Abstract: FR-PO630
ANKHD1 Has a Cytoprotective Role in Stressed Kidney Cells
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 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
- Mullenger, Jordan, The University of Sheffield, Sheffield, United Kingdom
- Zeidler, Martin, The University of Sheffield, Sheffield, United Kingdom
- Fragiadaki, Maria, Queen Mary University of London, London, United Kingdom
Background
ANKHD1 is a ubiquitously expressed RNA binding protein composed of ankyrin repeat domains for protein binding and a KH domain for nucleic acid binding. ANKHD1 is overexpressed in renal cancer, driving proliferation and growth. Clinically, increased expression of ANKHD1 is associated with increased metastasis and larger tumours, resulting in poorer patient prognosis. Our lab has predicted a similar function for ANKHD1 in autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common genetic form of renal failure and is characterised by excessive proliferation resembling early tumorigenesis. The signalling pathways activated in the proliferation of cancer cells and ADPKD closely resemble one another.
Methods
Immunohistochemistry of ANKHD1 was performed in kidney sections to examine protein localisation. Recombinant ANKHD1 was produced in HEK293T cells and elevated expression was validated using western blot and RT-qPCR. The resultant protein was purified via immunoprecipitation (IP), and its interacting partners were identified via mass spectrometry (MS). Bioinformatics analysis was performed on the experimentally validated ANKHD1 interactome from BioGrid, and analysis of function was performed using online platforms. Cells were exposed to cellular stress by growth in serum-free media for 24hrs, and viability was assessed via MTT assay.
Results
In ADPKD kidneys, ANKHD1 is localised to the cyst lining epithelial cells. Recombinant constructs of human ANKHD1 and its protein interactors were expressed and purified via IP; MS identified 267 interacting proteins. Combining our MS results with published protein-interaction databases revealed 17 overlapping ANKHD1-interacting proteins, 10 of which are involved in cellular responses to stress. Hence, we decided to test the putative role of ANKHD1 in protecting from stress. Serum starvation caused a 23% reduction in cell viability, while overexpression of ANKHD1 rescued this phenotype in a proliferation and apoptosis-independent manner. Serum-starved cells showed increased stress granules, which was suppressed by ANKHD1 overexpression. A physical interaction between ANKHD1 and HSPA8, a heat-shock chaperone involved in protein folding, was also demonstrated.
Conclusion
ANKHD1 interacts with proteins involved in the cellular stress response, and has a proposed novel role as a cytoprotective molecule during cellular stress.