Genetic diseases

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Introduction and Aims: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic disorders, affecting 1 in 1000 people. Polycystins 1 and 2 account for the majority of cases of ADPKD. We have identified a new protein (Samcystin, or Anks6) which contains a mutation (R823W) causing the development of renal cysts in the PKD/Mhm(Cy/+) rat model of ADPKD. This protein, which contains ankyrin repeats (ANK) and a sterile alpha motif (SAM), interacts with a RNA Binding protein (Bicc1) but its function remains unknown. To understand signaling mechanisms underlying disease pathogenesis, we focused on the identification of new Samcystin protein partners. Methods: In order to identify Samcystin partners, the yeast two hybrid assay was used and performed with a mouse 17 day embryo expression library fused to the GAL4, which controlled the expression of reporter genes involved in galactose utilization in yeast. Domains and full length cDNA of Samcystin was used as bait, inserted in a pGBD-B plasmid and transformed into yeast PJ69-A to produce a hybrid protein that binds the GAL4 promoter. Following mating with the PJ69-a transformed by the mouse embryo cDNA library, positive clones were rescued under suitable growth conditions and inserts of positive clones were sequenced. Homologies to known proteins were sought in Protein Data Bank (http://www.rcsb.org/pdb/). Results: A total of 4 preys were obtained with yeast two hybrid screening. These positive clones grow in the presence of yeast containing the wild type SAM domain, not with ANK or mutated SAM domains. These results indicate that the clones encode proteins which interact only with the SAM domain of Samcystin and these interactions disappear when the SAM domain is mutated and differs from the wild type domain by only one amino acid (change from arginine 823 to tryptophan). Clones were rescued and inserts were sequenced. We specifically focused further investigations to a clone sequence which shows strong evidence of homologies with the SAM domain of a predicted protein. Conclusions: Our preliminary results suggest that the SAM domain of Samcystin interacts with the SAM domain of a predicted protein of unknown function, which, with Bicc1, contributes to the construction of a functional network of proteins centered on Samcystin, and involved in cystogenesis. Co-immunoprecipitation assays will be used to verify protein-protein interaction and its modulation in the presence of the mutation. A critical question is whether R823W mutation in Samcystin, which abolishes this interaction, impairs Samcystin cellular functions and causes the PKD phenotype. In addition, we are exploring the hypothesis that the mutant Samcystin can interact with other proteins with a complementary approach based on the yeast two hybrid assay and the tandem affinity purification method.

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