E3 ligase recruitment by UBQLN2 protects substrates from proteasomal degradation

Ubiquilins are a family of proteins critical to cellular proteostasis that are also linked to several neurodegenerative diseases, with specific mutations in UBQLN2 causing dominant, X-linked ALS. Despite an initial characterization as proteasomal shuttle factors, Ubiquilins have paradoxically been reported to stabilize numerous substrates. The basis of this triage decision remains enigmatic. Many other fundamental aspects of Ubiquilin function are unclear at the mechanistic level, such as the physiological significance of Ubiquilin phase separation, the unique role of each Ubiquilin paralog, and the mechanistic defects of ALS mutants. To address these questions, we utilized a library of triple knockout (TKO) rescue cell lines with physiological expression of single Ubiquilin paralogs or disease mutants in an isogenic background. Our findings reveal that UBQLN2 has a unique ability to protect substrates from degradation and that substrate stabilization correlates with the recruitment of multiple E3 ligases, including SCF^bxo7. We propose that E3 ligase recruitment promotes UBQLN2 phase separation, which protects substrates from proteasomal degradation. Consistent with this model, we demonstrate that ALS mutants, which were previously shown to have altered phase separation properties, also show a defect in substrate stabilization. Finally, we show that substrate stabilization appears to be a general feature of proteins that interact with the UBQLN2 Sti1 domains as amyloid precursor protein (APP) is also protected from proteasomal degradation by the formation of biomolecular condensates. This proposal unifies many existing observations in the field and presents a new paradigm for understanding Ubiquilin function in neurodegenerative disease.