Inhibiting protein-protein interactions in the early stages of amyloid formation
Lead Research Organisation:
University of Leeds
Department Name: Sch of Molecular & Cellular Biology
Abstract
In this project we focus on aggregation of Islet Associated PolyPeptide (IAPP) (type II diabetes) and A-beta (Alzheimer's). Increasing evidence suggests that the intrinsically disordered monomers of these proteins are in dynamic equilibrium with aggregation-prone oligomers. Despite significant progress in the characterization of IAPP/A-beta aggregation, mechanistic understanding of their folding/aggregation mechanisms remains obscure and new fundamental knowledge is needed to inform the design of therapies.
Objectives:
Combining native MS with biochemical/biophysical assays we will:
1. Explore how small molecule inhibitors affect IAPP/A-beta aggregation: using MS, IMS and kinetic assays (ThT fluorescence) we will screen for new small molecule inhibitors of aggregation using 'hits' recently discovered in SER's lab (Nature Chem (2015) and Nature Chem Biol (2016)). Using MS mapping with ETD (FS: Anal Chem (2016)) combined with other solution-based assays we will elucidate which species are responsible for interactions with different inhibitors and characterize inhibitor binding interfaces. We will then use cell biological assays to determine whether the inhibitors affect cytotoxicity.
2. Explore the sequence-dependence of aggregation: using a novel screen for aggregation developed in SER's laboratory (Nature Chem Biol (2016)) we will select IAPP/A-beta sequences with altered aggregation properties. We will then select for mutations that enhance/retard disease progression and thus, elucidate how these mutations perturb folding and aggregation.
Novelty/Timeliness:
To our knowledge the project will reveal the first atomistic insights into the energy landscapes of folding/aggregation. By focusing on peptides that are the causative agent of amyloid associated with type II diabetes/Alzheimer's the results will also have impact on human health today.
Experimental Approach:
We will utilize native MS to the full for the project, and combine this with chemical biology, biophysics and cell biology, offering the student training in a wide range of techniques. All methods are up and running, enabling the student to make rapid progress.
Objectives:
Combining native MS with biochemical/biophysical assays we will:
1. Explore how small molecule inhibitors affect IAPP/A-beta aggregation: using MS, IMS and kinetic assays (ThT fluorescence) we will screen for new small molecule inhibitors of aggregation using 'hits' recently discovered in SER's lab (Nature Chem (2015) and Nature Chem Biol (2016)). Using MS mapping with ETD (FS: Anal Chem (2016)) combined with other solution-based assays we will elucidate which species are responsible for interactions with different inhibitors and characterize inhibitor binding interfaces. We will then use cell biological assays to determine whether the inhibitors affect cytotoxicity.
2. Explore the sequence-dependence of aggregation: using a novel screen for aggregation developed in SER's laboratory (Nature Chem Biol (2016)) we will select IAPP/A-beta sequences with altered aggregation properties. We will then select for mutations that enhance/retard disease progression and thus, elucidate how these mutations perturb folding and aggregation.
Novelty/Timeliness:
To our knowledge the project will reveal the first atomistic insights into the energy landscapes of folding/aggregation. By focusing on peptides that are the causative agent of amyloid associated with type II diabetes/Alzheimer's the results will also have impact on human health today.
Experimental Approach:
We will utilize native MS to the full for the project, and combine this with chemical biology, biophysics and cell biology, offering the student training in a wide range of techniques. All methods are up and running, enabling the student to make rapid progress.
