The amino acid proline is unique – within a polypeptide chain, proline adopts either a cis or trans peptide bond conformation while all other amino acids are sterically bound primarily in the trans configuration. In proteins, the isomeric state of a single proline can have dramatic consequences on structure and function. Consequently, cis-trans interconversion confers both barrier and opportunity – on one hand, isomerization is a rate limiting step in de novo protein folding and on the other can be utilized as a post-translational regulatory switch. Peptidyl-prolyl isomerases (PPIs) are a ubiquitous superfamily that catalyzes the interconversion between conformers. Although pervasive, the functions and substrates of most PPIs are unknown. The two largest subfamilies, FKBPs and cyclophilins, are the intracellular receptors of clinically relevant immunosuppressant drugs that also show promise in the treatment of neurodegenerative disorders and cancer. Therefore, narrowing the knowledge gap has significant potential to benefit human health.
FKBP25 is a high-affinity binder of the PPI inhibitor rapamycin and is one of few nuclear-localized isomerases. While it has been shown to bind DNA and associate with chromatin, its function has remained largely uncharacterized. I hypothesized that FKBP25 targets prolines in nuclear proteins to regulate chromatin-templated processes. To explore this, I performed high-throughput transcriptomic and proteomic studies followed by detailed molecular characterizations of FKBP25’s function. Here, I discover that FKBP25 is a multifunctional protein required for the maintenance of genomic stability. In Chapter 2, I characterize the unique N-terminal Basic Tilted Helical Bundle (BTHB) domain of FKBP25 as a novel dsRNA binding module that recruits FKBP25’s prolyl isomerase activity to pre-ribosomal particles in the nucleolus. In Chapter 3, I show for the first time that FKBP25 associates with the mitotic spindle apparatus and acts to stabilize the microtubule cytoskeleton. In this chapter, I also present evidence that this function influences the stress response, cell cycle, and chromosomal stability. Additionally, I characterize the regulation of FKBP25’s localization and nucleic acid binding activity throughout the cell cycle. Finally, in Chapter 4, I uncover a role for FKBP25 in the repair of DNA double-stranded breaks. Importantly, this function requires FKBP25’s catalytic activity, identifying for the first time a functional requirement for cis-trans prolyl isomerization by FKBP25.
Collectively, this work identifies FBKP25 as a multifunctional protein that is required for the maintenance of genomic stability. The knowledge gained contributes to the exploration of PPIs as important drug targets. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8473 |
| Date | 28 August 2017 |
| Creators | Dilworth, David |
| Contributors | Nelson, Christopher J |
| Source Sets | University of Victoria |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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