Design, synthesis and biological evaluation of peptides based on the TCP-1 ring complex and heat shock protein 27

Molecular chaperones are a group of proteins that are critical for maintaining the proper folding and function of proteins within the cell. This thesis describes the design, synthesis and biological evaluation of peptides that were structurally based on two chaperones: the TCP-1 ring complex (TRiC or CCT) and heat shock protein 27 (Hsp27). TRiC (or CCT) is chaperonin that consists of two rings, where each ring contains 8 subunits. This protein is essential for the folding of ~10% of newly synthesised proteins including actin and tubulin. Prefoldin (PFD) is another chaperone that is known to interact with TRiC and enhance its folding kinetics with actin. In 2019, Gestaut et al. demonstrated that the contact interface between TRiC and PFD was mainly electrostatic and can be disrupted by charge-reversal mutations at specific residues in TRiC. However, it is unknown whether this interaction can also be disrupted by inhibitors and lead to the same biological consequences as the mutations. Thus, Chapter 2 of this thesis describes the design, synthesis and biological evaluation of peptides that are structurally based on TRiC’s interaction site with PFD. Hsp27 is part of the small heat shock protein (sHsp) family and its main function is to prevent aggregation of misfolded proteins. In cancer, Hsp27 is overexpressed and facilitates cell maintenance. In neurodegenerative diseases, Hsp27 is inactive and is unable to prevent protein aggregation, leading to cell death. Thus, there is great interest in developing molecules that can regulate Hsp27 as potential therapeutics. In this thesis, peptides were designed from sequences of Hsp27 that are critical for protein function. At present, there are no readily available binding assay due to its self-aggregating property. Thus, Chapter 3 describes the multiple unsuccessful methods that were investigated in order to develop an effective method for assessing modulators of Hsp27 activity. Chapter 4 describes the success of native MS in evaluating the binding of the peptides to Hsp27 and their impact on Hsp27 function. Chapter 5 evaluates the function of the peptides when tested without Hsp27, where it was demonstrated that specific peptide sequences act as mini Hsp27 chaperones