TRIM9 and TRIM26 Interact with UBQLN2^P497H to Modulate Its Proteasomal Degradation

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.