Hypothesis

Protein aggregates function as transient redox buffers that sequester hyperoxidized cysteine residues, preventing aberrant disulfide‑driven toxicity. When the cellular reducing capacity falls below a threshold, these deposits mature into pathological fibrils; dissolving them without restoring thiol homeostasis releases reactive oxidants and accelerates damage.

Mechanistic Rationale

Recent work shows that RNA‑induced liquid‑liquid phase separation drives aggregation of hyperoxidized GAPDH【4】, linking cysteine over‑oxidation to phase separation. In yeast, Hsp42‑mediated deposits initially protect daughter cells by sequestering misfolded species【1】. We propose that the sequestered cargo includes not only misfolded polypeptides but also proteins bearing sulfinic or sulfonic acid cysteine modifications. By trapping these oxidized thiols, the aggregate lowers the free concentration of reactive species, buying time for NADPH‑dependent reductases (e.g., thioredoxin, glutaredoxin) to restore native thiols. If the reductive flux is insufficient, the aggregate undergoes a secondary transition: disulfide cross‑linking and β‑sheet stabilization convert the depot into a stable amyloid that resists chaperone remodeling. This shift explains why aggregate clearance can be deleterious—removing the redox sink unleashes the trapped oxidants before the cell has regenerated its reducing power.

Testable Predictions

1. In cells expressing a roGFP2‑Orp1 sensor, the formation of early Hsp42‑dependent deposits will correlate with a localized decrease in cytosolic H₂O₂ and a rise in the proportion of protein‑bound sulfinic cysteine (detected by dimedone‑based labeling).
2. Genetic or pharmacological enhancement of thioredoxin reductase activity will shift the equilibrium toward smaller, more dynamic deposits and delay the appearance of Thioflavin‑positive fibrils.
3. Acute dissolution of aggregates with Hsp104 overexpression, in the absence of concomitant NADPH regeneration, will cause a transient spike in intracellular oxidants and reduce viability, whereas co‑expression of glucose‑6‑phosphate dehydrogenase rescues survival.

Experimental Design

• Use a yeast strain expressing GFP‑tagged GAPDH(C152S) to monitor aggregation and a cytosolic roGFP2‑Orp1 for real‑time H₂O₂.
• Quantify sulfinic cysteine levels via biotin‑switch assay after dimedone treatment at 0, 2, 6, and 12 h post‑stress (e.g., 0.5 mM H₂O₂).
• Manipulate reductive capacity: overexpress TRR1 (thioredoxin reductase) or GPD1 (glycerol‑3‑phosphate dehydrogenase for NADPH) and compare deposit size (filter‑trap assay) and fibril formation (ThT fluorescence).
• Induce disaggregation with GAL‑Hsp104; measure oxidant spikes with roGFP2 and colony‑forming units after 4 h.
• Parallel mammalian cultures (neuronal SH‑SY5Y) transfected with tau‑K18‑C322S mutant to test conservation; assess protein‑sulfinicylation and cell death after tau aggregate dissolution with or without NAC supplementation.

If predictions hold, the data would support the view that aggregates are a controlled redox‑sink strategy, and that therapeutic clearance must be paired with redox‑restoration to avoid unleashing sequestered oxidants.