Modeling the impact of variants in the neuro-renal disease gene TRIM8
Embargo Date
2027-09-25
OA Version
Citation
Abstract
Nephrotic syndrome (NS) is a grave disorder of the kidney filtration system that can develop into chronic kidney disease (CKD) or end-stage renal disease (ESRD). TRIM8 is an E3 ubiquitin ligase of the ubiquitin-proteasome system (UPS), which is responsible for intracellular protein degradation. TRIM8 is also implicated in cancer and inflammation. Research by my mentor Dr. Majmundar has shown that de novo C-terminal truncating variants in the gene TRIM8 (tripartite motif containing 8) cause glomerular injury, NS, neurodevelopmental delays, and epileptic symptoms. More recently, the Majmundar laboratory has identified de novo missense variants in pediatric patients without renal disease suggesting these variants may cause distinct dysfunction in TRIM8 from the original truncating variants. TRIM8 is also implicated in other cellular processes that have yet to be further investigated in NS. For this thesis research, levels of TRIM8 proteins in wild type (WT), patient truncating variants (PVs), and missense variants (MVs) were modulated by MG132, a known 26S proteasome inhibitor. Transfection of immortalized human podocytes with GFP-tagged or mCherry-tagged TRIM8 and ubiquitin allowed for immunofluorescence studies to examine the conversion of nuclear morphological patterns and nuclear co-localization. FLAG-tagged TRIM8 proteins were assessed through western blotting to compare the protein expression levels at different concentrations of MG132.
MG132-treated GFP-tagged WT TRIM8 localized to nuclear condensates with increasing doses of plasmids. Diffuse staining at the lower dose of transfected plasmid steadily transitioned to a nuclear condensate pattern.
GFP-tagged WT TRIM8 was further analyzed with GFP-tagged TRIM8 PVs (Q411* and Y460*) under universal MG132 treatment. TRIM8 PVs did not form nuclear condensates but maintained diffuse and pan-nuclear morphology, unlike GFP-tagged WT TRIM8.
When immunofluorescent images of immortalized podocytes were transfected with GFP-tagged WT TRIM8 and mCherry-tagged TRIM8 PVs, the size and regularity of nuclear condensates were negatively impacted. The co-localization data between WT TRIM8 and TRIM8 PVs may putatively explain the dominant negative effect of C-terminal truncating mutation on the localization and enzymatic activity of WT TRIM8.
Immunoblotting of FLAG-tagged WT TRIM8 and FLAG-tagged TRIM8 MVs with different concentrations of MG132 yielded different results amongst different missense variants. WT TRIM8 protein expression appeared to be dose dependent on MG132 at each concentration. E12A and R152G TRIM8 were dose-dependent in a binary manner. Lastly, C52S and K105del did not establish dose dependency from MG132 treatments. Overall, these data showed that UPS-dependent regulation of TRIM8 is impacted by patient truncating variants and a subset of novel missense variants.
Description
2024