Dissecting disease pathways of TRIM8-associated nephrotic syndrome
Embargo Date
2028-02-26
OA Version
Citation
Abstract
Glomerular diseases are a leading cause of end-stage kidney disease and are frequently characterized by dysfunction in the key kidney filtering epithelial cells, podocytes. Genetic podocytopathies (GP) are the most severe subset, in which patients frequently develop treatment resistance and irreversible renal scarring (e.g. focal segmental glomerulosclerosis). Delineating the molecular pathways underlying these conditions can reveal therapeutic targets to reverse disease progression. The Majmundar lab, in which I have been conducting the research for this thesis, had previously discovered that de novo C-terminal truncating variants in TRIM8 (tripartite motif containing 8), encoding an E3 ubiquitin ligase, cause a pediatric GP and disrupt the formation of TRIM8 nuclear bodies. To understand the key proteins and pathways that are associated with TRIM8 and that are impaired in disease, the Majmundar lab had previously performed quantitative proteomics to determine critical protein interactors of TRIM8 abrogated by disease mutants. Mass spectrometry was performed to identify co-precipitated interaction partners. For this thesis, we stratified candidate interactions using orthogonal systems-level data. This showed that 884 of 2288 detected proteins were significantly more abundant in the wildtype (WT) group relative to both disease mutant groups (false detection rate <1%, fold change 3, minimum 3 peptides/protein length*1000), reflecting proteins that co-precipitated with the WT protein more so than the variants. To understand the potential biological functions of these TRIM8 interacting proteins, protein-protein interaction (PPI) network analysis was performed in STRING (string-db.org). Markov clustering algorithm (MCL) clustering based on experimental data yielded 152 PPI clusters. The top cluster contained 156 proteins enriched for protein translation functions. Within this cluster were two sub-clusters containing ribosomal proteins and ubiquitin proteosome system (UPS) proteins, respectively. This suggested that TRIM8 protein organizes into nuclear bodies containing protein translation and quality control machinery, and this is disrupted in genetic disease. To confirm localization of the proteasome in TRIM8 nuclear bodies, immunofluorescence (IF) studies were performed, demonstrating the co-localization of proteosome proteins 26S proteasome non-ATPase regulatory 4 (PSMD4) and 26S proteasome non-ATPase regulatory 12 (PSMD12) within TRIM8 nuclear bodies. Patient variants disrupted the formation of TRIM8 nuclear bodies as well as the accumulation of these UPS factors to nuclear bodies. To study the mechanism by which TRIM8 organizes into condensates, we hypothesized that the TRIM8 C-terminal intrinsically disordered region (IDR) plays a critical role. We generated synthetic TRIM8 constructs and conducted rescue studies. Immunofluorescence co-localization studies with mitogen-activated protein kinase kinase kinase 7 (TAK1), a known TRIM8 interactor implicated in kidney disease, showed that both TRIM family and non-TRIM family IDRs rescue TRIM8 condensate formation and co-localization with TAK1. Luciferase reporter assays with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is activated by TRIM8 and also implicated in kidney disease, showed that both TRIM family and non-TRIM family IDRs rescued NF-κB activation. We provide evidence that protein translation and quality control machinery are impacted in GPs, representing a potential therapeutic target for future investigation. We also elucidate the role of the IDR on TRIM8 condensate formation and function.
Description
2025