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Abstract: SA-PO1007

Nephrotic Syndrome-Associated TRIM8 Variants Impair the Proteasome-Dependent Turnover and Condensation of TRIM8 Protein

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Sharma, Vineeta, Boston Children's Hospital, Boston, Massachusetts, United States
  • Rubin, Alexander, Boston Children's Hospital, Boston, Massachusetts, United States
  • Liang, Lorrin, Boston University School of Medicine, 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
  • Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States
  • Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States
Background

Nephrotic syndrome (NS) is the second leading cause of chronic kidney disease. De novo C-terminal truncating variants in TRIM8 (tripartite motif containing 8) cause a novel form of NS. As N-terminal truncating TRIM8 variants present in gnomAD controls precluded haploinsufficiency, we hypothesized that NS-associated TRIM8 variants cause a podocytopathy through dominant-negative or gain-of-function mechanisms.

Methods

Protein-protein interactions were determined by co-immunoprecipitation (co-IP). The 26S proteasome inhibitor MG132 was used to modulate the levels of tagged wildtype (WT) and patient variant (PV) TRIM8 protein. The half-life of tagged TRIM8 protein was assessed by cycloheximide chase assay. Tagged WT and PV TRIM8 protein ubiquitination was measured by IP upon overexpression of HA-tagged Ubiquitin. GFP-tagged WT and PV TRIM8 protein cellular localization was evaluated by confocal microscopy.

Results

Wildtype and patient variant TRIM8 co-immunoprecipitated with WT TRIM8, indicating NS-associated TRIM8 variants do not impair TRIM8-TRIM8 interactions. MG132 treatment increased WT TRIM8 but not PV TRIM8 protein levels, demonstrating disease-associated variants impair 26S proteasome-dependent TRIM8 degradation. PV TRIM8 exhibited increased stability (half-life > 5 hours) relative to WT TRIM8 protein (half-life 3 hours) by cycloheximide chase assay. Tagged TRIM8 WT protein underwent polyubiquitination, which was impaired by NS-associated TRIM8 variants. GFP-tagged WT TRIM8 exhibited increased condensation to nuclear bodies upon proteasome inhibition, while PV TRIM8 maintained pan-nuclear localization in the presence or absence of MG132.

Conclusion

NS-associated variants in TRIM8 cause increased TRIM8 protein stability but reduced protein condensation, suggesting these variants have hyper- or neomorphic effects.

Funding

  • NIDDK Support