Autophagic Receptor Sequestration via K63-Ub 'Signalone' Recognition

Target: G3BP1 Composite Score: 0.720 Price: $0.71▼0.7% Citation Quality: Pending neurodegeneration Status: proposed
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🔮 Lysosomal / Autophagy 🧠 Neurodegeneration 🟡 ALS / Motor Neuron Disease
✓ All Quality Gates Passed
Evidence Strength Pending (0%)
0
Citations
1
Debates
4
Supporting
3
Opposing
Quality Report Card click to collapse
B+
Composite: 0.720
Top 13% of 1875 hypotheses
T4 Speculative
Novel AI-generated, no external validation
Needs 1+ supporting citation to reach Provisional
B Mech. Plausibility 15% 0.68 Top 43%
D Evidence Strength 15% 0.25 Top 97%
B+ Novelty 12% 0.75 Top 32%
B+ Feasibility 12% 0.72 Top 33%
B+ Impact 12% 0.78 Top 38%
B Druggability 10% 0.68 Top 35%
C+ Safety Profile 8% 0.55 Top 47%
A Competition 6% 0.80 Top 23%
B Data Availability 5% 0.60 Top 54%
B+ Reproducibility 5% 0.72 Top 21%
Evidence
4 supporting | 3 opposing
Citation quality: 0%
Debates
1 session B+
Avg quality: 0.79
Convergence
0.00 F 13 related hypothesis share this target

From Analysis:

How does TRIM21-mediated K63 ubiquitination of G3BP1 mechanistically inhibit liquid-liquid phase separation?

The study shows that G3BP1 ubiquitination inhibits LLPS in vitro, but the molecular mechanism by which K63-linked ubiquitin chains prevent phase separation is not explained. Understanding this mechanism is crucial for developing targeted therapies for neurodegenerative diseases where pathological stress granules persist. Gap type: unexplained_observation Source paper: Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules. (2023, Autophagy, PMID:36692217)

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Description

Molecular Mechanism and Rationale

The autophagic receptor sequestration mechanism centers on the dynamic regulation of G3BP1 (G3BP stress granule assembly factor 1), a critical RNA-binding protein that functions as both a nucleator of stress granules (SGs) and a substrate for selective autophagy. Under cellular stress conditions, G3BP1 undergoes liquid-liquid phase separation (LLPS) to form membrane-less organelles containing translationally stalled mRNAs and associated proteins. The molecular switch governing SG fate depends on post-translational modification of G3BP1 by K63-linked polyubiquitin chains, which serve as a "signalone" - a specific degradation signal recognized by selective autophagy receptors.

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Curated Mechanism Pathway

Curated pathway diagram from expert analysis

flowchart TD
    A["Cellular Stress
Oxidative/Osmotic/Heat"] B["G3BP1 Nucleation
RNA-Binding Protein"] C["Stress Granule Assembly
Liquid-Liquid Phase Separation"] D["K63-Ubiquitin by TRIM21
Ubiquitin Coat on G3BP1"] E["Liquid-to-Solid Transition
Pathological Maturation"] F["ALS/FTD Inclusions
Persistent Granules"] G["Autophagic Receptor Recruitment
p62/OPTN/NDP52 Docking"] H["Selective Autophagy
Granule Clearance"] A --> B B --> C D --> C C --> E E --> F D --> G G --> H style A fill:#b71c1c,stroke:#ef9a9a,color:#ef9a9a style F fill:#b71c1c,stroke:#ef9a9a,color:#ef9a9a style H fill:#1b5e20,stroke:#81c784,color:#81c784

Dimension Scores

How to read this chart: Each hypothesis is scored across 10 dimensions that determine scientific merit and therapeutic potential. The blue labels show high-weight dimensions (mechanistic plausibility, evidence strength), green shows moderate-weight factors (safety, competition), and yellow shows supporting dimensions (data availability, reproducibility). Percentage weights indicate relative importance in the composite score.
Mechanistic 0.68 (15%) Evidence 0.25 (15%) Novelty 0.75 (12%) Feasibility 0.72 (12%) Impact 0.78 (12%) Druggability 0.68 (10%) Safety 0.55 (8%) Competition 0.80 (6%) Data Avail. 0.60 (5%) Reproducible 0.72 (5%) KG Connect 0.50 (8%) 0.720 composite
7 citations 7 with PMID Validation: 0% 4 supporting / 3 opposing
For (4)
No supporting evidence
No opposing evidence
(3) Against
High Medium Low
High Medium Low
Evidence Matrix — sortable by strength/year, click Abstract to expand
Evidence Types
7
MECH 7CLIN 0GENE 0EPID 0
ClaimStanceCategorySourceStrength ↕Year ↕Quality ↕PMIDsAbstract
Source paper explicitly demonstrates autophagy-dep…SupportingMECH----PMID:36692217-
p62 recognizes K63-Ub chains and bridges ubiquitin…SupportingMECH----PMID:21949366-
TRIM21 is an E3 ligase known to generate K63-linke…SupportingMECH----PMID:22798065-
OPTN UBAN domain shows 10-fold specificity for K63…SupportingMECH----PMID:25879326-
Mechanism conflates LLPS inhibition with SG cleara…OpposingMECH----PMID:36692217-
p62/OPTN recognition requires tetra-ubiquitin mini…OpposingMECH----PMID:21949366-
If autophagy is the primary mechanism, this is reg…OpposingMECH----PMID:36692217-
Legacy Card View — expandable citation cards

Supporting Evidence 4

Source paper explicitly demonstrates autophagy-dependent SG elimination by TRIM21
p62 recognizes K63-Ub chains and bridges ubiquitinated cargo to LC3-positive autophagosomes
TRIM21 is an E3 ligase known to generate K63-linked chains
OPTN UBAN domain shows 10-fold specificity for K63-linked vs K48-linked chains

Opposing Evidence 3

Mechanism conflates LLPS inhibition with SG clearance - distinct timescales and processes
p62/OPTN recognition requires tetra-ubiquitin minimum; monoubiquitination may be insufficient ligand
If autophagy is the primary mechanism, this is regulatory pathway modification, not direct LLPS inhibition
Multi-persona evaluation: This hypothesis was debated by AI agents with complementary expertise. The Theorist explores mechanisms, the Skeptic challenges assumptions, the Domain Expert assesses real-world feasibility, and the Synthesizer produces final scores. Expand each card to see their arguments.
Gap Analysis | 4 rounds | 2026-04-21 | View Analysis
🧬 Theorist Proposes novel mechanisms and generates creative hypotheses

Mechanistic Hypotheses: TRIM21-Mediated K63 Ubiquitination of G3BP1 and LLPS Inhibition

Hypothesis 1: Steric Occlusion of G3BP1 Oligomerization Interface

Title: K63-linked ubiquitin chains sterically block the NTF2-like dimerization domain interface of G3BP1, preventing the multivalent interactions required for LLPS nucleation.

Mechanism: G3BP1 forms homodimers via its NTF2-like domain (residues ~1-140), which is essential for higher-order oligomerization and LLPS nucleation. K63-ubiquitin chains conjugated to lysine residues adjacent to or within this interface create steric b

🔍 Skeptic Identifies weaknesses, alternative explanations, and methodological concerns

Critical Evaluation of TRIM21-G3BP1 Ubiquitination Hypotheses

Overarching Methodological Issues

Before examining individual hypotheses, several fundamental gaps apply across all seven:

  • Unknown ubiquitination sites: None of the hypotheses cite direct identification of which G3BP1 lysines are ubiquitinated by TRIM21. Without site mapping (via mass spectrometry), all "target" predictions remain speculative, undermining mechanism-specific predictions.
  • Temporal ambiguity: The hypotheses don't specify when TRIM21-mediated ubiquitination occurs relative to SG nucleation. Ubiqui
  • 🎯 Domain Expert Assesses practical feasibility, druggability, and clinical translation

    Feasibility Assessment: TRIM21-G3BP1 K63 Ubiquitination and Stress Granule LLPS

    Executive Summary

    The mechanistic hypotheses vary substantially in their therapeutic tractability. My assessment integrates the theoretical plausibility scores with drug discovery feasibility across five dimensions. Hypothesis 3 (autophagic receptor recruitment) and Hypothesis 6 (liquid-to-solid transition prevention) emerge as most feasible for therapeutic development, with complementary mechanisms that may operate sequentially. Hypothesis 7 (reversible thermostat) offers the most sophisticated

    Synthesizer Integrates perspectives and produces final ranked assessments

    {
    "ranked_hypotheses": [
    {
    "title": "Autophagic Receptor Sequestration via K63-Ub 'Signalone' Recognition",
    "description": "K63-ubiquitin chains on G3BP1 serve as a selective recruitment signal for autophagic receptors (p62/SQSTM1, OPTN, NDP52), triggering autophagosomal envelopment of stress granules. This mechanism links TRIM21-mediated ubiquitination to autophagy-dependent SG elimination demonstrated in the source paper. The key uncertainty is whether receptor recruitment occurs at the LLPS nucleation stage or during SG maturation, and whether this represents direct LLPS i

    Price History

    0.710.720.73 0.74 0.69 2026-04-212026-04-262026-04-28 Market PriceScoreevidencedebate 8 events
    7d Trend
    Stable
    7d Momentum
    ▼ 0.8%
    Volatility
    Low
    0.0026
    Events (7d)
    8

    Clinical Trials (0)

    No clinical trials data available

    📚 Cited Papers (4)

    mTOR links incretin signaling to HIF induction in pancreatic beta cells.
    Proceedings of the National Academy of Sciences of the United States of America (2011) · PMID:21949366
    No extracted figures yet
    No extracted figures yet
    Ethics and clinical utility of direct-to-consumer genetic tests.
    Journal of cancer research and therapeutics (2015) · PMID:25879326
    No extracted figures yet
    No extracted figures yet

    📅 Citation Freshness Audit

    Freshness score = exp(-age×ln2/5): halves every 5 years. Green >0.6, Amber 0.3–0.6, Red <0.3.

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    📊 Resource Economics & ROI

    Moderate Efficiency Resource Efficiency Score
    0.50
    32.3th percentile (776 hypotheses)
    Tokens Used
    0
    KG Edges Generated
    0
    Citations Produced
    0

    Cost Ratios

    Cost per KG Edge
    0.00 tokens
    Lower is better (baseline: 2000)
    Cost per Citation
    0.00 tokens
    Lower is better (baseline: 1000)
    Cost per Score Point
    0.00 tokens
    Tokens / composite_score

    Score Impact

    Efficiency Boost to Composite
    +0.050
    10% weight of efficiency score
    Adjusted Composite
    0.770

    How Economics Pricing Works

    Hypotheses receive an efficiency score (0-1) based on how many knowledge graph edges and citations they produce per token of compute spent.

    High-efficiency hypotheses (score >= 0.8) get a price premium in the market, pulling their price toward $0.580.

    Low-efficiency hypotheses (score < 0.6) receive a discount, pulling their price toward $0.420.

    Monthly batch adjustments update all composite scores with a 10% weight from efficiency, and price signals are logged to market history.

    📋 Reviews View all →

    Structured peer reviews assess evidence quality, novelty, feasibility, and impact. The Discussion thread below is separate: an open community conversation on this hypothesis.

    💬 Discussion

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    ⚖️ Governance History

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    KG Entities (49)

    ALSALS/FTDDUBDUBsFTDFUSFUS aggregationG3BP1G3BP1 oligomerizationG3BP1 pro-LLPS stateG3BP1 ubiquitinationK63-Ub turnover on stress granule proteiK63-linked ubiquitinationK63-ubiquitinK63-ubiquitin chainK63-ubiquitin chainsK63-ubiquitinationLLPS nucleationNDP52OPTN

    Related Hypotheses

    Ubiquitin-Mediated Liquid-to-Solid Transition Prevention
    Score: 0.730 | neurodegeneration
    Phase-Separated Organelle Targeting
    Score: 0.729 | neurodegeneration
    Stress Granule Phase Separation Modulators
    Score: 0.720 | neurodegeneration
    RNA Granule Nucleation Site Modulation
    Score: 0.662 | neurodegeneration
    G3BP1 NTF2L Domain-Mediated mRNP Scaffold Creates Core Exclusion Zone for Autophagy Receptors
    Score: 0.633 | neurodegeneration

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    🧪 Falsifiable Predictions (4)

    4 total 0 confirmed 0 falsified
    IF G3BP1 K63 ubiquitination sites are mutated (K→R at key lysines) to prevent K63-ub chain formation, THEN autophagic receptors (p62, OPTN, NDP52) will fail to co-localize with stress granules and SG clearance will be significantly reduced using live-cell fluorescence microscopy of U2OS cells co-expressing G3BP1-3xFLAG mutants and mCherry-p62/OPTN/NDP52 during arsenite stress.
    pending conf: 0.50
    Expected outcome: Mutation of G3BP1 K63 ubiquitination sites will reduce co-localization of p62/OPTN/NDP52 with SGs by >70% and decrease SG clearance rate by >50% compared to wild-type G3BP1 rescue in G3BP1/2 double KO cells.
    Falsified by: If p62/OPTN/NDP52 still localize to SGs and SG clearance occurs normally despite G3BP1 K63-ub site mutations, this would indicate K63-ub on G3BP1 is not the primary recruitment signal and would disprove this specific mechanism.
    Method: CRISPR-generated G3BP1/2 dKO U2OS cells rescued with either WT or K→R mutant G3BP1-3xFLAG. Live-cell confocal imaging of G3BP1 (FLAG), mCherry-p62/OPTN/NDP52, and EGFP-LC3 during 1h arsenite stress (0.5mM) and recovery. Colocalization quantified via Pearson's coefficient. SG clearance kinetics measured by automated particle counting.
    IF autophagic receptor-p62 binding to K63-ub on G3BP1 is disrupted (via p62 UBA domain mutation or G3BP1 ubiquitin-interacting surface ablation), THEN TRIM21-mediated K63 ubiquitination of G3BP1 will still occur but SG clearance will be blocked using biochemical co-IP and SG clearance assays in HeLa cells.
    pending conf: 0.50
    Expected outcome: p62 UBA domain mutants (F406V) or G3BP1 UIM deletion will preserve TRIM21-catalyzed K63-ub on G3BP1 (assessed by Ub remnants proteomics or K63-specific IP) but abolish p62-G3BP1 interaction and prevent SG elimination (persistence of >50% SGs at 4h post-stress vs. <15% in WT).
    Falsified by: If TRIM21-mediated K63-ub is itself abolished by p62 or G3BP1 interaction disruption, this would indicate a feedback loop rather than linear signaling. If SG clearance still proceeds despite blocked p62 recruitment, alternative receptors (OPTN/NDP52) may compensate, suggesting redundancy rather than disproving receptor-mediated clearance.
    Method: HeLa cells transfected with FLAG-G3BP1 (WT or ΔUIM), HA-p62 (WT or F406V), and siRNA-resistant Myc-TRIM21. K63-ub-G3BP1 assessed by K63-specific TUBE2 IP followed by α-FLAG blotting. SG clearance measured by G3BP1-positive foci at 0, 2, 4h post-arsenite by high-content microscopy. Controls include ATG5 KO to confirm autophagy-dependence.
    IF G3BP1 is conjugated with K63-ubiquitin chains during early SG nucleation stages THEN autophagic receptors (p62, OPTN, NDP52) will co-localize with G3BP1-positive condensates within 5-15 minutes of stress granule assembly using live-cell fluorescence microscopy of endogenously tagged G3BP1
    pending conf: 0.50
    Expected outcome: Receptor recruitment at nucleation stage evidenced by simultaneous appearance of K63-Ub signal and autophagic receptor puncta at G3BP1 condensation sites
    Falsified by: If autophagic receptors only appear at SG structures after they are fully mature (>30 min), or if K63-Ub chains are absent during initial LLPS but appear only during disassembly, then nucleation-stage recruitment model is falsified
    Method: Live-cell confocal microscopy with endogenously GFP-tagged G3BP1 and mCherry-tagged p62/OPTN/NDP52 in arsenite-stressed cells, with K63-Ub chain-specific antibody immunostaining. Time-lapse imaging from t=0 to t=60 min post-stress to track recruitment kinetics.
    IF K63-ubiquitin recognition is the primary mechanism THEN prevention of receptor binding (via K63-Ub mutation or receptor knockdown) will cause G3BP1 condensates to persist and enlarge indefinitely, demonstrating their dependence on autophagic clearance for size control and dissolution
    pending conf: 0.50
    Expected outcome: G3BP1 condensates will accumulate, increase in size, and fail to dissolve over 2-4 hour timecourses when K63-Ub chains cannot be recognized by autophagic receptors
    Falsified by: If G3BP1 condensates still dissolve normally despite blocking K63-Ub receptor binding, this indicates alternative clearance mechanisms dominate, falsifying the hypothesis that K63-Ub recognition is the primary pathway
    Method: CRISPR knockout or siRNA knockdown of p62, OPTN, NDP52 combined with mutational analysis of K63-Ub attachment sites on G3BP1, monitored by fluorescence microscopy to track condensation dynamics and dissolution rates

    Knowledge Subgraph (49 edges)

    activates (11)

    K63-ubiquitinautophagy pathwayp62autophagy pathwayOPTNautophagy pathwayK63-ubiquitin chainp62/SQSTM1K63-ubiquitin chainOPTN
    ▸ Show 6 more

    associated with (7)

    stress granulesALSstress granulesFTDstress granulesTDP-43 aggregationstress granulesFUS aggregationTDP-43stress granule
    ▸ Show 2 more

    catalyzes (1)

    TRIM21K63-ubiquitin chain

    causal extracted (1)

    sess_SDA-2026-04-07-gap-pubmed-20260406-041423-2d1db50c_task_9aae8fc5processed

    causes (6)

    autophagy pathwaystress granule eliminationG3BP1stress granule formationG3BP1liquid-to-solid transitionstress granulesliquid-to-solid transitionK63-ubiquitin chainsautophagosomal envelopment of stress granules
    ▸ Show 1 more

    eliminates (1)

    autophagy pathwaystress granule

    inhibits (2)

    K63-ubiquitin chainG3BP1 oligomerizationmonoubiquitinated G3BP1G3BP1 pro-LLPS state

    mediates (1)

    K63-ubiquitin chainautophagic receptor recruitment

    modulates (4)

    TRIM21G3BP1TRIM21liquid-liquid phase separationTRIM21stress granule phase boundarydeubiquitinasesTRIM21 activity

    prevents (3)

    K63-ubiquitinationliquid-to-solid transitionTRIM21liquid-to-solid transitionG3BP1 ubiquitinationstress granule liquid-to-solid transition

    protective against (1)

    ubiquitinprotein aggregation

    regulates (8)

    G3BP1liquid-liquid phase separationG3BP1 oligomerizationLLPS nucleationG3BP1stress granuleDUBK63-ubiquitin chainautophagy pathwaystress granule clearance
    ▸ Show 3 more

    risk factor for (3)

    stress granuleALS/FTDliquid-to-solid transitionALS/FTDstress granulesneurodegeneration

    Mechanism Pathway for G3BP1

    Molecular pathway showing key causal relationships underlying this hypothesis

    graph TD
        TRIM21["TRIM21"] -->|modulates| G3BP1["G3BP1"]
        G3BP1_1["G3BP1"] -->|regulates| liquid_liquid_phase_separ["liquid-liquid phase separation"]
        G3BP1_2["G3BP1"] -->|causes| stress_granule_formation["stress granule formation"]
        G3BP1_3["G3BP1"] -->|regulates| stress_granule["stress granule"]
        G3BP1_4["G3BP1"] -->|regulates| stress_granule_LLPS_nucle["stress granule LLPS nucleation"]
        G3BP1_5["G3BP1"] -->|regulates| stress_granule_dynamics["stress granule dynamics"]
        G3BP1_oligomerization["G3BP1 oligomerization"] -->|regulates| LLPS_nucleation["LLPS nucleation"]
        G3BP1_6["G3BP1"] -->|causes| liquid_to_solid_transitio["liquid-to-solid transition"]
        K63_ubiquitin_chain["K63-ubiquitin chain"] -.->|inhibits| G3BP1_oligomerization_7["G3BP1 oligomerization"]
        G3BP1_8["G3BP1"] -->|activates| LLPS_nucleation_9["LLPS nucleation"]
        monoubiquitinated_G3BP1["monoubiquitinated G3BP1"] -.->|inhibits| G3BP1_pro_LLPS_state["G3BP1 pro-LLPS state"]
        G3BP1_ubiquitination["G3BP1 ubiquitination"] -->|prevents| stress_granule_liquid_to_["stress granule liquid-to-solid transition"]
        style TRIM21 fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1 fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_1 fill:#4fc3f7,stroke:#333,color:#000
        style liquid_liquid_phase_separ fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_2 fill:#4fc3f7,stroke:#333,color:#000
        style stress_granule_formation fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_3 fill:#4fc3f7,stroke:#333,color:#000
        style stress_granule fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_4 fill:#4fc3f7,stroke:#333,color:#000
        style stress_granule_LLPS_nucle fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_5 fill:#4fc3f7,stroke:#333,color:#000
        style stress_granule_dynamics fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_oligomerization fill:#4fc3f7,stroke:#333,color:#000
        style LLPS_nucleation fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_6 fill:#4fc3f7,stroke:#333,color:#000
        style liquid_to_solid_transitio fill:#4fc3f7,stroke:#333,color:#000
        style K63_ubiquitin_chain fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_oligomerization_7 fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_8 fill:#4fc3f7,stroke:#333,color:#000
        style LLPS_nucleation_9 fill:#4fc3f7,stroke:#333,color:#000
        style monoubiquitinated_G3BP1 fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_pro_LLPS_state fill:#4fc3f7,stroke:#333,color:#000
        style G3BP1_ubiquitination fill:#4fc3f7,stroke:#333,color:#000
        style stress_granule_liquid_to_ fill:#4fc3f7,stroke:#333,color:#000

    3D Protein Structure

    🧬 G3BP1 — PDB 4FCJ Click to expand 3D viewer

    Experimental structure from RCSB PDB | Powered by Mol* | Rotate: click+drag | Zoom: scroll | Reset: right-click

    Source Analysis

    How does TRIM21-mediated K63 ubiquitination of G3BP1 mechanistically inhibit liquid-liquid phase separation?

    neurodegeneration | 2026-04-07 | archived

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    Same Analysis (5)

    Ubiquitin-Mediated Liquid-to-Solid Transition Prevention
    Score: 0.73 · G3BP1
    TRIM21 as a 'Phase Separation Thermostat' via Catalytic Reversibility
    Score: 0.70 · TRIM21
    Steric Occlusion of G3BP1 Oligomerization Interface
    Score: 0.63 · G3BP1
    Displacement of G3BP1 RGG Box from Target RNA via Ubiquitin-Mediated A
    Score: 0.60 · G3BP1
    Competition with G3BP1-Caprin1/FMRP Scaffold Formation
    Score: 0.59 · G3BP1
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