Understanding and Controlling Protein Aggregation and Disease: Molecular Mechanisms in vitro and in vivo

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Background
Despite the increasing importance of amyloid diseases including Parkinson's and Alzheimer's in our ageing population, we lack fundamental knowledge about how and why protein aggregation causes disease. Such knowledge is needed to develop successful therapeutic strategies against these age-related diseases.
Objectives
In this project we will focus on a newly identified variant of beta2-microglobulin (beta2m) that causes fatal systemic amyloidosis. We will determine how and why this mutant aggregates; generate and characterize a C.elegans beta2m-disease model and screen for small molecules able to prevent its aggregation. We will then determine the mechanism of action of the small molecules in preventing aggregation in vitro and test their efficacy in a living organism exploiting C.elegans as a powerful model system.

Novelty
In recent work we have devised a novel and powerful screen in E.coli to identify small molecules that prevent protein aggregation. We will use this for a recently identified amyloid disease using a library of repurposed small molecules provided by our collaborator Richard Foster (School of Chemistry), and screen their efficacy and potential to enhance proteostasis in an intact living animal.
Timeliness
Combined with the exploitation of C.elegans as a screen for efficacy of small molecule hits in a living organism, the project will not only provide excellent training for a PhD student but it will also result in new knowledge of both fundamental and translational importance.
Experimental approach
The project combines biochemistry, protein chemistry, structural biology and biophysics with in vivo analyses and high-throughput screening methodologies using C.elegans. All methods and approaches needed are already well established in the applicants' laboratories. Hence the student will obtain excellent training in a wide variety of methods and will apply them to an exciting multidisciplinary project that is achievable within the 4 year tenure of this PhD.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1774723 Studentship BB/M011151/1 30/09/2016 31/01/2021 Sarah Good
 
Description Firstly, the research funded through this grant has been used to explore the role of transcellular chaperone signalling (TCS). Briefly, TCS is the response of one tissue to up- or down-regulation of chaperones in a different tissue; for example, in C. elegans when hsp-90 is specifically up-regulated in the neurons of the animal, this causes an upregulation of hsp90 in the body wall muscle of the animal. Research through this award found out that this upregulation can aid in protection of a aggregation-prone protein against the animal, through using amyloid disease models as a readout for the effects of this upregulation where amyloid disease models have been used as a readout for the effects of this upregulation.

Next, the research in this grant has funded work into the aggregation kinetics of the amyloidogenic protein that is involved in parkinson's disease, a-synuclein. Two regions of a-synuclein (P1 and P2) were discovered to be master controllers of aggregation, and by removing these regions the protein shows a dramatic decrease in it's ability to aggregate. This work was further confirmed through expressing the protein in the disease model C. elegans; where deletion of the P1 region or both the P1 and P2 regions inhibited the aggregation of the protein and rescued the toxicity phenotypes observed.

Further, this grant has funded work to create a new disease model expressing the amyloidogenic protein B2-microglobulin (B2m) that is associated with the disease Dialysis Related Amyloidosis. Wildtype B2m, and two variants D76N, a naturally occuring highly amyloidogenic variant, and DN6, a 6 amino acid truncated B2m that is found in ex vivo deposits, were expressed in the nematode C. elegans. Phenotypic characterisation uncovered that the expression of these proteins was associated with toxic phenotypes. It was also discovered that toxicity of this protein may be acting through the ER stress pathway, sequestering ER chaperones and disrupting the ER stress response.

In addition, this grant has also funded work to create a disease model expressing the type II diabetes associated protein Amylin. Three variants of Amylin have been expressing in the nematode C. elegans, the human wild type variant hIAPP, the highly amyloidogenic variant S20G IAPP, and the non-amyloidogenic variant rat IAPP. Both the S20G and hIAPP variants have been associated with toxic phenotypes in the animal; however, the rat IAPP variant has shown no observable phenotypes. Further, these strains were crossed into a C. elegans model expressing Q35-YFP, which acts as a proteostasis sensor. Surprisingly, the expression of both amyloidogenic proteins in the animal causes a delay in the aggregation of Q35-YFP and partially rescues it's paralysis phenotype.
Exploitation Route The implications of TCS on aggregation-prone proteins can be used in further study into the proteostasis network, possibly providing new strategies for therapeutics against protein misfolding diseases. Further, the two models set up expressing either an IAPP variant or a B2m variant will be used in further research in the research group.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology