Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron loss. ALS-linked mutations in UBQLN2 promote protein aggregation and disrupt proteostasis, yet the mutation-specific protein interactomes and their functional relevance remain poorly defined. We employed APEX2 proximity labeling, together with affinity enrichment of biotinylated peptides and LC-MS/MS analysis, to profile the interactomes of wild-type UBQLN2 and two ALS-linked variants, UBQLN2(P497H) and UBQLN2(P497S). We identified 785 unique biotinylated proteins, many of which exhibit augmented enrichment in the proximity proteomes of the two mutants over wild-type UBQLN2. Notably, the E3 ubiquitin ligases TRIM9 and TRIM26 were selectively enriched in the proximity proteome of UBQLN2(P497H), which we validated by coimmunoprecipitation followed by Western blot analysis. Fractionation analysis revealed coaccumulation of TRIM9 and TRIM26 with UBQLN2(P497H) in the insoluble fraction, consistent with its heightened aggregation propensity. Treatment of UBQLN2(P497H)-expressing cells with a proteasomal inhibitor led to elevated accumulation of a C-terminal UBQLN2 fragment that is absent in cells expressing wild-type UBQLN2 or its P497S mutant. Individual knockdown of TRIM9 and TRIM26 significantly increased the abundance of the fragment, establishing UBQLN2(P497H) as a substrate for TRIM9- and TRIM26-mediated ubiquitinylation and subsequent proteasomal degradation. These findings nominate TRIM9 and TRIM26 as specific interactors of UBQLN2(P497H) and as regulators of a previously underexplored C-terminal UBQLN2 fragment, suggesting that impaired clearance of this species may contribute to ALS pathogenesis.