Biology subjects

Tong, Z.

Publications and source records attributed to Tong, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Stabilizing gas in aqueous liquids with metal-organic frameworks for persistent acoustic transduction

Gas is the most efficient mediator of acoustic energy owing to its high compressibility, yet its thermodynamic instability in aqueous environments makes it inherently transient, limiting sustained ultrasound-biological interfacing. Here, we show that gas can be stabilized, confined, and manipulated in the liquid phase to function as a persistent acoustic transducer. By trapping gas within designed microporous metal-organic framework (MOF) nanocrystals, interfacial forces establish a stable ternary-phase equilibrium that suppresses water intrusion and prevents gas collapse, enabling reversible, non-destructive oscillations under ultrasound stimulation and providing design principles for stabilizing gas in liquid environments. By leveraging high-resolution ultra-high-speed digital image correlation (DIC), we unveil the previously inaccessible spatiotemporal dynamics of confined gas oscillations within these nanotransducers under ultrasound stimulation, revealing localized amplification of the surrounding acoustic field and establishing a quantitative structure-function relationship for programmable acoustic transduction. These aqueous-phase gas nanotransducers exhibit sustained acoustic amplification over millions of excitation cycles and remain functional after prolonged storage (>2 years) under physiological conditions. In vitro and in vivo, this platform enables long-term ultrasound imaging and sustained, non-invasive neuromodulation by converting confined gas oscillations into persistent and amplified mechanical actuation. Our findings establish a generalizable design framework for stabilizing gas in liquid environments to achieve persistent and programmable acoustic transduction, thereby enabling sustained ultrasound-biological interfacing and opening opportunities for gas-based therapeutics.

bioengineering↗

bioRxiv2026

A scalable human neuron model of Alzheimer's disease relevant tauopathy reveals mechanisms linking Tau fibrillization to synaptic dysfunction

Tauopathies, including Alzheimer's disease, are driven by pathological aggregation of hyperphosphorylated Tau, which disrupts synaptic integrity, impairs neuronal communication, and contributes to cognitive decline. To dissect tauopathy pathogenesis and enable therapeutic discovery, reliable and scalable human iPSC-neuron models are essential. Here, we developed two complementary iPSC-derived neuron models: an endogenous Tau seeding model, in which neurons are challenged with pre-formed Tau fragments that form paired helical filament (PHF)-consistent structures, and a Tau-0N3R overexpression seeding model to accelerate pathology. Both models recapitulate hallmark features of tauopathy, including the progressive formation of intracellular, hyperphosphorylated, sarkosyl-insoluble, and conformationally altered Tau aggregates (AT8, MC1 positive), along with synaptic and neuronal dysfunction. Cryogenic electron tomography (cryo-ET) further revealed the morphology of Tau fibrils within cells, as well as the ultrastructure of Tau fibrils trapping synaptic vesicles in situ. Using this platform, we performed integrated phosphoproteomics, high-content screening, and functional validation to identify key pathways driving Tau aggregation. MARK2-mediated phosphorylation within Tau's microtubule-binding domain emerged as an early trigger of aggregation, confirmed by site-specific mutagenesis. In parallel, small molecules targeting the PI3K/mTOR/GSK3 pathway reduced aggregation and restored synaptic function, with GSK3 inhibition lowering phosphorylation at critical aggregation-driving sites on Tau. Together, these findings establish a physiologically relevant, scalable platform for therapeutic screening that connects Tau seed uptake, site-specific phosphorylation, fibril formation, and synaptic disruption, ultimately identifying mechanistically separable intervention points along the aggregation cascade.

cell biology↗

bioRxiv2026

Label-free Pathogen Identification with Microscopy Imaging and Deep Learning

Rapid and accurate pathogen identification is crucial for the clinical management of infectious diseases, particularly sepsis and severe respiratory infections, yet standard clinical workflows remain slow and resource-intensive. Here, we developed an automated, high-throughput imaging platform built on standard, clinically accessible bright-field microscopy, and generated a large dataset comprising 24.9 million label-free bacterial cells across six focal pathogens. Leveraging this resource, we trained a neural network (ESKAPe-ResNet) to identify ESKAPe species at the single-bacterium level. The model achieved >92% accuracy in species-level classification and >82% accuracy in quantifying ESKAPe abundance in mock mixtures, with high specificity against non-ESKAPe bacteria. In clinical validation using sputum, bronchoalveolar lavage fluid and blood samples from patients with respiratory infections and sepsis, the approach correctly identified the dominant ESKAPe pathogen in >78% of samples after minimum broth culture enrichment. The imaging-to-identification pipeline was completed in under 10 minutes, and coupled with brief cultivation, the median time to accurate identification was reduced to 5-6 hours, compared with days for conventional blood culture-based workflows. This work establishes the proof-of-principle for label-free, hardware-minimal rapid pathogen identification, providing a clinically deployable workflow to expedite diagnosis and reduce mortality in severe bacterial infections.

microbiology↗

bioRxiv2026

Toward a networked central dogma: protein specificity reshapes molecular encounters across evolution

Since its formulation in 1958, the Central Dogma has provided the organizing framework for molecular biology, describing the informational relationships among DNA, RNA, and protein. Yet, the framework ends at protein synthesis: what proteins do after they are made, how their catalytic specificities determine which molecules actually meet inside the cell, lies beyond its scope. Using a zero-parameter physical model applied to three phylogenetically distant species, we show that physical encounter frequency and metabolic flux are decoupled: raw collision rates predict reaction importance only weakly and show no consistent link to gene essentiality. Enzymatic selectivity bridges this gap through three distinct, species-conserved rescue patterns that persist across more than a billion years of evolution. The cells most critical metabolic hubs are, paradoxically, its physically least conspicuous ones-- rescued from the collision background by enzymatic precision alone. We term this downstream, measurable layer the Networked Central Dogma: a complement to the classical schema that connects gene expression to the physical organization of cellular chemistry.

evolutionary biology↗

bioRxiv2026

Meditope-Enabled Chimeric Antigen Receptors Facilitate Plug-and-Play Control of T Cells

Chimeric antigen receptor (CAR) T cells have transformed cancer treatment, yet challenges for achieving broader clinical success remain, including overcoming tumor antigen heterogeneity and limited T cell fitness. To address these challenges and enhance CAR T cell functionality, we leveraged meditope technology, a lock-and-key platform where Fab regions of antibodies are modified to bind a small cyclic peptide termed meditope (meP). We developed a panel of meditope-enabled Fab-based CARs (meCARs), which show selective binding to the meP and comparable activity to traditional single-chain variable fragment (scFv)-based CARs. Focusing on HER2-targeted meCARs for evaluating platform utility, we exploited the modularity of the meditope platform to detect meCAR T cells using meP-fused fluorescent agents, promote meCAR T cell expansion via meP-fused IL-15 cytokine, and broaden tumor antigen targeting through meP-fused antibodies to address tumor heterogeneity. These findings establish the meditope technology as a versatile strategy to augment CAR T cell functionality and overcome key limitations of current CAR-based therapies.

bioengineering↗

bioRxiv2025

Steric Gating Dictates Selective Activation of BIRC6 by UBA6 Over UBA1

The giant E2-E3 chimera BIRC6 is specifically activated by the ubiquitin-activating enzyme UBA6, but not by UBA1. This specificity allows BIRC6 to ubiquitinate downstream substrates (e.g., caspases) and thereby regulate apoptosis. However, the molecular mechanism that underlies UBA6mediated specific activation of BIRC6 remains elusive. Here, we used a UbDha (Ub residue 76 dehydroalanine) probe to achieve one-step, activity-based trapping of the transient transthioesterification intermediate, and resolved the cryo-EM structures of two key complexes: 1) the doubly loaded UBA6-BIRC6UBC-UbT-UbA complex (3.4 [A] resolution) and 2) the singly loaded UBA6-BIRC6UBC-UbT complex (3.3 [A] resolution). Structural analysis reveals that the specific BIRC6-UBA6 pairing is dictated by steric compatibility between an insertion loop (residues 4649- 4653) in BIRC6s UBC domain and UBA6s gate helix. Unlike UBA1, UBA6 avoids steric clash with this insertion loop via two key conformational changes: a ~30{degrees} clockwise rotation and a 19 [A] displacement of its gate helix. These conformational changes collectively create a compatible cavity to accommodate the insertion loop. Biochemical validation confirms two key findings: 1) truncating BIRC6s insertion loop rescues UBA1-mediated ubiquitin charging of BIRC6UBC; 2) swapping UBA6s gate helix with that of UBA1 abolishes UBA6s transthioesterification activity towards BIRC6UBC. Our study provides key structural insights into the molecular basis of E1-E2 pairing specificity and advances our mechanistic understanding of apoptotic signalling.

biochemistry↗

bioRxiv2025

Rapid ecosystem collapse and biofilter formation following seabed methane leakage

Globally, vast amounts of methane are trapped within subseafloor gas hydrates. Recent evidence suggests that climate- and human-induced perturbations can destabilize gas hydrates and trigger methane release, yet the response of deep-sea ecosystems to these changes remains poorly understood. Here we show multi-year in situ monitoring of seabed ecosystem following methane leakage induced by hydrate exploration activities. Within just two years, benthic microbial and eukaryotic diversity in the affected areas declined significantly, while microbial and macrofaunal abundance increased. Integrated geochemical and omics analyses reveal the rapid successional shift of seabed ecosystem to a novel chemosynthetic system. Aerobic methanotrophs (Methyloprofundus) co-established with unexpectedly fast growing anaerobic methanotrophic archaea (ANME-3), accompanied by a rapid recruitment of opportunistic polychaetes that bioirrigated the seabed to >50 cm depths. The active animal-microbe interactions sustained exceptionally high rates of methane oxidation that utilized diverse electron acceptors. We demonstrate that methane hydrate destabilization can trigger rapid collapse of native seabed ecosystem while driving the formation of an effective methane biofilter consuming this potent greenhouse gas much faster than previously estimated. Understanding seabed ecosystem response and feedback is critical for predicting benthic methane cycling under ongoing global change and for developing sustainable strategies for methane hydrate resource management.

ecology↗

bioRxiv2025

High precision fluorescence tomography-guided system for pre-clinical radiation research: system design and validation

PurposeCone-beam computed tomography (CBCT), commonly used for image guidance in pre-clinical studies, is limited in soft tissue localization and lacks the functional information needed for image-guided radiation studies and treatment assessment. To address these limitations, we developed 3D fluorescence tomography (FT) for high precision functional image-guided research and integrated it with small animal irradiators. MethodsThe FT system, integrated with a commercial bioluminescence-guided platform in a standalone configuration, enabling compact multi-projection and multi-spectral imaging. A dual-axis galvo mirror scanner was used for laser spots scanning to excite internal fluorophore. A transportable mouse bed allows animals imaged in the optical system and transferred to irradiators for CBCT imaging and FT-guided irradiation. Spatial geometry-based methods were developed to map the laser spots and fluorescence images onto the animal numerical mesh surface, generated from the CBCT image, to serve as input data for FT reconstruction. A self-calibration method using Born-ratio data, fluorescence normalized to the excitation image, was adopted for reconstruction. Mouse phantom embedded with QD800 reporter and orthotopic glioblastoma (GBM) model injected with IRDye 800CW labelled U87-EGFR cells were used to verify the FT-guided system in target localization. ResultsThe laser spot mapping accuracy are within 0.7 mm maximum deviation. Our system can reconstruct the target QD800 as deep as 17 mm in the mouse phantom with single projection fluorescence imaging at localization accuracy < 1 mm deviation. Using the Born ratio approach, we effectively eliminated excitation light leakage and autofluorescence, enabling our FT system to localize IRDye 800CW-labeled EGFR-overexpressing GBM cells in the mouse brain with approximately 1 mm accuracy. ConclusionWe developed a compact FT system integrated with a small animal irradiator, achieving sub-millimeter localization accuracy in phantom and in vivo models. Its precision, flexibility, and compatibility position it as a powerful tool for advancing functional imaging-guided pre-clinical radiation research.

bioengineering↗

bioRxiv2025

Unique Gluing Effect of ASXL1 K351 Monoubiquitination Stimulates PR-DUB-Mediated Nucleosomal H2AK119Ub Deubiquitination

Protein ubiquitination plays a critical role in regulating fundamental biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for its role in targeting proteins for degradation, ubiquitination can also modulate enzymatic activity. However, all of the known mechanisms of such modulation involve spatially constrained mechanisms that take place near the enzyme-substrate interfaces. Here, we report an unprecedented regulatory paradigm mediated by ubiquitination that is located distal to the enzyme-substrate interface but can still activate the polycomb repressive deubiquitinase (PR-DUB) complex. Using cryo-electron microscopy, molecular dynamics (MD) simulations, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and kinetics assays, we determined that the ASXL1 K351 monoubiquitination promotes PR-DUB-mediated nucleosomal H2AK119Ub deubiquitination by stabilizing the catalytic pocket of PR-DUB, causing significant increase in maximal catalytic velocity (Vmax) but almost no change in the substrate binding affinity (Km). Structural analysis revealed that the ubiquitin moiety at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, acting as an intramolecular "glue" that constrains the conformational dynamics of these subunits without altering the nucleosome binding interface. Further MD and HDX-MS results demonstrated the monoubiquitination of ASXL1 K351 enables PR-DUB to effectively maintain a spatial arrangement of the catalytic conformation that closely aligns with substrate cleavage. This study expands our understanding of the mechanisms of ubiquitin function to include intramolecular fastening with a molecular gluing effect and provides a deeper mechanistic understanding of PR-DUB activation in nucleosomal H2AK119Ub removal.

biochemistry↗

bioRxiv2025

Inertial interface cavitation creates complex, flow-like structures within a soft solid

BackgroundInertial cavitation near soft material interfaces generates highly asymmetric bubble dynamics, intense stress localization, and complex fluid-structure interactions. However, the subsurface deformation fields within soft solids remain poorly resolved due to limitations in ultrafast, full-field measurement techniques. ObjectiveThis study aims to quantify the spatiotemporal deformation of soft hydrogels during laser-induced inertial cavitation near a gel-water interface. MethodsWe integrate single-pulse laser-induced inertial cavitation, an embedded internal Digital Image Correlation (DIC) speckle patterning method, and DIC to resolve in situ, full-field subsurface kinematics at 1-2 million frames per second. ResultsAmong all the tested different non-dimensional stand-off distances, four distinct cavitation-interface interaction regimes are identified, spanning symmetric bulk-like oscillations to strongly asymmetric collapses accompanied by interface indentation, jet reversal, and bubble penetration. Full-field measurements reveal stagnation points, vortex-like deformation patterns, and large localized strains that depend on the non-dimensional bubble stand-off distance. ConclusionsThis work establishes an experimental framework for quantifying inertial cavitation dynamics near a compliant gel-water interface. Using ultrafast imaging and DIC, we captured full-field deformation, strain localization, and jet formation across a wide range of stand-off distances near gel-water interfaces.

bioengineering↗

bioRxiv2024

Optimizing Angiopep-2 Density on Polymeric Nanoparticles for Enhanced Blood-Brain Barrier Penetration and Glioblastoma Targeting: Insights from In Vitro and In Vivo Experiments

The blood-brain barrier (BBB) poses a formidable challenge to efficient drug delivery into the brain. One promising approach involves leveraging receptor-mediated transcytosis facilitated by Angiopep-2 peptide (Ang-2)-conjugated nanoparticles. However, the precise impact of Ang-2 density on BBB penetration remains poorly understood. In this study, we developed a versatile polymeric nanoparticle system with tuneable Ang-2 surface density and systemically examined its influence on BBB penetration through various in vitro assays and an in vivo study. Our findings revealed a nuanced relationship between Ang-2 surface density and BBB penetration across the different experimental setups. In 2D cell culture, we observed a positive correlation between Ang-2 surface density and cellular association in hCMEC/D3 cells, characterized by a distinctive inflection point. Conversely, in the Transwell model, higher Ang-2 density correlated negatively with BBB penetration, whereas the BBB-GBM-on-a-chip showed the opposite trend. These disparities may arise from differences in avidity under static versus dynamic conditions, potentially modulating nanoparticle interactions due to fluidic forces. In vivo studies revealed that higher Ang-2 densities facilitated nanoparticle transport across the BBB, consistent with the findings of the BBB-GBM-on-a-chip model. Furthermore, loading doxorubicin into nanoparticles with optimal Ang-2 density resulted in controlled pH-responsive release and enhanced anticancer effect against U87 GBM cells in both 2D cell cultures and a 3D BBB-GBM-on-a-chip model. These results underscore the critical importance of optimizing Ang-2 surface density for efficient BBB penetration and emphasize the utility of dynamic models in preclinical in vitro assessment of novel nanoparticle formulations for targeted delivery to the brain.

bioengineering↗

bioRxiv2024

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally-defined K63-Polyubiquitylated Linker Histone H1

The chemical synthesis of histones with homogeneous modifications is a potent approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). Here, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine-Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on ubiquitylated chromatosomes with different ubiquitin lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally-defined polyubiquitylated H1.0 by CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

biochemistry↗

bioRxiv2024

Integrated multi-omics analyses reveal the pro-inflammatory and pro-fibrotic pulmonary macrophage subcluster in silicosis

BackgroundSilicosis is a lethal occupational disease caused by long-term exposure to respirable silica dust. Pulmonary macrophages play a crucial role in mediating the initiation of silicosis. However, the phenotypic and functional heterogeneities of pulmonary macrophages in silicosis have not been well-studied. MethodsThe silicosis mouse model was established by intratracheal administration of silica suspension. Bronchoalveolar lavage fluids (BALFs) of mice were collected for the multiplex cytokine analysis. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were performed to reveal the heterogeneity and spatial localization of macrophages in the lung tissues. The formation of the fibrotic nodules was characterized by histology, hydroxyproline assay, and immunohistochemical staining, respectively. The expression of the pro-inflammatory or pro-fibrotic genes was investigated by quantitative polymerase chain reaction (qPCR). ResultsWe found that the level of pro-inflammatory cytokines and chemokines is significantly increased in the BALFs of silicosis mice. Apparent collagen deposition can also be observed in the silicotic lung tissues. By scRNA-seq, we have identified a subpopulation of Mmp12hi macrophages significantly expanding in the lung tissues of mice with silicosis. Spatial transcriptomics analysis further confirmed that the Mmp12hi macrophages are mainly enriched in silicosis nodules. Pseudotime trajectory showed that these Mmp12hi macrophages, highly expressing both pro-inflammatory and pro-fibrotic genes are derived from Ly6c+ monocytes. Additionally, 4-octyl itaconate (4-OI) treatment, which can alleviate pulmonary fibrosis in silicosis mice, also reduces the enrichment of the Mmp12hi macrophages. Moreover, we found a subset of macrophages in BALFs derived from patients with silicosis exhibited similar characteristics of Mmp12hi macrophages in silicosis mice models. ConclusionsOur study suggested that a group of Mmp12hi macrophages highly express both pro-inflammatory and pro-fibrotic factors in silicosis mice, and thus may contribute to the progression of fibrosis. The findings have proposed new insights for understanding the heterogeneity of lung macrophages in silicosis, suggesting that the subset of Mmp12hi macrophages may be a potential therapy target to further halt the progression of silicosis.

bioinformatics↗

bioRxiv2024

Blockade of TREM2 ameliorates pulmonary inflammation and fibrosis by modulating sphingolipid metabolism

Pulmonary fibrosis is a chronic interstitial lung disease involving systemic inflammation and abnormal collagen deposition. Dysregulations in lipid metabolism, such as macrophage-dependent lipid catabolism, have been recognized as critical factors for the development of pulmonary fibrosis. However, little is known about the signaling pathways involved and the key regulators. Here we found that triggering receptor expressed on myeloid cells 2 (TREM2) plays a pivotal role in regulating the lipid handling capacities of pulmonary macrophages and triggering fibrosis. By integrating analysis of single-cell and bulk RNA sequencing data from patients and mice with pulmonary fibrosis, we revealed that pulmonary macrophages consist of heterogeneous populations with distinct pro-fibrotic properties, and found that both sphingolipid metabolism and the expression of chemotaxis-related genes are elevated in fibrotic lungs. TREM2, a sensor recognizing multiple lipid species, is specifically upregulated in a subset of monocyte- derived macrophages. Blockade of TREM2 by gene knock-out or soluble TREM2 administration can both attenuate bleomycin-induced pulmonary fibrosis. By utilizing scRNA Seq and lipidomics, we found that Trem2 deficiency downregulates the synthesis of various sphingomyelins, and inhibits the expression of chemokines such as Ccl2. Together, our findings not only reveal the alterations in lipidomic profiles and the atlas of pulmonary macrophages during pulmonary fibrosis, but also suggest that targeting TREM2, the crucial regulator affecting both pulmonary sphingolipid metabolism and the chemokines secretion, can benefit pulmonary fibrosis patients in the future.

pharmacology and toxicology↗

bioRxiv2024

Capturing Snapshots of Nucleosomal H2A K13/K15 Ubiquitination Mediated by the Monomeric E3 Ligase RNF168

AbstractsThe DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, plays a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent Lys13 and Lys15 sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to ubiquitinate H2AK13/15 site-specifically has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c[~]Ub in complex with nucleosomes. Here, we developed mechanism-based chemical trapping strategies and determined the cryo-EM structures of the RNF168/UbcH5c-Ub/NCP complex captured in transient H2AK13/15 monoubiquitination and adjacent dual-monoubiquitination reactions. Our structural analysis revealed that RNF168 stably binds to the nucleosomal H2A-H2B acidic patch through a basic helix with multiple interactions, which positions the UbcH5c active centre directly over the H2A N-terminus, providing a "helix-anchoring" mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the "compass-binding" mode of dimeric E3 ligases. Furthermore, our chemically synthesized ubiquitinated histones have enabled the elucidation of the efficiency of Ub installation and the interplay between the initial and subsequent Ub modifications on the adjacent H2A K13 and K15 sites. Overall, our work not only provides structural snapshots of H2A K13/K15 site-specific monoubiquitination and adjacent dual-monoubiquitination, but also offers a near-atomic resolution structural framework for understanding how pathogenic mutations or physiological modifications affect the molecular function of RNF168 in H2A K13/15 ubiquitination.

biochemistry↗

bioRxiv2023

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFN{gamma})-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFN{gamma}-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFN{gamma}R expression, tumor intrinsic IFN{gamma} signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFN{gamma} receptor influences outcomes of ICB.

cancer biology↗

bioRxiv2023

A Cryptic Basic Groove formed by Ubiquitin and Histone H3 Mediates the Selective Recognition of H2AK119Ub Nucleosomes by Synovial Sarcoma X Breakpoint 1 Protein

The cancer-specific fusion oncoprotein SS18-SSX1 disturbs chromatin accessibility by hijacking the BAF complex from the promoters and enhancers to the polycomb-repressed chromatin regions. This process relies on the selective recognition of H2AK119Ub nucleosomes by SSX1. However, the mechanism by which SSX1 selective recognition of H2AK119Ub nucleosomes in the absence of ubiquitin (Ub)-binding capacity remains unknown. Here we report the cryo-EM structure of SSX1 bound to H2AK119Ub nucleosomes at 3.1 [A] resolution. Combined in vitro biochemical and cellular assays revealed that the Ub recognition by SSX1 is unique and depends on a cryptic basic groove formed by H3 and the Ub motif on the H2AK119 site. Moreover, this unorthodox binding mode of SSX1 induces DNA unwrapping at the entry/exit sites. Together, our results describe a unique mode of site-specific ubiquitinated nucleosome recognition that underlies the specific hijacking of the BAF complex to polycomb regions by SS18-SSX1 in synovial sarcoma.

molecular biology↗

bioRxiv2023

Structural Visualization of HECT-E3 Ufd4 accepting and transferring Ubiquitin to Form K29/K48-branched Polyubiquitination on N-degron

The K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal Ub to the active cysteine of Ufd4 for K29-linked branched ubiquitination. These structures not only provide mechanistic insights into the architecture of the Ufd4 complex but also provide structural visualization of branched ubiquitin chain formation by a HECT-type E3 ligase.

biochemistry↗