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Biochemical and Structural Characterisation of Pathological Alpha1-Antitrypsin polymers

Elliston, Emma Louise Kate; (2019) Biochemical and Structural Characterisation of Pathological Alpha1-Antitrypsin polymers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The hereditary disease alpha1-antitrypsin deficiency (AATD), most prevalent in those of Northern European ancestry, is a leading genetic cause of early-onset emphysema and liver cirrhosis. Currently, the only effective treatment is a transplant of the lungs or liver. The disease is caused by mutations in the gene encoding alpha1-antitrypsin (AAT), which promote misfolding and aggregation into polymer chains. These polymers accumulate and induce cellular damage to hepatocytes, and the resultant lack of AAT in the circulation permits uncontrolled remodelling, and eventual breakdown, of lung tissue. The mechanism of polymerisation and the linkage between individual subunits of the polymer found in affected individuals is unknown. Current structural models of polymerisation are based on artificially-induced in vitro polymers, but their relevance to in vivo systems is disputed. Polymers formed by heating are believed to represent polymers found in patients. A structural understanding of the mechanism by which AAT polymerises will be useful for the development and evaluation of therapeutic strategies. Conformational-specific monoclonal antibodies and electron microscopy have been used in combination to study the structure of heat-induced and patient-derived polymers. Negative stain-electron microscopy (NS-EM) of polymers, with Fab fragments attached to each AAT subunit, has generated similar reconstructions of a dimer for both polymer types. This supports the notion that heat-induced polymers are a suitable model for ex vivo polymers. An initial cryo-EM reconstruction of the labelled ex vivo dimer and a single subunit has also been obtained, in preparation for a higher-resolution cryo-EM dataset. Fitting of the polymerisation models into the EM data best supports the C-terminal model of polymerisation. Lastly, a novel monoclonal antibody that is selective for AAT monomer over polymer, called 1D9, has been developed. X-ray crystallography reveals it binds to a region of AAT most exposed in the monomer; a characteristic which could mean it has future applications in diagnosing AATD.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Biochemical and Structural Characterisation of Pathological Alpha1-Antitrypsin polymers
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
URI: https://discovery.ucl.ac.uk/id/eprint/10072628
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