The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl isomerase enzyme (PPIs) that catalyze the cis-trans inter-conversion of peptidyl-prolyl bonds in proteins. This non-covalent post-translational modification is a reversible mechanism to modulate protein structure and function. PPIs have been implicated in a wide variety of processes from protein folding to signal transduction. Despite these enzymes being ubiquitous, the substrates and functions of most PPIs have yet to be described.
FKBP25 is a nuclear FKBP that has been shown to associate with transcription factors and chromatin modifying enzymes, however its functions and substrates remain largely unresolved. FKBP25 is the human ortholog of S. cerevisiae Fpr4, which has been shown to regulate the chromatin landscape by two distinct mechanisms: 1. Acting as a histone chaperone at ribosomal DNA, and 2. Isomerizing histone prolines. Based on these observations, I hypothesized FKBP25 regulates chromatin and/or ribosome biogenesis through isomerization of histone prolines and a discrete collection of substrate proteins.
While small molecule inhibitors exist for FKBPs, applying them to dissect the specific function(s) of any given FKBP is confounded by the fact that multiple FKBPs are found in each organism, and several are inhibited by these molecules. In Chapter 2, I biochemically and structurally characterize a set of FKBP25 loss-of-function mutants, yielding a toolset capable of distinguishing between catalytic and non-catalytic functions. These reagents provide the tools necessary to analyze potential substrates of FKBP25 identified in my research going forward. In Chapter 3, I present the first unbiased proteomic screen of FKBP25 associated proteins and show that it interacts with a large number of ribosomal proteins, ribosomal processing factors and a smaller subset of chromatin proteins. I focus on the interaction between FKBP25 and nucleolin, a multi-functional nucleolar protein, and show that FKBP25 interacts with nucleolin and the pre-60s ribosomal subunit in an RNA dependent fashion. In Chapter 4, I gain insight into the role of FKBP25 in ribosome biology, and demonstratex that FKBP25 regulates RNA binding activity of nucleolin, however this does not appear to involve cis-trans prolyl isomerization.
Collectively, my work establishes FKBP25 as the first human FKBP to be implicated in the maturation of the pre-60S ribosomal subunit in the nucleus. My data supports a model whereby FKBP25 associates with the assembling large ribosomal subunit, where it is likely to chaperone protein-RNA interactions. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/7684 |
| Date | 21 December 2016 |
| Creators | Gudavicius, Geoffrey |
| Contributors | Nelson, Christopher J. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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