Expression and characterisation of cardiovascular amyloid proteins

Davies, Hannah
Expression and characterisation of cardiovascular amyloid proteins. Doctor of Philosophy thesis, University of Liverpool.

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There are currently 30 extracellular proteins known to form pathogenic amyloid within the human body. These proteins result in a wide range of diseases described as amyloidoses. These amyloidoses can be either systemic, affecting multiple organs, or localised to a single tissue. Almost all systemic amyloidoses can have cardiac manifestations and cardiac involvement correlates with poor prognosis. Cardiac infiltration is most commonly associated with AL amyloidosis, with 50 % of patients presenting with heart failure at the time of diagnosis. In addition to the myocardium, amyloid deposits are frequently reported in the surrounding vasculature. The most common form of localised amyloid – aortic medial amyloid (AMA) - is primarily localised to the internal elastic laminae of the ascending aorta. It is estimated to be present in 97 % of the Caucasian population of 50 years of age but despite its prevalence there remains very little information available about the structural, biophysical and aggregation properties of the main protein component, a 5.5 kDa peptide termed medin. Clinically, it has been suggested that AMA may have a role in aortic aneurysm and dissection through binding to the elastic structures of the aorta and reducing elasticity. This work aimed to further our molecular-level understanding of amyloid aggregates formed by proteins, known to affect the cardiovascular system; medin, and a model peptide for AL amyloidosis, SMA. Initially, this work investigated the structural and biophysical properties of three medin derived peptides with a view to identifying key amyloidogenic regions that could be targeted therapeutically. Furthermore, this work established that small ii medin peptides were not suitable structural models for full length medin. It was therefore necessary to develop a reliable method for the production of full-length medin. This work describes for the first time a procedure for the expression and purification of soluble medin in both unlabelled and 13C/15N labelled forms in E.coli for future biophysical and structural investigations. The recombinant medin was then characterised using a variety of techniques commonly used for the characterisation of amyloid proteins. These results indicated that medin aggregation proceeds via a nucleation dependent mechanism with a lag time of 30 hours and forms amyloid-like fibrils visible by transmission electron microscopy. Structural and biophysical studies were coupled with computational techniques to generate a structural model of medin fibrils, stabilised by a salt bridge between residues D25 and K30. Salt bridge mutations gave rise to aggregates with altered aggregation mechanisms and morphology. It is not known what causes some immunoglobulin light chain fragments involved in AL amyloidosis to become pathogenic. It is possible that structure underlies the differences. This work demonstrates a protocol for producing isotopically labelled immunoglobulin light chains, in particular a model protein SMA, and subsequent NMR analysis to detect inter-nuclear contacts within aggregates. Collectively this work has identified key regions and features of medin that could be targeted in future for therapeutic or diagnostic purposes. Furthermore, it paves the way for further structural studies of aggregates formed by immunoglobulin light chains.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2013-06 (completed)
Uncontrolled Keywords: Aortic medial amyloid, medin, cardiovascular amyloid, AL amyloidosis, SMA, Circular dichroism, solid state NMR, thioflavin T
Subjects: ?? QH301 ??
Divisions: Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 07 Aug 2013 10:40
Last Modified: 16 Dec 2022 04:39
DOI: 10.17638/00012075
  • Middleton, David
  • Yates, Ed