Kamuda, Kamila; (2024) Molecular changes in the cellular landscape elicited by expression of the Z variant α1-antitrypsin. Doctoral thesis (Ph.D), UCL (University College London).

Text PHD_THESIS_KK_corrected.pdf - Accepted Version Access restricted to UCL open access staff until 1 April 2026. Download (81MB)

Abstract

Alpha-1-antitrypsin (α1AT) is a 52 kDa glycoprotein, predominantly synthesized and secreted from the liver into the circulation, where it acts as a protease inhibitor. Single mutations in the SERPINA1 gene can lead to alpha-1-antitrypsin deficiency (AATD), a genetic disorder where the lack of protein in the circulation is associated with liver cirrhosis and early onset emphysema. The susceptibility of native α1AT to point mutations is a result of the metastability of the protein. Most commonly associated with severe disease is the Z variant (E342K), which self-associates into long-chain polymers through a domain swap. These oligomers are retained within the endoplasmic reticulum (ER) of the liver, leading to the formation of inclusion bodies (IBs) and chronic ER stress. While there is an association between these inclusions and liver cirrhosis, the mechanisms behind this process have not been fully elucidated. In my research I have used electron microscopy imaging to determine the effect of α1AT-Z on the cellular landscape. This study established strong association of ER-derived IBs with mitochondria. The organelle ultrastructure analysis indicated profound morphological changes in mitochondria induced by chronic ER stress, and consequently an alteration in mitochondrial function. Furthermore, I have established a workflow for the use of cryoFIB/ET, a technique allowing for in situ visualisation of cells in a near-native state. Tomograms showed direct interaction of IBs with other cellular components informing of the cellular response to the polymer accumulation. The EM data contributes to the knowledge of the liver disease progression and provides basis for structural studies of cellular components. Naturally occurring rare mutations have played an essential role in understanding the molecular mechanisms underlaying AATD. I characterised a novel α1AT variant (G192C) using crystallography and biochemical assays. This new variant named α1AT-Sydney, represents a potential tool to characterise the role of altered conformation dynamics in accumulation and secretion from mammalian cells.

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