Persulfidation-dependent phase separation of IDPs disrupts TRiC chaperone function and drives mitochondrial interactome decay in aging

Hypothesis

Age‑related loss of protein persulfidation promotes aberrant liquid‑liquid phase separation (LLPS) of specific intrinsically disordered proteins (IDPs). These condensates sequester subunits of the TRiC/CCT chaperone complex, reducing its availability for folding nascent polypeptides and mitochondrial assembly factors. Consequently, TRiC deficiency leads to misassembly of electron‑transport‑chain complexes I and IV, reproducing the interactome and respiration defects observed in aged mouse skeletal muscle.

Mechanistic Basis

Persulfidation modifies cysteine residues on many IDPs, keeping them soluble and preventing sticky interactions. Recent work shows endogenous protein persulfidation declines with age [3], coinciding with increased LLPS propensity. TRiC is known to prevent aggregation of client proteins and is preserved in cognitively healthy centenarians [4]. If persulfidation loss drives IDPs into LLPS droplets that trap TRiC, the chaperone’s network connectivity drops, mirroring the universal decline of ATP‑dependent chaperone PPI specificity reported in aging [2]. Sequestered TRiC cannot assist the folding of mitochondrial import factors and ETC subunits, leading to the specific disruptions in complex I and IV assembly, glutamate dehydrogenase activity, and CoA‑binding enzymes identified by XL‑MS [1]. This links a molecular‑scale PTM defect to organelle‑scale interactome remodeling.

Testable Predictions

1. In aged C. elegans and Drosophila, pharmacological restoration of persulfidation (e.g., NaHS or CSE overexpression) will reduce IDP‑positive LLPS foci, increase soluble TRiC levels, and rescue mitochondrial complex I respiration.
2. Genetic disruption of LLPS‑prone IDPs (e.g., deletion of low‑complexity domains) will prevent TRiC sequestration and ameliorate age‑related interactome changes even without persulfidation rescue.
3. Conversely, acute induction of LLPS (via optogenetic clustering of an IDP) in young worms will mimic aged TRiC loss, decrease complex I activity, and shorten lifespan.
4. Centenarian‑derived fibroblasts will show higher basal persulfidation and lower IDP‑LLPS burden compared with age‑matched controls, persisting after in‑vitro aging stress.

Experimental Approach

• Measure persulfidation: Use biotin‑switch assay or MS‑based persulfidomics in young vs. old worms, flies, and mouse muscle.
• Visualize LLPS: Express GFP‑tagged candidate IDPs (e.g., FUS‑like, TIA‑1 homologs) and quantify droplet number/size by confocal microscopy.
• Assess TRiC availability: Perform TurboID‑based proximity labeling of TRiC subunits followed by AP‑MS to quantify changes in interacting partners under persulfidation modulation.
• Mitochondrial readouts: Seahorse XF analysis of OXPHOS capacity, BN‑PAGE for complex I/IV assembly, and glutamate dehydrogenase activity assays.
• Lifespan/healthspan: Track survival and motility in C. elegans after pharmacological or genetic manipulations.

A falsifiable outcome would be that enhancing persulfidation fails to alter LLPS, TRiC interactome, or mitochondrial respiration despite verified chemical rescue, thereby refuting the causal link proposed.