The chromosome passenger complex (CPC) is an essential mitotic regulator with key roles in mitotic processes such as chromosome condensation, spindle dynamics, chromosome bi-orientation, the spindle checkpoint and cytokinesis. The Aurora B kinase is the CPC’s catalytic subunit. Its targeting and activation are dependent on interactions with the other components of the complex: inner centromere protein (INCENP), survivin and borealin/Dasra B. INCENP serves both as a scaffolding subunit for the CPC as a whole and as an activator of Aurora B via its highly conserved INbox domain. Aurora B is a putative anti‐cancer target; several inhibitors of the kinase are currently in clinical trials. All these are ATP-analogues targeting the kinase active site. The protein-protein interaction between Aurora B and the INCENP INbox is also essential for CPC function. Earlier studies have demonstrated that INCENP INbox mutants unable to bind and/or activate Aurora B cannot rescue lethality in the absence of endogenous INCENP. The first goal of this study was to test the in vivo effects of disrupting the interaction between endogenous wild type INCENP and Aurora B. For this, a cell-based CPC function assay was developed in HeLa cells. Using this assay, I show that expression of soluble INbox in HeLa cells produces a significant increase in multinucleated and micronucleated cells: both effects consistent with Aurora B loss of function. Expression of soluble INbox bearing the mutations W845G and/or F881A does not elicit this effect suggesting that those mutants cannot bind to Aurora B and occlude INCENP binding. The result concerning the F881A mutant contrasts with earlier reports that equivalent mutants could bind, but not activate, Aurora B. Expression of an INbox mutant lacking the C-terminal TSS motif reported to be involved in Aurora B activation but not binding has effects similar to those of the wild type INbox. Using the INbox/Aurora B interaction as a model, a secondary goal of this study was to develop and evaluate a novel approach to identify small peptides capable of dissociating intracellular protein‐protein interactions. For this, a library of small (5-9 residues long) circular peptides (CPs) mimicking the INbox was generated using the split intein circular ligation of proteins and peptides (SICLOPPS) methodology and assayed using the cell-based CPC function assay. Over two successive rounds of screening, a small number of CPs were identified that caused a significant increase in rates of multinucleated and micronucleated cells. Although statistically significant, these increases were very modest. Furthermore, due to high heterogeneity in SICLOPPS processing efficiencies, it was not practicable to compare the effects of different peptides side-by-side by transfection. The level of variation in processing efficiency – thus, CP production – was unexpectedly high and puts into question the functional complexity of more commonly used combinatorial cyclic peptide libraries derived using current SICLOPPS methodology. The results of this study are divided into three sections. The first is a methods section concerning the testing of SICLOPPS in HeLa cells and the development of a cell‐based CPC function assay. In the second, the effects of expressing soluble INbox and mutants thereof in HeLa cells are presented. The final results section presents the results of the feasibility study of the rationally-designed genetically encoded library approach.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656170 |
| Date | January 2015 |
| Creators | Gohard, Florence Helen |
| Contributors | Earnshaw, Bill; Walkinshaw, Malcolm; Tyers, Mike |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10456 |
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