HUWE1 interactomes reveal sex-specific networks of nucleic acid-binding proteins

HUWE1 (HECT, UBA, and WWE domain-containing protein 1) is an X-linked E3 ubiquitin ligase that regulates a broad range of substrates through both degradative and non-degradative ubiquitination. The essential role of HUWE1 is underscored by the neonatal lethality of Huwe1 knockout mice. Although HUWE1 has been extensively studied in cancer and neurodevelopment, its functions in brain aging and neurodegeneration remain poorly understood. Here, we used cross-linking-assisted immunoprecipitation coupled with mass spectrometry to characterize HUWE1 interactomes in 26-month-old wild-type and Tg-SwDI mouse brains. HUWE1 interactomes were enriched for proteins involved in gene expression, translation, synaptic signaling, and nucleic acid metabolism. HUWE1-associated protein complexes contained an extensive network of nucleic acid-binding proteins, including the G-quadruplex-associated helicases DDX5, DDX3X, DDX3Y, and DDX17, the nucleic acid-binding proteins nucleolin and FUS, and the DNA topology regulator TOP2B. These interactomes exhibited both disease- and sex-specific organization. Independent biochemical validation confirmed the association between HUWE1-containing complexes and the G-quadruplex helicase DDX5. In addition, APOE was uniquely detected in Tg-SwDI HUWE1 interactomes and was more abundant in females than in males, suggesting a disease- and sex-specific association between HUWE1-containing complexes and APOE. Taken together, these findings identify HUWE1 as a component of sex-specific nucleic acid-binding protein networks in the aging brain and provide new insights into its potential roles in neurodegeneration.