All ETDs from UAB

Advisory Committee Chair

Lisa M Guay-Woodford

Advisory Committee Members

Bradley Yoder

Kai Jiao

David Schneider

Paul Sander

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Autosomal recessive polycystic kidney disease (ARPKD; MIM 263200) is a ma-jor cause of pediatric morbidity and mortality. Typically, orthologous animal models are the mainstay for pathogenic studies of human diseases. However, gene-targeting of Pkhd1, the mouse ortholog of the ARPKD gene, results in mutants with little or no kidney disease. In contrast, disruption of the non-orthologous gene, Cys1, in the cpk mouse model closely phenocopies human ARPKD. We speculate that this phenotypic similarity suggests that the Pkhd1 and Cys1 genes encode proteins (FPC and cystin, respectively) that share, at least in part, common molecular pathways. Our laboratory, as well as others, has shown that both proteins are cilia-associated and involved in regulated trafficking from the cilia to the nucleus, where they modulate specific gene transcription. The current study was designed to identify cystin-binding partners and character-izing the pathways for cystin intracellular trafficking and nuclear function. To identify interacting partners, we undertook an agnostic approach using tandem affinity purification (TAP). A stable mIMCD-3 cell line was generated expressing a cystin TAP construct. TAP was performed (n=4) on cell lysate from cells grown to optimize cilia expression. A combination of SDS-PAGE, Spyro Ruby staining, western blotting, and mass spectrometry was used to identify putative interacting partners. GST-pulldown experiments have confirmed a direct interaction between cystin and Importin α1, Importin α2, and Importin β2. We also present data that implicates cystin in the regulation of Pkhd1 splicing. Finally, we were able to identify and characterize a novel functional interaction between cystin and the ciliary GTPase, Arl3. Together, these data extend our initial observations regarding cystin interactions and implicate its function in cilia-associated and nuclear transport-related protein complexes. Taken together, these data suggest that cystin and its putative interacting partners e.g., Arl3, define one or more protein complexes and/or pathways that are critical for renal tubular homeostasis and are involved in recessive PKD pathogenesis.

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