Biochemical and Biophysical Studies of the UPR Kinase PERK and its Interactors in Health and Disease

The unfolded protein response (UPR) is a protective cellular mechanism that restores protein folding homeostasis under conditions of endoplasmic reticulum (ER) stress. The PERK-eIF2α branch of the UPR is activated by the accumulation of misfolded proteins in the ER lumen. PERK, upon activation through autophosphorylation, recruits its substrate eIF2α for phosphorylation, initiating a transcriptional and translational response. Overactivation of this signalling pathway is a major pathological feature in a range of neurodegenerative diseases. However, the mechanism by which the PERK pathway is activated in these conditions, where misfolded proteins are predominantly deposited in the cytoplasm rather than the ER, is still unknown. Recently, evidence for novel functions and modulation of PERK signalling has emerged. This has led to the working hypothesis that in disease, cytoplasmic disease-specific aggregates may directly interact with the cytoplasmic face of PERK, modifying its signalling in a non-conventional manner and resulting in the observed increase of PERK pathway activation. The overarching aim of this thesis is to further understand mechanisms of PERK pathway modulation and how these may potentially be altered in disease. This was addressed by developing a combination of biochemical techniques and biophysical methods, namely microfluidic diffusional sizing and Förster resonance energy transfer (FRET), to investigate interactions of PERK in solution and live cells. In-solution interaction studies confirmed recruitment of eIF2α by the unique insert loop situated within PERK’s kinase domain, but also indicated the presence of additional eIF2α binding sites in PERK, which may play a significant, but currently unknown, role in PERK signalling. Additionally, the interaction between PERK and its newly identified binding partner, the mitochondrial protein ATAD3A, was found to be direct, with ATAD3A’s tertiary and quaternary structure potentially being necessary for its modulatory effect on PERK signalling. Next, the question was posed whether disease-specific protein aggregates that accumulate in the cytoplasm, specifically α-synuclein oligomers, can act directly on the PERK pathway. In-solution techniques provided evidence to support the existence of a non-canonical mechanism of PERK signalling activation driven by these cytotoxic oligomers. Oligomeric α-synuclein interacted directly with PERK’s cytosolic domain and, strikingly, with PERK’s substrate eIF2α. This increased or stabilised the recruitment of eIF2α to PERK and resulted in elevated levels of phosphorylated eIF2α. Importantly, this was not observed with the monomeric α-synuclein protein. These findings could potentially explain the increased levels of PERK signalling and eIF2α phosphorylation observed in synucleinopathies and, possibly, other neurodegenerative diseases. To delve deeper into PERK:eIF2α interaction dynamics, FRET biosensors to monitor the interaction between the two proteins in live cells were developed. These successfully reported the specific, kinase insert loop-dependent, interaction of PERK with its substrate eIF2α. In the future, these biosensors hold potential for resolving local and temporal aspects of PERK:eIF2α binding dynamics under physiological and pathological conditions. Taken together, this work contributes to the understanding of PERK signalling modulation in health and disease. Whilst the proposed model for direct aggregate-mediated activation of the PERK pathway in neurodegenerative diseases requires further validation in cell-based and in vivo models, it may represent a unique therapeutic target which could enable specific inhibition of pathology-driven PERK pathway signalling.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Rieder, Lara-Sophie

Advisors dc:contributor.advisor

• Mallucci, Giovanna
• Knowles, Tuomas
• Avezov, Edward

• FRET
• microfluidic diffusional sizing
• neurodegeneration
• PERK signalling
• unfolded protein response

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e5982470-827b-45c5-b3b5-61363902b435/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.112599

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/374572