HERC1-mediated orphan subunit quality control maintains proteasome homeostasis and is disrupted in HERC1-associated neurodevelopmental disease
Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC1 cause a neurological disorder clinically characterized by macrocephaly, dysmorphic facies, and psychomotor delay. Identifying the substrates of HERC1 is essential for understanding its biological functions and the mechanisms underlying disease-associated variants. To address this, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC1 targets and examine their regulation in cells expressing HERC1 with or without ubiquitin ligase activity. This approach revealed a broad set of HERC1-dependent ubiquitylation substrates, including proteins from major multiprotein complexes such as the proteasome, the translation initiation machinery, and pathways involved in cell metabolism. Focusing on the proteasome, the most prominently represented complex, we identified several regulatory particle subunits, including PSMC6, PSME1, and PSME2. Specifically, within the 19S regulatory particle, we demonstrated that ubiquitylation of the PSMC6 subunit involves the formation of K48-linked polyubiquitin chains and depends on HERC1 activity. Other 19S subunits, such as PSMC2, PSMC4, and PSMC5, undergo a similar modification. Mechanistically, we show that HERC1 selectively recognizes unassembled subunits, promotes their ubiquitylation, and targets them for proteasomal degradation. Notably, in HERC1-deficient cells, disruption of this mechanism impaired proteasomal activity. At the disease level, quantitative proteomic analysis of the pathogenic HERC1 variant c.1448G>A (p.Gly483Glu) likewise identified altered ubiquitylation of proteasome subunits and components of the translation initiation machinery. Biochemical analyses further revealed impaired ubiquitylation of PSMC6 by the mutant HERC1 protein, associated with a loss of interaction between HERC1 and PSMC6. Together, these findings establish a link between HERC1-associated neurological disorders and defective ubiquitylation pathways. They elucidate the molecular mechanisms by which HERC1 regulates proteostasis and demonstrate how disruption of these processes contributes to disease pathogenesis, thereby highlighting potential avenues for therapeutic intervention.