<p>Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC2 cause a neurodevelopmental disorder clinically resembling Angelman syndrome, characterized by global developmental delay, intellectual disability, autism spectrum features, and movement abnormalities. Defining the target substrates of HERC2 is essential for understanding its biological role and the mechanisms underlying its pathological variants. To this end, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC2 targets and assess their regulation in cells expressing HERC2 with or without ubiquitin‑ligase activity. This approach revealed extensive sets of subunits from major multimeric complexes, including the proteasome, the tRNA–biosynthesis machinery, microtubule‑associated assemblies, vesicle‑coat complexes, centrosomes, and the Ski and GATOR2 complexes, as substrates of HERC2-dependent ubiquitylation. Among these, the proteasome emerged as the most prominently affected complex. We identified up to eleven proteins required for assembly of the 19S regulatory particle whose ubiquitylation depends on HERC2. Mechanistically, we show that HERC2 recognizes unassembled subunits via chaperone-mediated interactions and targets them for proteasomal degradation. Loss of this mechanism in HERC2‑deficient cells alters proteasomal activity. It is noteworthy that fibroblasts derived from patients carrying the common pathogenic variant c.1781 C &gt; T (p.Pro594Leu) exhibit impaired processing of 19S subunits and an aberrant increase in proteasome activity. Our findings establish a link between HERC2-related neurodevelopmental disorder and impaired proteasome activity, elucidate the molecular mechanisms through which HERC2 regulates proteostasis and how its disruption contributes to human pathology, and underscore potential therapeutic strategies for affected individuals.</p>