Investigating the interactions between Wilms' tumor suppressor protein and the protein ligands par4, p53, Ciao 1 and U2AF65

• Main Author: Weiss, Tristen Carla
• Other Authors: Romaniuk, Paul John
• Language: English; en
• Published: 2010
• Subjects: Zinc-finger proteins; UVic Subject Index::Sciences and Engineering::Chemistry::Biochemistry
• Online Access: http://hdl.handle.net/1828/2215

Wilms' tumor suppressor protein (WTI) is a key regulatory factor involved in controlling the development and normal physiology of the genitourinary tract. Mutations within WT1 result in multiple syndromes affecting the kidney and gonads with the most severe effects being Wilms' tumor, a pediatric kidney cancer. The WTI protein is composed of two distinct functional domains; the amino terminus is a proline and glutamine rich regulatory domain, while the carboxyl terminus is a DNA binding domain which contains four C2H2 zinc fingers. Although the zinc finger motif is small in size, proteins containing zinc fingers are extremely diverse in their functions. The functional diversity of WT1 is exemplified through its interactions with a wide range of ligands, such as DNA, RNA and proteins. The interaction between WT1 and DNA has been well characterized, while the interactions with RNA and proteins still require intensive investigation. Recent studies have identified a diverse group of WT1 protein partners but the characterization of the protein-protein interactions has been limited and inconclusive. Therefore, the experiments conducted in this study focused on investigating the mechanism of interaction between WTI with the protein ligands Ciao 1, p53, par4, and U2AF65. To identify which WTI zinc finger(s) are critical in protein binding, a series of finger swap and deletion mutant proteins were created using site directed mutagenic PCR. The effects the mutant proteins had on the protein interactions were analyzed qualitatively using GST pulldown assays. Two different approaches were used for the GST pulldown assays. The first approach utilized bacterially expressed and purified proteins. None of the mutant WTI proteins exhibited a decrease in protein binding in these assays. Numerous pulldown trials involving various zinc fmger proteins revealed non-specific protein-protein interactions were occurring. The second approach employed in vitro translated 35S-labelled proteins. The results from these assays demonstrate a clear role for WT lzf3, and a possible role for WTI zf4 in the WT 1-par4 interaction. The replacement of WT 1 zinc fingers 3 and 4 with those from YY1 caused a distinct reduction in binding to par4 which was exclusive for the WT1-par4 interaction. YY1 is a transcription factor from yeast that contains four C2H2 type zinc fingers. A decrease in binding between the chimeric proteins WTI :YY1 and the protein partners Ciao 1 and U2AF65 was also observed, although to a much lesser extent. This difference in binding ability may indicate that the interactions between WT1 and its protein ligands involve different zinc fingers.