Investigating the protein disaggregation machinery in the early secretory pathway

Abstract

Protein misfolding and subsequent aggregation represent key pathological features in various forms of dementia, notably Alzheimer’s disease (AD). AD is characterised by the presence of extracellular plaques, comprised of aggregated amyloid beta (Aβ) peptides, and intracellular neurofibrillary tangles, consisting of hyperphosphorylated tau protein aggregates within brain tissue. Therefore, a comprehensive investigation into the molecular mechanisms driving and preventing protein aggregation is important for gaining molecular understanding of the development and progression of dementia. A primary contributor to AD pathogenesis, Aβ, originates from a membrane protein and traverses the secretory pathway. While tau is also implicated in AD, particularly affecting the ER-Golgi secretory pathway, recent research highlights the significance of Aβ due to its initial extracellular processing and subsequent re-entry into neurons. Additionally, emerging evidence suggests that compromised intracellular protein folding quality control mechanisms may be part of facilitating the accumulation of toxic protein aggregates. The scientific evidence underlines the importance of exploring the intricate system responsible for managing aggregation-prone proteins. Recent findings from our laboratory have illuminated the inherent capability of the early secretory pathway, specifically the endoplasmic reticulum (ER), in resolving preformed protein aggregates, particularly under conditions of pharmacologically-induced ER stress. These discoveries have led us to postulate that the induction of this system could potentially mitigate the pathology associated with protein aggregation. Conversely, any dysfunction within this system may pose an increased risk for the development of AD-related pathology. Hence, the primary objective of this research was to investigate the molecular mechanisms and precise interactors comprising the intrinsic disaggregation system within the ER. This aim was achieved through a series of proteomics investigations involving the isolation of surrogate metastable proteins and its interaction partners under disaggregation conditions. Additionally, a parallel mission to engineer an aggregation biosensor, which will facilitate the detection and characterisation of aggregation-prone proteins in their various molecular states was undertaken. The outcomes of the proteomics investigation identified various chaperones important to ER folding processes. Consequently, this discovery prompted a more in-depth exploration of their potential involvement in the disassembly of ER aggregates. Additionally, success in the construction of closed and open conformation versions of a FRET-FLIM paired biosensor, which demonstrated its efficacy upon detection through Fluorescence Lifetime Imaging Microscopy (FLIM).