Targeting C-terminal binding proteins (CtBPs) using genetic selection

Abstract

There are many protein-protein interactions that are vital for cellular processes such as signal transduction, structural organisation and apoptosis. In this study we decipher the role of the protein-protein interaction of C terminal Binding Proteins (CtBPs). CtBPs function as transcriptional co-repressors in the nucleus playing key roles in tumorigenesis and metastasis by regulating cellular processes, critical to cell survival, cell migration and senescence. CtBP proteins also play a role in the cytoplasm in regulating mitotic Golgi membrane fission Studies in which the expression or function of CtBPs has been inhibited have independently identified roles for CtBPs in both suppressing apoptosis and promoting cell cycle progression. Modulation of these interactions with small molecules is a potential therapeutic strategy with benefits over current methods. Our approach in studying protein-protein interactions and uncovering potential inhibitors involves constructing a bacterial Reverse Two Hybrid System (RTHS) linking the dimerisation of the target protein partners to the expression of reporter genes, whose regulation can be monitored via host survival. Subsequent screening of a cyclic peptide library for potential inhibitors was then carried out. The libraries were produced using Split Intein-mediated Circular Ligation Of Peptides and Proteins (SICLOPPS) technology, developed for intracellular synthesis of cyclic peptides. We have used this methodology to identify inhibitors of CtBP dimerisation and better understand the roles of this protein interaction in cell cycle regulation. Chapter 1 provides an introduction to the work carried out to study protein-protein interactions and finding potential inhibitors. Since our investigations involved the extensive use of the RTHS and SICLOPPS system, the background and work performed by others has been described in detail. A detailed review of CtBPs has also been carried out. Chapter 2 details our work investigating the homodimeric and heterodimeric protein-protein interaction of CtBPs using the RTHS. This work allowed us to optimise selection conditions and find cyclic peptide inhibitors of the homodimerisation of CtBP1 and CtBP2 using the SICLOPPS process. The synthesis of these inhibitors is described. Chapter 3 details our work carried out to develop ELISAs for in vitro analysis of the selected cyclic peptides. This involved the purification of His- and GST-tagged CtBP1 and CtBP2 proteins. The ELISA conditions were optimised to carry out CtBP homodimeric and hetrodimeric analysis. This work showed that the peptides lead to a reduction in CtBP homdimerisation and heterodimerisation in vitro. Chapter 4 details the in vivo effects of the uncovered CtBP dimerisation inhibitors. Using these cyclic peptide inhibitors we have demonstrated that CtBP dimerisation is essential for the regulation of mitotic fidelity, and that inhibition of CtBP dimerisation by the cyclic peptides leads to aberrant segregation of chromosomes during mitosis. We have also shown that inhibition of CtBP dimerisation leads to a reduction in migration of MCF-7 breast cancer cells. Chapter 5 details the experimental procedures used in this work and presents spectroscopic and analytical data for the compounds prepared.

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Identifiers

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

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