Chemical Mutagenesis for the Modulation and Study of Protein Function and Dysfunction within Alzheimer’s Disease

Chemical mutagenesis is defined as the post-expression interconversion of amino-acid (AA) sidechains. This approach enabled the first ever point-mutations in proteins and has seen a resurgence of interest over the last two decades. This thesis reports on two disparate applications of chemical mutagenesis within the context of Alzheimer’s disease (AD). First, exploring the capabilities of utilizing chemical mutagenesis to enable a structure-activity relationship study on a single-domain antibody (SdAb) beyond the 20 canonical AAs. The starting SdAb was designed to target the Amyloid-b peptide (Ab) and has a nascent ability to inhibit its aggregation, a key pathological process in AD. Utilizing the synthetically versatile non-canonical AA dehydroalanine (Dha), we explore diverse sidechains at key residues along the binding loop of the SdAb and identify a non-canonical sidechain that potently enhances the inhibition of Ab’s primary nucleation. The second application focuses on the other major protein implicated in the pathology of AD: Tau. In AD, tau aggregates into ordered amyloid fibrils and contains a plethora of post-translational modifications (PTMs), many of which are speculated to have an impact on the pathogenesis of AD as well as other so-called tauopathies. In order to facilitate the accurate study of tau PTMs, an easily accessible method for their site- selective installation is needed. To this end, we again explore the utilisation of Dha to install accurate PTM mimetics for phosphorylation, lysine acetylation, and lysine dimethylation at diverse sites along tau. This approach produced homogenous products that accurately recapitulated known behaviours and allowed for the initial characterisation of poorly investigated PTMs. The characterisation of S199 phosphorylation, a known AD associated modification, through this approach provides the first evidence for its potential role in regulating tau’s primary function of microtubule polymerisation and stabilisation.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Lindstedt, Philip

Advisors dc:contributor.advisor

• Vendruscolo, Michele
• Dobson, Christopher

• Alzheimer's
• Protein modification
• Amyloid
• Antibody

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/03747ab9-398c-4c7c-9de4-4774d56629a4/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.68392

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/321268