The ubiquitylation system regulates diverse substrates and is closely linked to human pathology. While the proteasome efficiently degrades soluble substrates, challenging targets ─ such as protein complexes and aggregates ─ often require additional factors like the ubiquitin (Ub)-dependent unfoldase VCP/p97. How the ubiquitylation system recognizes challenging substrates and generates Ub signals to recruit VCP/p97 remains unclear.
We identified HUWE1 and its “Ub Signal Amplification” (UbSA) as a key regulator in degrading challenging substrates. HUWE1, a HECT-family E3 ligase, recognizes pre-existing Ub chains on substrates and rapidly amplifies Ub signal. Beyond elongating Ub chains, HUWE1 conjugates new Ub chains directly on substrate. The latter activity, termed “Ubiquitin-Directed ubiquitin Ligase” (UDL), is necessary for UbSA. HUWE1-amplified Ub signals effectively stimulate substrate unfolding by the purified p97–Npl4–Ufd1 complex.
We examined HUWE1’s role in clearing challenging substrates, particularly protein aggregates. HUWE1 colocalizes with Alzheimer's biomarkers, such as tau aggregates and ASC specks, in mouse brains and patient samples. In HEK293 cells lacking UDL and/or conventional E3 activity, UDL was required for clearing protein aggregates induced by synphilin, tau or AgDD (a chemically-inducible aggregation system), poly-Q inclusion bodies and stress granules. HUWE1 depletion reduces Ub and VCP/p97 localization at aggregates, while HUWE1 overexpression or induced proximity enhanced aggregate clearance.
In summary, the HUWE1-VCP/p97 axis is a critical regulator of protein homeostasis, mediating the clearance of challenging substrates─including protein complexes, condensates, and aggregates─and holds therapeutic potential for diseases involving disrupted proteostasis.