Hypothesis

In aged tissues, the redox‑sensitive cysteines within GLI transcription factors undergo oxidative thiol hyperoxidation (sulfinylation/sulfonylation) that promotes liquid‑liquid phase separation (LLPS) and subsequent aggregation. Initially, this aggregation acts as a protective sequestration mechanism—GLI is chaperoned into compact, HSP70/HSP40‑enriched aggregates that limit aberrant Hedgehog signaling and preserve the soluble proteome. When oxidative stress exceeds a critical threshold, these aggregates transition to a pathological state: they grow beyond ~500 nm, expose hydrophobic surfaces, adopt smaller oligomeric cores (~30 nm), and trigger cytokine release, thereby sequestering GLI away from target genes and collapsing morphogen gradients essential for tissue repair.

Mechanistic Reasoning

1. Thiol Oxidation Triggers LLPS – GLI’s 15‑20 zinc‑finger cysteines are prone to sulfonylation under chronic ROS, reducing zinc affinity and increasing surface hydrophobicity, which drives LLPS (see 3).
2. Chaperone‑Mediated Triage – HSP70/HSP40 bind early LLPS droplets, forming compact, reversible aggregates that sequester excess GLI and reduce noise in signaling (2). This represents proteostatic triage: the cell prefers rapid insolubilization over degradation when proteasomal/lysosomal capacity wanes (4).
3. Switch to Pathology – Persistent sulfonylation overwhelms chaperone capacity, leading to aggregate maturation, exposure of hydrophobic patches, and activation of NLRP3 inflammasome‑like pathways, converting GLI aggregates into inflammatory seeds (2).
4. Functional Outcome – Protective GLI aggregates attenuate basal Hedgehog output, preventing ectopic proliferation; pathological aggregates deplete nuclear GLI, flattening the gradient and impairing fibroblast‑to‑myofibroblast transition during wound healing.

Testable Predictions

• Prediction 1: In aged human dermal fibroblasts, inducing GLI cysteine sulfonylation (e.g., with low‑dose diamide) will increase GLI‑positive aggregates >500 nm, reduce nuclear GLI, and diminish PTCH1 transcriptional response to SHG; co‑treatment with N‑acetylcysteine or overexpressing HSP70 will reverse these effects.
• Prediction 2: Expressing a redox‑insensitive GLI mutant (Cys→Ser in zinc‑finger domains) in aged mice will lower aggregate burden, restore SHH‑dependent gene expression, and accelerate excisional wound closure compared with wild‑type GLI overexpression.
• Prediction 3: Pharmacological enhancement of the ubiquitinome (e.g., rapamycin) will decrease overall GLI insolubility but will not affect the redox‑sensitive switch; only thiol‑reducing agents will shift aggregates from the pathological to the protective state.

Experimental Approach

1. Biochemical: Quantify GLI sulfonylation via dimedone‑based chemoproteomics; assess aggregate size distribution by filtration and fluorescence‑correlation spectroscopy.
2. Cellular: Use GLI‑GFP reporters to measure nuclear/cytoplasmic ratios and transcriptional activity (luciferase under Gli‑responsive promoter).
3. In vivo: Employ aged GLI‑GFP reporter mice; administer topical diamide or NAC, monitor wound closure rates, histology, and Gli‑target expression via qPCR and immunostaining.
4. Modulation: CRISPR knock‑in of Cys‑Ser GLI; HSP70 overexpression via AAV; rapamycin treatment to test ubiquitinome effects.

If the data show that modulating cysteine redox state—not just overall proteostasis—dictates whether GLI aggregates are protective or pathogenic, this hypothesis will be substantiated. Conversely, if GLI aggregation correlates solely with total insolubility independent of specific thiol modifications, the hypothesis will be falsified.