Hypothesis

When proteostasis capacity is exceeded, cells direct misfolded proteins to aggresomes where they are converted into highly ordered, disulfide‑cross‑linked amyloid fibrils. These fibrils are not inert debris; they function as redox sinks that bind free thiol groups, lower the pool of labile cysteines, and thereby buffer oxidative stress. By sequestering cysteines, aggresome fibrils influence glutathione synthesis and recycling, shifting the redox balance toward a more reduced state that supports autophagy and delays proteostasis collapse. Dissolving these fibrils without augmenting antioxidant capacity should increase labile cysteine, elevate reactive oxygen species, and accelerate proteotoxic damage, even if overall protein load remains unchanged.

Mechanistic reasoning

1. Disulfide‑driven fibril maturation – In long‑lived species, aggresome formation is accompanied by upregulation of ER oxidoreductases (e.g., PDIA1) and peroxiredoxins that promote disulfide bond formation within aggregating proteins 2. The resulting fibrils are enriched in cystine bonds, making them thermodynamically stable and capable of scavenging thiols.
2. Thiol sequestration alters glutathione homeostasis – Free cysteines are precursors for glutathione (GSH). When fibrils bind these thiols, the cytosolic cysteine pool drops, feedback‑inhibiting γ‑glutamylcysteine synthetase and reducing GSH synthesis. Conversely, the cell compensates by upregulating glutathione reductase and import, creating a tighter redox buffer 3.
3. Redox state regulates autophagy flux – A reduced cytosol favors activation of ATG4 and LC3 lipidation, enhancing autophagosome formation 4. Thus, aggresome fibrils indirectly promote clearance of damaged organelles and protein aggregates by shaping the redox environment.
4. Transition to pathology – If fibril formation outpaces thiol sequestration capacity, excess disulfides can form aberrant mixed disulfides with essential proteins, inhibiting their activity. Simultaneously, depleted cysteine limits GSH replenishment, tipping the balance toward oxidative stress and impairing autophagy—a feed‑forward loop that converts a protective sink into a source of toxicity 6.

Testable predictions

• Prediction 1: In cells from long‑lived species (e.g., naked mole rat, human fibroblasts), aggresome isolates will show higher cystine content and greater ability to bind exogenous thiol‑reactive probes compared with aggregates from short‑lived species (e.g., mouse).
• Prediction 2: Pharmacological inhibition of disulfide bond formation (using DCAD or bacitracin) during aggresome induction will reduce fibril thiol‑binding capacity, increase labile cysteine measured by thiol‑specific fluorescent probes, and elevate ROS levels without changing total aggregate load.
• Prediction 3: Enhancing glutathione synthesis (via N‑acetylcysteine overexpression) will rescue the proteolytic defect caused by fibril dissolution, whereas boosting autophagy alone will not prevent ROS‑mediated damage if cystine scavenging is compromised.
• Prediction 4: In vivo, mice expressing a mutant form of a model aggregation‑prone protein that cannot form disulfide‑linked fibrils will exhibit earlier onset of motor deficits and higher oxidative markers, despite similar aggregate burden, relative to wild‑type fibrillating controls.

Experimental approach (outline)

1. Aggregate purification – Induce aggresome formation with proteasome inhibitor MG132 in primary fibroblasts from species of differing lifespan. Isolate aggresomes via centrifugation, quantify cystine using Ellman’s assay under reducing/non‑reducing conditions.
2. Redox readouts – Measure labile cysteine (ThiolTracker), GSH/GSSG ratio, and ROS (CellROX) in parallel cultures treated with disulfide‑formation inhibitors or activators.
3. Functional assays – Assess autophagic flux (LC3‑II turnover with bafilomycin A1) and cell viability after aggregate dissolution with HSP110/Hsp70 disaggregase mix.
4. In vivo validation – Generate knock‑in mice expressing a cysteine‑to‑serine mutant of HTT‑exon1 that blocks disulfide‑linked fibrillization; monitor behavior, oxidative stress markers, and survival.

Falsifiability

If aggresome fibrils do not sequester labile cysteines or modulate glutathione/ROS levels—as shown by unchanged thiol pools and oxidative stress after fibril disruption—then the redox‑sink mechanism is refuted. Conversely, consistent elevation of labile cysteine and ROS upon fibril loss, coupled with rescue by antioxidant supplementation, would support the hypothesis.

Keywords: aggresome, disulfide bonds, cysteine sequestration, glutathione redox buffer, proteostasis threshold