The many faces of amyloid fibres: their detection and regulation by molecular chaperone proteins
Creators: name: Kumar, Manjeet
Publisher: name: The Australian National University
Year: 2017
Subjects: Protein aggregation
amyloidoisis
neurodegenerative diseases
macromolecular crowding
protein homeostasis
monitoring protein oligomers

description: Proteins are the molecular machines that control and regulate most of the vital cellular functions in living organisms, and account for about half of the total dry mass of the cell. For a protein to function properly, it must attain its correct conformation and location within the crowded milieu of the cell. A complex set of molecular chaperone proteins assist polypeptides to acquire their native functional fold within the relevant biological timescale. However, the intricacy and diverse nature of the protein folding process and various environmental factors present numerous opportunities for error, which may lead to its misfolding and aggregation. Misfolded, aggregated, non-functional proteins and peptides within the cell are implicated with more than 55 pathological conditions including various neurodegenerative diseases such as Alzheimer’s,Parkinson’s and haemodialysis-related amyloidosis, which together affect more than 24 million people worldwide. Collectively, these disorders are classified as protein conformational or protein aggregation diseases. Over the past few years, significant progress has been made to understand the various types and processes of protein aggregation, identify the toxic species, structurally characterise various species generated during their assembly and comprehend the underlying mechanisms which regulate protein conformational diseases. This thesis has attempted to address some of the issues discussed above, to assist in advancing the understanding of the complexities of protein misfolding, aggregation and their mitigation. In this thesis, I have found that various fragments of mouse Acyl co-enzyme thioesterase 7 (mAcot7) and human full-length ACOT7 form amyloid fibrils under physiological conditions. Acot7 is a brain cytosolic protein, involved in fatty acid metabolism, and has a putative role during inflammation. Using various mouse Acot7 constructs, I demonstrated that mAcot7 undergoes nucleation-independent, multi-stranded polymerisation, leading to the formation of globular oligomeric species and subsequently amyloid fibrils. Arachidonoyl-CoA, the substrate of mAcot7 did not prevent the fibrillation of mAoct7, and the protein remained enzymatically active throughout the assembly process. Understanding the biological significance of fibrillar and other forms of Acot7 is one of the avenues to be explored in the future. After putting forth a unique molecular model of mouse Acot7 fibrillation, the next objective was to identify and characterise the heterogeneous population of protein aggregates formed during polymerisation. Bis(Triphenylphosphonium) tetraphenylethene (TPE-TPP), a novel aggregation-induced emission luminogen aided in detecting early-stage protein aggregates; the species that are considered to be the most toxic entities during the development and progression of protein conformational diseases. Compared to traditional amyloid binding fluorescent dyes such as Thioflavin T (ThT), TPE-TPP showed a broader applicability in monitoring the process of fibrillation in various conditions such as acidic pH, elevated temperature, presence of potential amyloid inhibitors, and its ability to detect variations in amyloid fibril morphologies. Once I could differentiate between the different species generated during protein aggregation, I attempted to explore ways to modify these entities. Utilising the intra- and extra-cellular molecular chaperones αB-crystallin (αB-C), clusterin, α2-macroglobulin and haptoglobin, I observed that αB-C and clusterin stabilise various forms of D76N β2-microglobulin (a potent amyloidogenic variant of β2-microglobulin) generated during the fibrillation process. It is likely that these chaperones prevent primary and secondary nucleation of D76N β2-microglobulin (D76N β2m) fibrillation in vivo and decelerate the proliferation of amyloid fibril plaques. Experiments performed in vitro are usually less confined compared to the crowded cellular environment, and such crowding conditions further confound the process of protein aggregation. Moreover, oligomers of various amyloid proteins such as α-synuclein (αS) are believed to be the primary cause of cellular damage, for example in Parkinson’s disease. Utilising the highly identical presynaptic α- and β-synuclein and crowding agents such as Ficoll 400, I analysed the impact of crowding on the association of the synuclein proteins. Under physiologically relevant conditions, I found that β-synuclein (βS) exists as positively charged oligomers, which gradually polymerise to form self-assembled, non-amyloid fibril-like structures. Furthermore, βS destabilises αS and induces its aggregation, which is significantly moderated under the crowded cell-like environment. The results documented in this dissertation have assisted in advancing our knowledge and understanding of the molecular, biophysical and biochemical mechanisms of amyloid fibril formation. In addition, this thesis determined the broader applicability of TPE-TPP compared to the standard amyloid dye (i.e. ThT) as a fluorescent probe to monitor amyloid fibril formation, detect early-stage aggregates, variations in fibril morphologies, and can be deemed useful in screening amyloid inhibitors. Part of the aim to identify and characterise various protein aggregate species was to develop a method for regulating potentially toxic entities. I showed that intra- and extra-cellular molecular chaperones were capable of acting on different stages of D76N β2m aggregation, thereby reducing the effects of undesirable, misfolded toxic protein forms. Furthermore, I demonstrated that there could be other potentially hazardous forms of unwanted protein aggregates (i.e. βS self-assembled structures) that are not necessarily amyloid fibrils. Greater comprehension of protein aggregation will not only shed light on protein conformational disorders and aid in the development of therapeutics against their toxic effects, but may also offer insights into opportunities for exploiting stable, non-toxic protein conformations to our advantage.