Dissecting disease pathways of NOS1AP variants that contribute to nephrotic syndrome
Embargo Date
2028-03-04
OA Version
Citation
Abstract
Glomerular diseases are a leading cause of end-stage kidney disease and characterized by dysfunction in the key kidney filtering 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. We previously discovered that NOS1AP (nitric oxide synthase 1 adaptor protein) is a Mendelian glomerular disease gene that regulates the actin cytoskeleton, a major hub of disease proteins in GPs. Disease variants localize to its phosphor-tyrosine binding (PTB) domain and cause defective actin remodeling in immortalized podocytes. In this investigation, we strive to understand the key proteins and pathways through which NOS1AP regulates the podocyte actin cytoskeleton and that are impaired in disease. Quantitative proteomics was performed to determine the critical protein interactors of NOS1AP abrogated by disease and biological mutants. Wildtype and mutant tagged NOS1AP constructs were transiently transfected in immortalized human podocytes. Immunoprecipitation (IP) of NOS1AP proteins was performed followed by a non-denaturing elution, protein trypsinization and tandem mass tagging. Liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) was performed on biological triplicates. Peptide and protein calling was performed, and aggregate peptide signal was normalized per 100 peptides and to the signal of the bait. Co-expression of candidate interaction partners in podocytes with NOS1AP was assessed using kidney single cell mRNA sequencing datasets (z-score of % cell expression>1). Top candidates were confirmed by subsequent IP studies. Additionally, commercial PTPN14 antibodies were assessed through immunoblotting and immunofluorescence. PTPN14 antibody testing was also conducted with immortalized podocytes exhibiting PTPN14 overexpression or PTPN14 knockdown.
Quantitative-IP proteomics of wildtype NOS1AP, patient variant NOS1AP C143Y, and a biological mutant lacking the PTB domain (NOS1AP δPTB) resulted in the detection of 12761 peptides in 2351 proteins. Of these proteins, PTPN14 (protein tyrosine phosphatase non-receptor type 14) and KANK1 (KN motif and ankyrin repeat domains 1) were highly expressed (z-score>1) in the majority of datasets with NOS1AP in podocytes, suggesting these proteins may interact with NOS1AP in the disease-relevant cell type. Confirmatory co-IP studies showed that tagged PTPN14 as well as KANK1 co-precipitated with wildtype NOS1AP, and this co-precipitation was reduced with the disease mutant C143Y and biological mutant δPTB.
Overall, this data highlights new potential signaling pathways in genetic podocyte disorders (e.g. protein tyrosine phosphorylation) and suggests therapeutic targets for future investigation.
Description
2025