Abstract: SA-PO617
Nephrotic Syndrome-Associated Variants Dysregulate TRIM8 Functions in Biomolecular Condensates, Cell Adhesion, and Glomerular Injury
Session Information
- Genetic Kidney Diseases: Models, Mechanisms, and Therapies
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1202 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Sharma, Vineeta, Boston Children's Hospital, Boston, Massachusetts, United States
- Singh, Hari R., Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
- Hong, Sunwoo, Boston Children's Hospital, Boston, Massachusetts, United States
- Rubin, Alexander, Boston Children's Hospital, Boston, Massachusetts, United States
- Ball, David A., Boston Children's Hospital, Boston, Massachusetts, United States
- Gauntner, Victoria C., Boston Children's Hospital, Boston, Massachusetts, United States
- Salant, David J., Boston University Chobanian & Avedisian School of Medicine, Boston, Massachusetts, United States
- Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
- Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States
Background
C-terminal truncating TRIM8 variants (tripartite motif containing 8) cause human nephrotic syndrome (NS) and mislocalization of TRIM8 from nuclear bodies. However, the effects of these variants at the physicochemical, molecular, cellular and whole organism levels are poorly understood.
Methods
Tagged TRIM8 constructs were expressed in U2OS cells or immortalized human podocytes. Time lapse fluorescence microscopy, immunofluorescence staining, confocal microscopy, coimmunoprecipitation and immunoblotting were performed. CRISPR/Cas9-mediated gene editing in immortalized human podocytes and in mice was performed to generate disease mimicking C-terminal truncating variants.
Results
Tagged TRIM8 nuclear bodies exhibited rapid fluorescence recovery after photobleaching (T-half=10s) and fusion consistent with liquid-like condensates in U2OS cells. Supporting this classification, as bulk tagged TRIM8 concentration increased to 4 µM, its concentration within nuclear bodies increased up to 20 µM while remaining low outside (<2 µM). In silico algorithms (ESpritz, IUPred, PrDOS, PONDR) detected intrinsically disordered region (IDR) features in the TRIM8 C-terminus. In correlation, truncation of the IDR impaired TRIM8 condensate formation in vitro. In immortalized podocytes, NS-associated C-terminal IDR truncating variants impaired tagged TRIM8 polyubiquitination and 26S-dependent degradation, suggesting potential gain-of-function. Endogenous proteasome components (PSMD4, PSMD12) co-localized with tagged wildtype TRIM8—but not NS mutants—in condensates, implicating these condensates in proteasome-dependent TRIM8 regulation. TAK1 is a published TRIM8 substrate that regulates cell adhesion and murine podocyte homeostasis. TAK1 co-localized with wildtype TRIM8 in condensates and coimmuoprecipitated, which NS variants reduced. Rather, TRIM8 NS mutant protein caused reduced TAK1 protein levels. At the cellular level, CRISPR-mediated TRIM8 variants led to reduced immortalized podocyte adhesion. As compared to wildtype controls, Trim8 mutant mice developed increased albuminuria after nephrotoxic serum injury.
Conclusion
TRIM8 forms proteasome-associated biomolecular condensates that may impact its regulation of TAK1, cell adhesion, and podocyte injury.
Funding
- NIDDK Support