Hypothesis

Aging shifts the cysteine redox balance toward sulfonylation, driving pathogenic liquid‑liquid phase separation and irreversible aggregation. We propose that aggregates act as redox sinks that sequester sulfonylation‑prone cysteines, temporarily preventing oxidative damage to essential proteins. The proteostasis network deliberately enrolls vulnerable proteins into these aggregates, converting a dangerous soluble species into a protease‑resistant, amyloid‑like depot. When persulfide (PSSH) levels are restored, the sink releases its cargo, allowing refolding or degradation; without this redox reset, dissolution merely exposes liberated sulfonylated proteins to further oxidative stress, exacerbating dysfunction.

Mechanistic Rationale

1. Redox‑Driven LLPS – Declining PSSH and rising sulfonylation promote formation of static, insoluble condensates from proteins that normally engage in dynamic stress‑response droplets (PMC9909609). Sulfonylated cysteines increase hydrophobicity and β‑sheet propensity, biasing phase separation toward amyloid‑like assemblies.
2. Aggregate Composition – Insoluble fractions enrich for proteins involved in proteostasis, synaptic signaling, and metabolism, many of which contain redox‑sensitive cysteines (PMC6260915). Sequestration of these cysteines limits their availability for aberrant sulfonylation, thereby lowering global oxidative load.
3. Dynamic Release – Enhancing persulfidation (e.g., via H₂S donors or CBS/CSE activation) reverses sulfonylation on aggregate‑resident cysteines, reducing crosslink strength and permitting solubilization by chaperones and the proteasome (2023.11.07.566021). This mirrors the observation that caloric restriction and rapamycin remodel the ubiquitinome and lower insoluble protein burden (10.1093/gerona/glx047).
4. Risk of Premature Clearance – Senolytic or reprogramming strategies that dissolve aggregates without restoring persulfidation release sequestered sulfonylated proteins, causing a surge in oxidative modifications, mitochondrial ROS, and secondary aggregation (10.1126/science.aag3048; 10.1038/nn.4325).

Testable Predictions

• Prediction 1: In aged murine neurons, pharmacological elevation of H₂S (using NaHS) will decrease detergent‑insoluble tau and α‑synuclein levels without altering proteasome activity, measurable by sequential extraction and filter‑trap assay.
• Prediction 2: Knockdown of cystathionine γ‑lyase (CSE) in young cells will increase sulfonylated cysteine content and accelerate formation of protease‑resistant aggregates under mild oxidative stress (H₂O₂ 50 µM).
• Prediction 3: Treating aged microglia with an aggregate‑disaggregase (e.g., HSP110/HSP70/NEF cocktail) in the presence of a persulfidation enhancer will reduce IL‑1β secretion and senescence markers (p16^INK4a^, SA‑β‑gal) compared with disaggregase alone.
• Prediction 4: Mass‑spectrometric redox profiling of isolated aggregates will show enrichment for persulfide‑modified peptides under conditions of high H₂S, whereas sulfonylated peptides dominate when H₂S synthesis is inhibited.

Experimental Approach

• Use sequential detergent fractionation to quantify insoluble protein fractions.
• Employ antibody‑based sulfonylation detection (anti‑SO₂H/SO₃H) and persulfide‑specific labeling (SSP4) followed by immunoblotting or dot‑blot.
• Monitor proteasome activity with fluorogenic substrates to ensure changes are not secondary to degradation capacity.
• Assess cell viability, ROS (MitoSOX), and senescence phenotypes after aggregate manipulation.

If aggregates function as redox sinks, boosting persulfidation should lower aggregate burden and oxidative damage, whereas forcing disaggregation without redox correction will worsen pathology. This reframes therapeutic aim: restore cysteine redox homeostasis before attempting aggregate clearance.