Proteomic Analysis of TRIM8 Biomolecular Condensates Reveals Proteasome and NFκB Pathways Disrupted in Steroid-Resistant Nephrotic Syndrome/FSGS

Background: C-terminal truncating variants of TRIM8 (tripartite motif-containing 8) cause steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis (SRNS/FSGS) by disrupting TRIM8 localization to undefined nuclear bodies. We previously demonstrated that disease variants disrupt phase separation of TRIM8 into biomolecular condensates, its proteasome-dependent turnover and recruitment of NFκB signaling protein TAK1. However, the complete composition of these novel condensates and how this is altered in SRNS/FSGS is unknown. Methods: TRIM8 interactors were identified in immortalized human podocytes by tandem mass tagging-based quantitative immunoprecipitation (IP) proteomics (LC/MS/MS). Interactors were stratified by protein-protein interaction (PPI) network analysis (STRING by MCL based clustering) and pathway enrichment analysis. Localization of TRIM8 interactors was assessed by indirect immunofluorescence. NF-κB transcriptional activity was evaluated via luciferase assays. Results: IP proteomics identified 13,198 peptides and 2,288 proteins that co-precipitated with either the wildtype or disease mutant forms of TRIM8. 884 proteins were significantly enriched in wildtype TRIM8 groups compared to mutant variants (FDR<1%, fold-change≥3, ≥3 peptides/protein length*1000), indicating the interaction of these proteins was disrupted by disease variants. PPI analysis revealed three large clusters (>20 proteins), of which the largest contained a significant fraction of proteins from the 26S proteasome (38/47). In addition to confirming colocalization of proteasome proteins PSMD4/PSMD12, we demonstrated that the proteasomal activity probe Me4BodipyFL-Ahx3Leu3VS accumulated within TRIM8 nuclear bodies, establishing these condensates house functional proteasomes to mediate TRIM8 turnover. Moreover, TRIM8 regulates TAK1/NFκB signaling, which is required for murine podocyte homeostasis. 15/49 NFκB signaling proteins were enriched in wildtype TRIM8 eluates from IP proteomics data. Consistently, wildtype TRIM8, but not disease mutants, recruit TAK1 into condensates and induced NFκB luciferase activity. Conclusion: NS disease variants disrupt TRIM8 biomolecular condensation and cause aberrant protein quality control and NFκB signaling. Funding: NIDDK Support