverse strain distributions on membrane surfaces. Cyclic stretching showed that cells maintained viability up to 14 h. Additionally, cells responded to the applied strain by perpendicular alignment to the stretch axis at 6 h. ConclusionsThe PDMS based stretching platform offers a biocompatible and tunable mechanical environment that mimics physiological and pathophysiological elastic properties. It enables systematic investigations on how elastic properties and mechanical strain modulate AF cell behavior in IVD disease progression. Finally, this study raises the awareness of non-uniform and transverse strain components within the stretching chambers and highlights the discrepancy of effective strain transfer to the cell interaction surface.

bioengineering↗

bioRxiv2025

Characterization of full-length and cytoplasmic tail-truncated envelope glycoproteins incorporated into human immunodeficiency virus (HIV-1) virions and virus-like particles

During transport to the surface of infected cells, the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is cleaved to produce the mature functional Env trimer ((gp120/gp41)3). Env cleavage stabilizes the pretriggered Env conformation (PTC), the major target for broadly neutralizing antibodies. Although the mature Env is relatively enriched in virions and virus-like particles (VLPs), conformationally flexible uncleaved Envs typically contaminate preparations of these particles. In non-permissive cells, the long [~]149-residue gp41 cytoplasmic tail (CT) is necessary for Env incorporation into virions. In a minority of HIV-1 strains, the gp41 CT is clipped in virions by the viral protease. Here, we compare Envs with CT truncations and CT alterations that increase or decrease protease clipping in permissive cells. Changes in protease clipping affected amphotericin B sensitivity, but did not alter other viral phenotypes. By contrast, a corresponding CT truncation (L748STOP) increased cell-surface and virion Env levels, cell-cell fusion, and virus infectivity and cytotoxicity. Notably, in diverse HIV-1 strains, the ratio of cleaved/uncleaved Envs in preparations of virions and extracellular vesicles was increased by this CT truncation. ESCRT and ALIX-binding region (EABR) vesicles incorporated significantly more uncleaved CT-truncated Env than HIV-1 VLPs. Env CT deletion/truncation did not qualitatively alter the viral neutralization profile; however, increased antibody concentrations were required to neutralize viruses with the higher levels of cleaved Env that resulted from CT truncation. Specific CT truncations provide a means of enriching the PTC and limiting the incorporation of nonfunctional and conformationally heterogeneous uncleaved Envs into preparations of virions and VLPs. IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry of the virus into host cells. The pretriggered conformation (PTC) of Env is the major target for protective broadly neutralizing antibodies, but the PTC is unstable and therefore difficult to study. The cleavage of the flexible Env precursor stabilizes the PTC. Therefore, the presence of uncleaved Env compromises the purity of the PTC in Env preparations. We found that certain truncations of the Env cytoplasmic tail resulted in improved ratios of cleaved:uncleaved Env in preparations of HIV-1 viruses or virus-like particles. In some contexts, cytoplasmic tail truncation increased the level of Env in virus preparations. Although higher concentrations of antibodies were required to neutralize these viruses, Envs with specific truncations of the cytoplasmic tail retained the PTC. Thus, cytoplasmic tail truncation could assist efforts to purify and characterize the Env PTC on the viral membrane.

microbiology↗

bioRxiv2025

Thrombin-preconditioned Mesenchymal Stem Cell-Derived Exosomes For Wound Healing in vitro and in vivo

Background and ObjectivesThe skin is the largest organ of the human body, capable of protecting it from external harm. However, due to trauma, paralysis and other external factors, skin damage can occur, scars may form. Exosomes have regenerative functions and, as a cell-free therapy, show great potential for wound healing. In this study, we investigate that whether different umbilical cord mesenchymal stem cell exosomes (UCMSCs-Exos) can accelerate the healing of skin. MethodsIn our study, UCMSCs were cultured from newborns and Cultured with thrombin. Exosomes were isolated from mesenchymal stem cells culture supernatant by ultracentrifugation. The impact of exosomes on Hacat cell migration was evaluated in vitro. Additionally, the wound healing capacity of exosomes was evaluated in vivo using a mouse skin injury model. ResultsCompared to UCMSCs-Exos, T-UCMSCs-Exos significantly promoted cell proliferation and migration of cells. In vivo experiments demonstrated that T-UCMSCs-Exos can accelerate wound closure and enhance collagen maturation, promoting angiogenesis in the vascularized wound area. These results indicate that T-UCMSCs-Exos have a good potential for accelerating wound healing and minimizing scar formation. ConclusionsOur research indicate that thrombin pre-UCMSCs, the production of exosomes significantly increased. The prepared T-UCMSCs-Exo can accelerate skin wound healing during the process of skin injury repair, promote angiogenesis, and facilitate the reconstruction of epidermis and dermis as well as hair follicle regeneration. These findings demonstrate that T-UCMSCs-Exos for skin wounds are a promising cell-free therapy that can be applied in the treatment of skin injuries.

cell biology↗

bioRxiv2025

Copper-Containing ROS-Scavenging Nanozyme Paradoxically Drives Alpha-Synucleinopathy by Amplifying Nitrosative Stress

Reactive oxygen and nitrogen species (RONS) are implicated in neurodegeneration, but their specific pathogenic roles remain unclear. Here, we developed a pair of iridium-based nanozymes with opposing functionalities to dissect these pathways. We show that a copper-tuned iridium nanozyme (Ir{square}Cu), despite being a superior ROS scavenger, paradoxically and dramatically exacerbated -synuclein (Syn) pathology in vivo. This pathology was causally linked to its ability to amplify RNS, as pharmacological inhibition of nitric oxide synthase (NOS) with L-NAME completely abrogated the pathology and reversed a human Parkinsons disease (PD)-like transcriptomic signature. In contrast, a copper-free, broad-spectrum RONS-scavenging iridium (Ir) nanozyme demonstrated substantial therapeutic efficacy across diverse brain-first, body-first, and Alzheimers disease with Lewy body co-pathology models. Our findings uncover the importance of the RNS pathway in driving -synucleinopathies and establish a critical design principle for nanomedicine, mandating caution in the use of redox-active copper for neuroprotective applications.

pathology↗

bioRxiv2025

Signal Detection of Point Targets Using Eigen-Images for Super-Resolution Ultrasound Imaging and Gas Vesicle Localization

Accurate signal detection of ultrasound contrast agents, such as microbubble (MB) and gas vesicle (GV), in the presence of clutter and noise is essential to increase image quality in super-resolution ultrasound imaging (SRUS) and achieve precise GV localization. We developed and evaluated an eigen-image based signal detection method using singular value decomposition (SVD) and changepoint detection to automatically segment the data that are closely related to physical events such as MB flow and GV collapse. Eigen-image based method was compared with the elbow point and hard thresholding method when selecting MB signals after SVD of raw data, acquired from phantom and in vivo experiments. Image reconstructed by eigen-image based method was also compared with unregistered difference image for GV localization when moving GVs in a phantom were collapsed by ultrafast plane waves. The eigen-image based MB signal detection method resulted in higher vessel density (VD) visualization in both the phantom and in vivo mouse tumor. It also achieved increased signal-to-noise ratio (SNR) in both cases. Moreover, this method localized moving GVs more efficiently than the difference imaging method, without requiring pixel registration based on landmarks. The eigenimage based method offers a reliable and automated approach to MB and GV signal detection for both SRUS and point target localization. This approach is a valuable tool for medical imaging providing high-quality vessel images along with accurate locations of moving ultrasound contrast agents, which can be potentially translatable to clinical diagnosis and pre-clinical research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/662077v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d03b0corg.highwire.dtl.DTLVardef@d4b318org.highwire.dtl.DTLVardef@3a2894org.highwire.dtl.DTLVardef@3e2962_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAn approach to efficiently detect point targets including microbubbles and gas vesicles is developed using eigenimages that describe the changes of point targets over time for super-resolution ultrasound imaging and gas vesicle localization. C_LIO_LISignals from point targets were automatically identified from clutter and noise by the changepoint detection method without prior Information. C_LIO_LIThis approach enhances microvasculature visualization of tumors in mice and improves the localization of gas vesicles in a phantom. C_LI

bioengineering↗

bioRxiv2025

Spatio-temporal mass spectrometry in living cells reveals regulators of cuproptosis

Cuproptosis is a unique form of regulated cell death triggered by excessive copper accumulation in mitochondria. To decipher the mechanisms of cuproptosis, we develop Spatio-Temporal Mass Spectrometry (STMS) technique to characterize dynamic subcellular proteomic changes during its early stage. STMS analysis reveals distinct subcellular proteome alterations and unique trafficking patterns over time. Through a genetic functional screen, we discover that copper stress reduces OXA1L in mitochondria, disrupting MRPL20 import and impairing oxidative phosphorylation complex I synthesis. This mitochondrial dysfunction promotes MRPL20 nuclear translocation, triggering the mitochondrial unfolded protein response as an adaptive mechanism to restore mitochondrial proteostasis during early cuproptosis. Meanwhile, AMPK phosphorylates ISCU at serine 29, stabilizing ISCU protein, and increasing the abundance of Fe-S cluster-containing proteins, which, in turn, enhances cells susceptible to cuproptosis. Building on this mechanistic insight, we develop AMPK-SPARK, a phase separation-based EGFP reporter for real-time visualization of endogenous AMPK activity in living cells during cuproptosis. Additionally, we identify COMMD4 as a cuproptosis sensor and establish a GFP-tagged COMMD4 cell model for high-content drug screening. Using this platform, we identify three FDA-approved drugs including Plerixafor, Dithranol, and Bleomycin as potential cuproptosis inhibitors. Overall, our work advances the understanding of cuproptosis regulation and provides valuable molecular tools and data resource for future research in this area.

cell biology↗

bioRxiv2025

Structure and iron-transporting mechanism of brain organic cation transporter 1

Brain organic cation transporter 1 (BOCT1), also known as the solute carrier family 22 member 17 (SLC22A17) or the receptor for lipocalin-2 (LCN2), plays critical roles in health and disease. Its deficiency in mice results in early postnatal mortality and severe neurogenesis impairments. Despite its importance in physiology and pathophysiology, BOCT1s structure and transport mechanism remain elusive. Here, we integrate cryo-electron microscopy (cryo-EM), functional assays, biochemical experiments, and molecular dynamics simulations to elucidate the structure, substrate recognition, and transport mechanism of mouse BOCT1 (mBOCT1). The high-resolution cryo-EM structure reveals a distinctive N-terminal domain with a unique folding pattern dominated by a transmembrane loop atop TM6 (TML6), diverging from both known structures of SLC22 transporters and AlphaFold predictions. Notably, mBOCT1 functions as a high-capacity, low-affinity iron transporter independent of LCN2 binding. Iron transport is facilitated by a substrate gating mechanism involving TML6. These findings establish a structural basis for BOCT1s role as an independent iron transporter, enhancing our understanding of the transport mechanisms within major facilitator superfamily (MFS) transporters and providing new insights into brain iron homeostasis.

biophysics↗

bioRxiv2025

Accurate Prediction of ecDNA in Interphase Cancer Cells using Deep Neural Networks

Oncogene amplification is a key driver of cancer pathogenesis and is often mediated by extrachromosomal DNA (ecDNA). EcDNA amplifications are associated with increased pathogenicity of cancer and poorer outcomes for patients. EcDNA can be detected accurately using fluorescence in situ hybridization (FISH) when cells are arrested in metaphase. However, the majority of cancer cells are non-mitotic and must be analyzed in interphase, where it is difficult to discern extrachromosomal amplifications from chromosomal amplifications. Thus, there is a need for methods that accurately predict oncogene amplification status from interphase cells. Here, we present interSeg, a deep learning-based tool to cytogenetically determine the amplification status as EC-amp, HSR-amp, or not amplified from interphase FISH images. We trained and validated interSeg on 652 images (40,446 nuclei). Tests on 215 cultured cell and tissue model images (9,733 nuclei) showed 89% and 97% accuracy at the nuclear and sample levels, respectively. The neuroblastoma patient tissue hold-out set (67 samples and 1,937 nuclei) also revealed 97% accuracy at the sample level in detecting the presence of focal amplification. In experimentally and computationally mixed images, interSeg accurately predicted the level of heterogeneity. The results showcase interSeg as an important method for analyzing oncogene amplifications.

cancer biology↗