In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model

Abstract

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.

Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney Disease Progression

“…One important question that remains unanswered is how the multiple metabolic alterations observed in ADPKD originate and what is the connection with the Polycystins function. Recent studies have described that the cleaved C-tail of PC-1 translocates to mitochondria and interacts with multiple metabolic enzymes (Lin et al , 2018; Onuchic et al , 2023; Lin et al , 2023; Pellegrini et al , 2023), although the molecular details of how these multiple interactions might result in the observed metabolic phenotype remain currently unexplained.…”