Design and synthesis of molecular tools for the ubiquitin proteasome system

Alzheimer's disease (AD) has become one of the biggest global public health challenges, with 0.5% of the population living with dementia. The ubiquitin proteasome system (UPS), in particular the interaction between a mutant form of ubiquitin, Ubb+1, and the E2-conjugating enzyme E2-25K, has been identified as playing a possible pathophysiological role in the early and late stage progression of AD. The E2-25K/Ubb+1 protein-protein interaction (PPI) is regulated by the α-helix 9 of E2-25K. Creating a short helical peptide analogous to α-9 of E2-25K with the technique of peptide stapling could potentially block the action of Ubb+1 and provide a tool to investigate the role of the UPS in AD.Following synthesis of the requisite alkenyl amino acids for peptide stapling, an initial palette of nine peptides, containing the wild-type E2-25K sequence, and eight stapled analogues, were successfully synthesized and ring-closed on the solid phase. Circular dichroism spectroscopy was used to analyse secondary structure, revealing the peptides exhibited a range of 16 - 91% helicity. Further NMR studies demonstrated the more precise helical nature of short helical peptides in solution. Preliminary biological assays indicated that the synthesised peptides are capable of inhibiting Ubb+1 incorporation into long polyubiquitin chains. Remarkably, these antagonists proved to be selective towards Ubb+1 over ubiquitin despite the high degree of similarity in the relative binding sites of the two proteins. Further in silico design led to the development of a second generation of six stapled peptides. Further biological evaluation revealed an additional sequence potentially capable of activating E2-25K mediated Ubb+1 capped PolyUb chain formation.Overall, this preliminary study has shown that short helical analogues of E2-25K can be successfully synthesized and used to block or activate the E2-25K/Ubb+1 PPI. Further optimisation of these stapled peptides could provide valuable tool compounds into the investigation of Ubb+1 mediated inhibition of the UPS, and the downstream effects on the pathogenesis of AD.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2016

Author dc:creator

• Watson, Morag Elspeth

DOI

10.48730/gvke-s625

Identifier

T14420

Author Identifier

201286014

OAI identifier oai:identifier

oai:strathclyde:h128nd689