Plessa, Eleni; (2023) Studies of protein folding and misfolding on the ribosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Protein folding is one of the most important processes for maintaining cellular homeostasis. It begins co-translationally on the ribosome, however, it also competes with protein misfolding. Under severe conditions, protein misfolding can result in the accumulation of toxic aggregates, which are known to give rise to lethal conformational diseases including those linked to neurodegeneration, respiratory diseases, diabetes, and cancer. Very little is understood of co-translational protein misfolding mechanisms as they first begin in the cell, and also of their links to human disease. Alpha-1 deficiency, for example, results from pathological variants of the alpha-1-antitrypsin (AAT) protein, forming toxic polymer aggregates in the endoplasmic reticulum in hepatocytes, where the protein is synthesised. To investigate aspects of co-translational misfolding, this Thesis examines the biosynthesis of AAT and of a pathological disease variant (Z AAT), on the ribosome, in a mammalian cell-free system, by applying quantitative biochemistry and structural biology techniques. These studies have revealed that ribosome-bound AAT nascent chains (NCs) form co-translational folding intermediates which are important for guiding correct folding (Chapter 3). Also observed are novel circumstances under which this process is hindered, as indicated by the presence of naturally-paused ribosomes at the full-length of AAT sequence; these induce ribosome collisions and the formation of higher-order ribosome-associated assemblies (Chapter 4), which are evident in biochemical assays and visualised using negative stain electron microscopy. The formation of ribosome-bound mis-assemblies, as investigated using sucrose density gradients and quantitative biochemistry, reveals that both “co-post” and “co-co” mechanisms are possible for the formation of AAT polymers (Chapter 5). Finally, a high-resolution cryo-EM structure of the AAT ribosome nascent chain complex (RNC), gives insights into the NC-ribosome interactions that could contribute to its natural stalling (Chapter 6). A better understanding of the origins of the AAT toxic aggregates that lead to disease, will provide a foundation for therapeutics centred on early-intervention strategies.

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