Biology subjects

Hur, S.

Publications and source records attributed to Hur, S..

14 recordsLinked to original sources

bioRxiv2026

A dynamic molecular glue targets a plastic MDA5 filament interface to stimulate innate immunity

Innate immune signaling is a double-edged sword: uncontrolled activation drives autoinflammatory diseases, while controlled activation enhances antiviral and antitumor immunity. MDA5, a cytosolic dsRNA sensor that activates type I interferon signaling, mediates both immunopathology and protective immunity, yet small-molecule modulators have remained elusive. Here we report MA2, a first-in-class selective MDA5 agonist that acts as a molecular glue. MA2 binds a cryptic inter-protomer pocket within MDA5 filaments that undergoes ATPase-associated conformational remodeling. Rather than rigidly locking the interface, MA2 remains bound while accommodating and reshaping these structural transitions, strengthening MDA5-dsRNA and inter-protomer interactions and attenuating ATP hydrolysis. MA2 does not induce new filament assembly but potentiates pre-existing filaments, enabling context-dependent activation in dsRNA-enriched cells. MA2 enhances antiviral activity and synergizes with chemotherapeutic agents and natural killer cells to promote tumor cell killing. These findings establish dynamic molecular glues as a strategy for targeting conformationally plastic interfaces previously considered undruggable.

immunology↗

bioRxiv2026

Identification of highly immunogenic endogenous dsRNAs from cellular MDA5 filaments

ADAR1 converts adenosine to inosine in endogenous double-stranded RNAs (dsRNAs) to prevent excessive MDA5-driven interferon-stimulated gene expression. The source of endogenous immunogenic dsRNAs remains enigmatic because only a small fraction of ADAR1 substrates activate MDA5, and cellular MDA5 filaments have not been isolated. Here, we couple affinity purification of cellular MDA5 filaments with RNA sequencing to define immunogenic endogenous dsRNAs. Greater than 84% of dsRNAs suppressed by combined DDX3X RNA helicase and ADAR1 base-editing activities were present in MDA5 filaments, compared to less than 1% of dsRNA substrates acted on by ADAR1 alone. Dual substrate dsRNAs consisted of inverted repeats embedded in 3-UTRs with high base-pair complementarity and longer intervening sequences between repeats, with a minor contribution coming from intermolecular dsRNAs formed by sense and antisense transcripts. Moreover, the majority of dual substrate immunogenic dsRNAs were hyperedited in DDX3X mutant cancers. This reveals the identity of endogenous immunogenic dsRNAs and quality control mechanisms underlying their suppression.

cell biology↗

bioRxiv2026

RIG-I-like receptors sense replication stress through ribosomal DNA integrity to drive inflammation

Replication stress, a major source of DNA damage and vulnerability in cancer, activates innate immunity through both the cGAS-STING and RIG-I-like receptor (RLR) pathways, yet the mechanism for RLR activation remains poorly understood despite frequent suppression of cGAS-STING in cancer. Here, we show that replication stress induces genomic instability at ribosomal DNA (rDNA), accompanied by aberrant RNA polymerase I-dependent sense and antisense transcription that generates immunostimulatory RNA activating RLR signaling. RLR activation required CDK1 activity but occurred independently of mitosis or micronuclei formation. Cas9-mediated disruption of transcribed rDNA regions was sufficient to induce dysregulated rDNA transcription and RLR activation. Human tumor genomes also showed increased structural variations at rDNA, consistent with rDNA instability. Together, these findings reveal how genomic instability drives inflammation through rDNA-derived immunostimulatory RNA and identify a previously unappreciated consequence of damage-induced transcription.

molecular biology↗

bioRxiv2026

MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity

Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA51,2, a conserved innate immune receptor that recognizes viral dsRNA by forming filaments3. Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies--beyond antibody affinity or nucleic acid presence alone--govern innate immune activation.

biochemistry↗

bioRxiv2024

Ultrastable and Versatile FoxP3 Ensembles on Microsatellites

Microsatellites are essential genomic components increasingly linked to transcriptional regulation. FoxP3, a transcription factor critical for regulatory T cell (Treg) development, recognizes TTTG repeat microsatellites by forming multimers along DNA. However, FoxP3 also binds a broader range of TnG repeats (n=2-5), often at the edges of accessible chromatin regions. This raises questions about how FoxP3 adapts to sequence variability and the potential role of nucleosomes. Using cryo-electron microscopy and single-molecule analyses, we show that FoxP3 assembles into distinct supramolecular structures depending on DNA sequence. This structural plasticity enables FoxP3 to bridge 2-4 DNA duplexes, forming ultrastable structures that coordinate multiple genomic loci. Nucleosomes further facilitate FoxP3 assembly by inducing local DNA bending, creating a nucleus that recruits distal DNA elements through multiway bridging. Our findings thus reveal FoxP3s unusual ability to shapeshift to accommodate evolutionarily dynamic microsatellites and its potential to reinforce chromatin boundaries and three-dimensional genomic architecture.

biochemistry↗

bioRxiv2024

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKRs ability to scan along dsRNA--a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKRs movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.

biochemistry↗

bioRxiv2024

SARM1 orthosteric base exchange inhibitors cause subinhibitory SARM1 activation

SARM1, an octameric NADase, is a key regulator of axon degeneration and an emerging target in small molecule drug discovery to treat a wide range of neurodegenerative diseases. Recently, a structurally diverse series of adduct-forming, orthosteric SARM1 inhibitors have been discovered. Here, we show that subinhibitory concentrations of these orthosteric inhibitors, under mildly SARM1 activating conditions, cause sustained SARM1 activation. This synergistic adverse effect leads to increased nicotinamide adenine dinucleotide (NAD) consumption, neurodegeneration and release of the biomarker neurofilament-light (NfL) in cultured cortical neurons. In two distinct animal models, we found that low-dose treatment with these orthosteric SARM1 inhibitors results in increased plasma NfL and adverse events when combined with cellular stress or injury conditions. This may present a critical liability for orthosteric SARM1 inhibitors in certain patient populations.

neuroscience↗

bioRxiv2023

A new mode of transcriptional hub assembly by autoimmune regulator Aire

Transcriptional condensates play a crucial role in gene expression and regulation1-4, yet the mechanisms governing their assembly at the correct location and time remain poorly understood2,5-7. We here report a multi-layered mechanism for condensate assembly by the Autoimmune regulator (Aire), an essential transcriptional regulator (TR) that orchestrates gene expression reprogramming for central T-cell tolerance8,9. Previous studies demonstrated that Aire utilizes its polymerizing domain CARD to form nuclear condensates critical for its transcriptional activity10,11. Our current data show that Aire condensates assemble on enhancers, stimulating local transcriptional activities, while also connecting disparate inter-chromosomal genomic loci. This process of functional condensate formation hinges upon the coordination between three Aire domains: the CARD, the histone binding domain PHD1 and the activation domain C-terminal tail (CTT). Specifically, CTT directly binds CBP/p300 coactivators, recruiting Aire to CBP/p300-rich enhancers and promoting CARD-mediated condensate assembly. Conversely, PHD1 restrains spontaneous Aire polymerization by binding to the ubiquitous unmethylated H3K4, ensuring Aire remains dispersed throughout the genome until it nucleates on enhancers. Accordingly, deletion of CTT or PHD1 leads to a complete loss of Aires functions through distinct mechanisms--deletion of CTT abrogates Aire condensate formation, whereas deletion of PHD1 leads to dysfunctional Aire condensates detached from chromatin. Thus, our findings highlight the balance between PHD1-mediated suppression and CTT-mediated stimulation of Aire polymerization to form transcriptionally active condensates at appropriate target sites, providing new insights into controlled polymerization of TRs.

cell biology↗

bioRxiv2023

Ladder-like multimerization of FoxP3 enables microsatellite recognition and DNA bridging

FoxP3 is a transcription factor (TF) essential for development of regulatory T cells (Tregs), a branch of T cells that suppress excessive inflammation and autoimmunity1-5. Molecular mechanisms of FoxP3, however, remain elusive. We here show that FoxP3 utilizes the Forkhead domain--a DNA binding domain (DBD) that is commonly thought to function as a monomer or dimer--to form a higher-order multimer upon binding to TnG repeat microsatellites. A cryo-electron microscopy structure of FoxP3 in complex with T3G repeats reveals a ladder-like architecture, where two double-stranded DNA molecules form the two "side rails" bridged by five pairs of FoxP3 molecules, with each pair forming a "rung". Each FoxP3 subunit occupies TGTTTGT within the repeats in the manner indistinguishable from that of FoxP3 bound to the Forkhead consensus motif (FKHM; TGTTTAC). Mutations in the "intra-rung" interface impair TnG repeat recognition, DNA bridging and cellular functions of FoxP3, all without affecting FKHM binding. FoxP3 can tolerate variable "inter-rung" spacings, explaining its broad specificity for TnG repeat-like sequences in vivo and in vitro. Both FoxP3 orthologs and paralogs show similar TnG repeat recognition and DNA bridging. These findings thus reveal a new mode of DNA recognition that involves TF homo-multimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

biochemistry↗

bioRxiv2022

FoxP3 can fold into two distinct dimerization states with divergent functional implications for T cell homeostasis

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrate that, unlike other known forkhead TFs, FoxP3 forms a head-to-head dimer using a unique linker (Runx1-binding region, RBR) preceding the forkhead domain. Head-to-head dimerization confers distinct DNA-binding specificity and creates a docking site for the cofactor Runx1. RBR is also important for proper folding of the forkhead domain, as truncation of RBR induces domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impairs FoxP3 function, as demonstrated with the disease-causing mutation R337Q, while a swap-suppressive mutation largely rescues R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap-dimerization with impaired functions.

immunology↗

bioRxiv2021

Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, we find that host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independent of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis-by dampening both toxic immune response and viral replication.

immunology↗

bioRxiv2020

Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance

Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, is known to form large aggregate-like assemblies visualized as nuclear foci. We demonstrate that Aire utilizes Caspase Activation Recruitment Domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization is a double-edged sword as it also makes Aire susceptible to interaction with PML bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs Aires transcriptional activity, while dispersing PML bodies with a viral antagonist restores it. Thus, our study reveals a new regulatory role of PML bodies in Aire function and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated quality control.

immunology↗

bioRxiv2018

Non-canonical role of E3 ligase RIPLET in innate immunity as a co-receptor for RIG-I

Ubiquitin (Ub) and its E3 ligases play diverse biological roles, from proteasomal degradation to innate immune signaling. The conventional view posits that E3 ligases function primarily through conjugating Ub to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET utilizes its dimeric structure and the bivalent binding mode to preferentially recognize RIG-I preoligomerized on dsRNA. RIPLET can also adopt an alternative binding mode for filamentous oligomers of RIG-I assembled on longer dsRNAs, cross-bridging RIG-I filaments in both ubiquitin-dependent and-independent manners. The resultant receptor clustering leads to the formation of aggregate-like cytosolic granules and the stimulation of RIG-I-mediated antiviral signaling in a RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. This study also highlights previously unrecognized mechanisms by which an E3 ligase induces receptor clustering and signal amplification, and offers insights into the unique cellular function of membrane-less granule assembly.\n\nOne-sentence summaryRIPLET functions as a molecular glue to assemble signaling scaffolds for the antiviral innate immune receptor RIG-I.

immunology↗

bioRxiv2018

An origin of the immunogenicity of in vitro transcribed RNA

The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.

molecular biology↗