Aggregates as Redox Sinks

Protein aggregation in aging may represent a controlled redox‑buffering system rather than mere waste. Hyperoxidation of cysteine residues (to sulfinic/sulfonic acids) promotes liquid‑liquid phase separation (LLPS) that sequesters these reactive thiols into insoluble deposits, lowering cytosolic oxidative pressure. When the ubiquitin‑proteasome system is overwhelmed, the cell actively shuttles hyperoxidized proteins to microtubule‑organized inclusions, converting a dangerous soluble pool into a thermodynamically stable solid phase.

Mechanistic Extension

We hypothesize that aggregates function as redox sinks: they store oxidizing equivalents derived from cysteine hyperoxidation, and their reversible persulfidation can release reducing equivalents back to the cytosol on demand. Persulfidation (‑SSH) decreases with age, tipping the balance toward hyperoxidation and aggregation. Restoring persulfidation should dissolve aggregates without releasing toxic oligomers, because the sequestered cysteines are already in their highest oxidation state and cannot regenerate harmful soluble intermediates.

• Step 1: Cysteine thiols become hyperoxidized (Cys‑SO₂H/Cys‑SO₃H) under chronic oxidative stress.
• Step 2: Hyperoxidized cysteines promote LLPS via increased polarity and hydrogen‑bonding capacity, nucleating chaperone‑rich hydrogels.
• Step 3: Hydrogels mature into amyloid‑like aggregates that sequester the oxidized species, preventing aberrant disulfide‑mediated cross‑links with vital proteins.
• Step 4: Persulfidating enzymes (e.g., cystathionine γ‑lyase, 3‑mercaptopyruvate sulfurtransferase) donate thiol groups to aggregate surfaces, converting Cys‑SO₂H/‑SO₃H back to Cys‑SSH, which destabilizes the solid phase and promotes solubilization.

Testable Predictions

1. Genetic augmentation of persulfidation pathways in aged C. elegans or human myotubes will increase Cys‑SSH levels on aggregate‑associated proteins, reduce insoluble protein fraction, and improve motility without elevating soluble oligomer load.
2. Pharmacological boosting of persulfidation (using Na₂S or H₂S donors) will shift the equilibrium from Cys‑SO₂H/‑SO₃H toward Cys‑SSH, detectable by mass‑spec‑based redox proteomics, and concomitantly decrease aggregate‑associated ROS production measured with HyPer sensors.
3. Inhibition of persulfidation (with AOAA or PAG) in young cells under oxidative challenge will accelerate hyperoxidation‑dependent LLPS and aggregate formation, confirming the causal link.
4. Falsification: If enhancing persulfidation fails to reduce insoluble protein or instead increases soluble oligomer toxicity, the redox‑sink model is refuted.

Experimental Outline

• Use CRISPR‑knock‑in of a Cys‑SO₂H‑specific antibody to visualize hyperoxidized substrates in vivo.
• Perform fractionation assays (Triton‑X100 soluble vs. SDS‑insoluble) before and after persulfidation manipulation.
• Measure lifespan, stress resistance, and proteotoxicity reporters (e.g., polyQ‑YFP aggregation) in C. elegans.
• In human skeletal‑muscle cultures, assess myotube diameter and contractility after aggregate modulation.

Broader Implications

If aggregates serve as redox reservoirs, therapeutic strategies should aim not at wholesale clearance but at regulated redox exchange—modifying the thiol chemistry on aggregate surfaces to safely mobilize stored equivalents when needed. This perspective reframes amyloid‑like deposits as dynamic, physiologically regulated compartments whose malfunction arises from impaired redox cycling rather than from their existence per se.