Hierarchical selective autophagy of HAS2 dictates HA molecular weight and dermal fibroblast senescence

Hypothesis

In aged dermal fibroblasts, the selective autophagy machinery prioritizes HAS2-containing complexes for degradation based on ubiquitin chain topology and lysosomal calcium signaling, thereby shifting hyaluronan synthesis from high‑molecular‑weight (HMW) to low‑molecular‑weight (LMW) fragments. This hierarchical targeting is not a passive consequence of bulk autophagy decline but an active triage mechanism that determines the extracellular matrix’s signaling output and drives fibroblast senescence.

Mechanistic Rationale

1. Ubiquitin code determines cargo rank – HAS2 resides in Golgi‑derived vesicles that acquire K63‑linked ubiquitin chains under oxidative stress. The density of these chains, amplified by the E3 ligase TRAF6, confers high affinity for the autophagy adaptor p62/SQSTM1.
2. Lysosomal Ca2+‑TFEB axis sets the threshold – Lysosomal calcium release via MCOLN1 activates calcineurin, which dephosphorylates TFEB, promoting its nuclear translocation and transcription of selective autophagy genes (including p62 and LC3). In aged fibroblasts, basal lysosomal Ca2+ is elevated, lowering the ubiquitin‑density threshold required for HAS2 engulfment.
3. Hierarchy over bulk flux – When autophagy is induced (e.g., by serum starvation), cargos are sorted by a "ubiquitin score": K63‑chains > K48‑chains > non‑ubiquitinated. HAS2 complexes, stress‑induced and heavily K63‑ubiquitinated, outcompete other membrane proteins (e.g., integrin β1) for limited phagophore nucleation sites, leading to their preferential sequestration.
4. Feedback via LMW‑HA – Reduced HMW‑HA synthesis diminishes CD44‑mediated anti‑survival signaling, while hyaluronidases HYAL1/2 continue to generate 2‑6mer oligosaccharides that act as DAMPs through TLR2/4, reinforcing NF‑κB activity and further increasing oxidative stress, thereby amplifying the ubiquitin‑dependent targeting of HAS2.

Testable Predictions

• Prediction 1: Inhibition of TRAF6‑mediated K63 ubiquitination (using siRNA or a dominant‑negative TRAF6 mutant) will reduce HAS2 autophagic flux without altering overall LC3‑II levels, resulting in preserved HMW‑HA secretion and delayed senescence markers (SA‑β‑gal, p16).
• Prediction 2: Pharmacological blockade of MCOLN1 (with ML-SI1) will raise the ubiquitin‑density threshold for autophagic capture, selectively sparing HAS2 while still allowing degradation of bulk cargo; this should rescue HMW‑HA levels and attenuate TLR‑dependent inflammatory signaling.
• Prediction 3: Proximity ligation assays will show increased K63‑ubiquitin‑p62‑HAS2 triads in fibroblasts from old donors compared with young counterparts; disrupting this triad (via p62 UB‑domain mutation) will shift the autophagy cargo hierarchy toward non‑ubiquitinated proteins.
• Prediction 4: Overexpression of a non‑ubiquitinatable HAS2 mutant (lysine‑to‑arginine at predicted ubiquitination sites) will resist autophagic degradation, maintain HMW‑HA synthesis, and suppress senescence even under chronic oxidative stress.

Falsification

If any of the above interventions fail to modify HAS2 turnover, HA size distribution, or senescence readouts while leaving general autophagy flux unchanged, the hypothesis that a ubiquitin‑dependent, lysosomal Ca2+‑regulated hierarchy dictates HAS2 fate would be refuted. Conversely, consistent support across these experiments would establish selective autophagy as a rheostat that translates intracellular stress into extracellular matrix aging.