Abstract: FR-PO278
Disruption of Cystin Myristoylation Causes Autosomal Recessive Polycystic Kidney Disease (ARPKD)
Session Information
- Genetic Diseases of the Kidneys: Cystic - II
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1101 Genetic Diseases of the Kidneys: Cystic
Authors
- Yang, Chaozhe, Children's National Hospital Center for Translational Research, Washington, District of Columbia, United States
- Harafuji, Naoe, Children's National Hospital Center for Translational Research, Washington, District of Columbia, United States
- Caldovic, Ljubica, Children's National Hospital Center for Genetic Medicine Research, Washington, District of Columbia, United States
- Bebok, Zsuzsanna M., The University of Alabama at Birmingham Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, United States
- Burrill, Natalie, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
- Hartung, Erum Aftab, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
- Moldenhauer, Julie, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
- Guay-Woodford, Lisa M., Children's National Hospital Center for Translational Research, Washington, District of Columbia, United States
Background
Approximately 80% of patients with typical autosomal recessive polycystic kidney disease (ARPKD; MIM 263200) have pathogenic variants in PKHD1, and DZIP1L variants account for less than 1% of the remainder. In mice, renal disease resulting from mutations in Cys1 (encoding cystin), but not in either Pkhd1 or Dzip1l, closely phenocopies human ARPKD.
Methods
Identification and clinical characterization of successive fetuses from a consanguineous union with CYS1 mutations and ARPKD phenotype. In silico analysis of mammalian cystin genes to identify putative functional domains. Analysis of mouse Cys1 by mutagenesis, fluorescence staining, affinity purification/mass spectrometry, and phosphorylation/dephosphorylation assays.
Results
Exome sequencing of the affected siblings and parents revealed CYS1 c.4G>A as a homozygous variant in both fetuses, which is predicted to disrupt the G2 myristoylation site within the cystin MGxxxSx N-terminal myristoylation motif. In mouse cell lines, we have confirmed that the G2 site is myristoylated and glycine-to-alanine substitution at G2 disrupts cystin ciliary localization (Tao 2009). Sequence alignment of 97 mammalian cystin protein sequences identified a conserved arginine-rich stretch of amino acids flanked by serine-8 and -17 residues. Serine-17 (S17) is a predicted phosphorylation target, suggesting that cystin membrane association is regulated by a myristoyl-electrostatic switch mechanism. Treatment with 8-bromo-cAMP attenuated the ciliary localization of endogenous cystin, but did not directly alter S17 phosphorylation, indicating a role for other cAMP-dependent kinase pathways, e.g. EPAC. In addition, we identified protein phosphatase PPM1A as a cystin-interacting protein and demonstrated that sanguinarine (a PPM1A inhibitor) regulates the phosphorylation status of cystin S17. Finally, we demonstrated that cystin interacts with α1, α2, and β2 importin subunits, suggesting that the importin complex mediates cystin trafficking between the cilium and nucleus.
Conclusion
Our data provide further evidence that CYS1 mutations cause ARPKD and provide new insights about the role of regulated ciliary trafficking in ARPKD pathogenesis.
Funding
- NIDDK Support