Abstract: SA-PO0758
Proteomic Analysis of TRIM8 Biomolecular Condensates Reveals Proteasome and NFκB Pathways Disrupted in Steroid-Resistant Nephrotic Syndrome/FSGS
Session Information
- Glomerular Diseases: Profiling Through Multiomics
November 08, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Sharma, Vineeta, Boston Children's Hospital, Boston, Massachusetts, United States
- Yu, Guanghao, Boston Children's Hospital, Boston, Massachusetts, United States
- Ranga, Arathi, Boston Children's Hospital, Boston, Massachusetts, United States
- Hong, Sunwoo, Boston Children's Hospital, Boston, Massachusetts, United States
- Rubin, Alexander, Boston Children's Hospital, Boston, Massachusetts, United States
- Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States
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