Kidney Week

Abstract: SA-PO610

Disruption of the Human Cystin-1 Myristoyl-Electrostatic Switch Causes Autosomal Recessive Polycystic Kidney Disease (ARPKD)

Session Information

• Cystic Kidney Diseases: Genetic Causes, Modifiers, and Extrarenal Manifestations
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Yang, Chaozhe, Children's National Hospital, Washington, District of Columbia, United States

Chaozhe Yang, MD

• Harafuji, Naoe, Children's National Hospital, Washington, District of Columbia, United States

Naoe Harafuji, MS, PhD

• Watts, Jacob, Children's National Hospital, Washington, District of Columbia, United States

Jacob Watts, PhD

• Moran, Claire, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Claire Moran

• Clements, Jenna, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Jenna Clements

• Price, Kailyn M., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States

Kailyn M. Price

• Burrill, Natalie, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Natalie Burrill, MS

• Gebb, Juliana S., The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Juliana S. Gebb, MD

• Soni, Shelly, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Shelly Soni, MD

• Oliver, Edward R., The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Edward R. Oliver, MD, PhD

• Savla, Jill, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Jill Savla, MD, MS

• Santani, Avni, Veritas Genetics, Danvers, Massachusetts, United States

Avni Santani, PhD

• Hartung, Erum Aftab, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States

Erum Aftab Hartung, MD, MS

• Karl, Rudolfo, Universitatsklinikum Bonn, Bonn, Nordrhein-Westfalen, Germany

Rudolfo Karl

• Wachten, Dagmar, Universitatsklinikum Bonn, Bonn, Nordrhein-Westfalen, Germany

Dagmar Wachten

• Bebok, Zsuzsanna M., The University of Alabama at Birmingham Department of Cell Developmental and Integrative Biology, Birmingham, Alabama, United States

Zsuzsanna M. Bebok, MD

• Wilson, Landon Shay, The University of Alabama at Birmingham Department of Pharmacology and Toxicology, Birmingham, Alabama, United States

Landon Shay Wilson

• Caldovic, Ljubica, Children's National Hospital, Washington, District of Columbia, United States

Ljubica Caldovic, PhD

• Guay-Woodford, Lisa M., Children's National Hospital, Washington, District of Columbia, United States

Lisa M. Guay-Woodford, MD

Background

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is primarily caused by pathogenic variants in PKHD1. The Cys1^cpk/cpk (cpk) mouse expresses a renal lesion that closely phenocopies ARPKD. Cys1 encoded cystin is a myristoylated protein that traffics to the primary cilium and the nucleus, where it regulates gene expression.

Methods

Clinical characterization of siblings with ARPKD and Cystin-1_G2S variant. Bioinformatics analysis of mammalian cystin sequences. Immunofluorescence (IF) staining and optogenetic stimulation assay to determine subcellular localization of wild-type and mutant cystin. Tandem affinity purification (TAP) and mass spectroscopy (MS) to identify cystin-binding partners. Evaluation of cystin serine-17 (S17) phosphorylation.

Results

The homozygous Cystin-1_G2S variant identified in the siblings is predicted to disrupt the G2 myristoylation site within the cystin MGxxxSx N-terminal motif. Alignment of 97 mammalian cystin sequences showed high conservation of a putative myristoyl-electrostatic switch that can regulate reversible protein binding to membranes. The conserved region includes the N-myristoylation site and an adjacent arginine-rich stretch flanked by S17 residues. Using IF staining and site-directed mutagenesis, we confirmed that S17 phosphorylation modulates cystin membrane association and intracellular trafficking. In turn, optogenetic activation of ciliary cAMP signaling reduced the cystin ciliary localization in a PKA-dependent manner. TAP-MS identified the protein phosphatase PPM1A as a cystin-interacting partner. Inhibition of PPM1A with sanguinarine impeded cystin S17 de-phosphorylation confirming functional interaction.

Conclusion

Our study demonstrates that cystin intracellular trafficking and nuclear function are regulated by a myristoyl-electrostatic switch mechanism, and further supports CYS1 as a disease-causing gene for human ARPKD, providing the first mechanistic insight for disease pathogenesis.

Funding

• NIDDK Support