The structure, function and interactions of holdase molecular chaperones in neurodegenerative diseases

Abstract

Neurodegenerative diseases are characterised by the progressive loss of cells in the nervous system, resulting in a decrease in cognitive, behavioural and motor function. Currently, no treatments are available to prevent, halt or reverse neuronal loss. A majority of these disorders such as Parkinson's (PD) and Alzheimer's diseases are associated with the misfolding and aggregation of proteins within the brain, which causes neurotoxicity. Holdase-like molecular chaperones are key players of the cell's intrinsic protection machinery which prevent protein aggregation. This thesis examines the structure, function and interactions of holdase molecular chaperones. The first study investigated the effect of oxidised dopamine (DA), a pathological hallmark in PD on the structure and function of small heat-shock proteins (sHsps), which are ATP-independent, holdase molecular chaperones. Oxidised DA promoted the cross-linking of sHsps which formed well-defined dimer, trimer, tetramer, etc., species as monitored by SDS-PAGE. When modified with a molar equivalent of DA, the chaperone function of the sHsps was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant A53T alpha-synuclein (aS), whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In a cellular context, retention of significant chaperone functionality by mildly oxidised DA-modified sHsps would contribute to protein homeostasis (proteostasis) by preventing protein aggregation, particularly of aS, the putative causative agent in PD. The second study revealed that irradiation with far-UV light in a circular dichroism spectrophotometer promoted the unfolding of sHsps. Recent studies have reported that sHsps unfold upon dissociation from the oligomeric state, which likely results in their most chaperone-active species. The polyproline type-II (PPII) helix is a secondary structural motif postulated to be a common structural motif in sHsps, and to be of importance in their function. However, monitoring and stabilising the unfolded or PPII helical state of sHsps has been experimentally challenging. This study suggests that far-UV light (180 - 260 nm) leads to the unfolding of sHsps and the adoption of an unfolded or PPII helical structure. Potentially, these findings provide a means to undertake detailed investigations of how sHsps prevent protein unfolding and aggregation within the cell. The third study investigated the cytosolic interactome of beta-synuclein (bS), a small presynaptic intracellular protein co-expressed with aS. Recently, novel functions of bS have been discovered, suggesting that bS may possess roles other than its putative molecular chaperone activity to stabilise aS and prevent its aggregation. However, in comparison to aS, bS has been under-investigated, deeming it challenging to understand its function. This study employed APEX2 labelling and found 590 novel proteins within the close spatial proximity and hence assumed to interact with bS, including 58 identified with higher confidence. Autophagy, antigen processing, copper binding, ubiquitin and ubiquitin-like conjugation, poly(A) tail binding and translational control, liquid-liquid phase separation and amyotrophic lateral sclerosis were a subset of enriched functional terms associated with these novel, putative interactors of bS. These findings provide an overview of the roles that bS may have in the cell, along with insights into the potential links between bS and neurodegenerative diseases. Overall, this thesis may contribute to the understanding of post-translational modifications, mechanism of chaperone activity and additional intracellular functions of molecular chaperones, which may aid in understanding the pathogenesis of neurodegenerative diseases that are associated with protein aggregation and hence disruption of proteostasis. Show more