Proteomic Analysis of Podocytopathy Protein NOS1AP Reveals Novel PTPN14 Interaction Disrupted in Disease

Background: Genetic forms of glomerular disease often result from dysfunction in the key kidney filtering cells, podocytes. These genetic podocytopathies (GPs) frequently progress to end-stage kidney disease. Delineating the molecular pathways underlying these conditions can reveal therapeutic targets. Disease variants in the GP gene NOS1AP (nitric oxide synthase 1 adaptor protein) localize to its phospho-tyrosine binding (PTB) domain and cause defective actin remodeling through unclear molecular mechanisms. Methods: Wildtype (WT) and mutant tagged NOS1AP constructs were transiently transfected in immortalized human podocytes. Immunoprecipitation (IP) of NOS1AP proteins, elution of co-precipitating proteins, and tandem mass tagging-based quantitative immunoprecipitation (IP) proteomics (LC/MS/MS) were performed. Interactors were stratified by co-expression in podocyte clusters of kidney single cell mRNA sequencing (scRNAseq) datasets. Confirmatory IP and indirect immunofluorescence (IF) studies were performed. Results: Quantitative-IP proteomics of WT NOS1AP, patient variant NOS1AP C143Y, and biological mutant NOS1AP lacking the PTB domain (NOS1AP dPTB) yielded 2351 proteins (12761 peptides). 28 proteins were significantly enriched in the WT group relative to both mutant groups (fold change 1.5, FDR 1%, minimum 10 peptides), indicating PTB mutations abrogated these interactions. We focused on NOS1AP interactions potentially occurring in the disease-relevant cell type and found 2/28 proteins were co-expressed (z-score of % cell expression>1) with NOS1AP in podocyte clusters of multiple (3+) kidney scRNAseq datasets: PTPN14 (protein tyrosine phosphatase non-receptor type 14) and KANK1 (KN motif and ankyrin repeat domains 1). Published Ptpn14 knockout mouse studies indicate a causal role in podocyte injury (Lin BBRC 2021). We confirmed that PTB mutations impaired NOS1AP:PTPN14 interactions through bi-directional co-IP studies with over-expressed proteins as well as an endogenous PTPN14 antibody. We determined that Ptpn14 co-localizes with podocyte marker Nephrin in a linear pattern in rat kidney glomerular podocytes by indirect IF. Conclusion: Quantitative proteomics reveals a novel NOS1AP:PTPN14 interaction that is disrupted in genetic disease. Funding: NIDDK Support