Nephrotic syndrome (NS) is the second leading cause of pediatric chronic kidney disease. Pathogenic de novo C-terminal truncating variants in the gene TRIM8 (tripartite motif containing 8) causes a syndrome of NS and epilepsy. In contrast, N-terminal truncating variants are observed in gnomAD control subjects, indicating haploinsufficiency does not cause disease. Based on previously reported TRIM8-TRIM8 interactions, we hypothesized that NS-associated TRIM8 variants may impact wildtype TRIM8 through dominant-negative effects. Alternatively, these variants may have gain-of-function mechanisms in the development of NS.
MG132, a 26S proteasome inhibitor, was used to modulate wildtype (WT) and patient variant containing (PV) TRIM8 protein levels. Protein-protein interactions were determined using co-immunoprecipitation of WT- and PV-TRIM8 protein upon transfection of immortalized podocytes with tagged cDNA constructs. Ubiquitination of WT and PV TRIM8 protein was measured by immunoprecipitation and western blotting upon overexpression of tagged Ubiquitin and TRIM8 cDNA constructs in immortalized podocytes.
MYC-tagged WT and PV TRIM8 half-lives were determined through cycloheximide (CHX) treatment over a five-hour timed trial. CHX, used to inhibit the elongation step in eukaryotic protein translation, was administered to prevent protein synthesis. CHX-chase assay was performed to determine steady state protein stability of PV-TRIM8 relative to WT.
Tagged TRIM8 WT protein levels increased upon treatment with MG132 whereas protein levels of tagged TRIM8 PV proteins were not. Flag-tagged TRIM8 PV proteins, but not flag-tagged WT protein, co-immunoprecipitated with MYC-tagged TRIM8 WT protein. MG132 rescued co-immunoprecipitation of FLAG- and MYC-tagged WT TRIM8 proteins. Tagged TRIM8 WT protein undergoes polyubiquitination, which is impaired by TRIM8 patient variants. PV-TRIM8 had a significantly longer half-life when compared to the WT.
TRIM8 PV cause reduced TRIM8 polyubiquitination, impaired 26S proteasomal degradation, and increased TRIM8-TRIM8 interactions, conferring a gain-of-function mechanism of disease. / 2026-03-07T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48370 |
| Date | 08 March 2024 |
| Creators | Rubin, Alexander |
| Contributors | Beck, Laurence, Majmundar, Amar |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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