Biochemical Studies of the ER Resident Kinase PERK and Implications for Local Translational Control

The unfolded protein response (UPR) is a highly conserved protein quality control mechanism that becomes activated in response to misfolded protein accumulation in the endoplasmic reticulum (ER) resulting in ER stress. The ER resident kinase protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is able to couple ER stress to translational repression and transcriptional reprogramming through phosphorylation of eIF2α. This is achieved through the recruitment of eIF2α by a unique cytoplasmic loop in PERK’s kinase domain. PERK signalling has been shown to play an important role in disease pathology notably in neurodegenerative diseases. Increased levels of phosphorylated PERK and eIF2α have been observed in the brains of patients with Alzheimer’s disease, Parkinson’s disease, other tauopathies, and prion disease. In many of these conditions, the associated disease-specific misfolded proteins accumulate in the cytoplasm instead of the ER. Currently, the mechanism for PERK activation by cytoplasmic misfolded proteins, as opposed to those in the ER, remains elusive. To gain insight into novel aspects of cytoplasmic PERK signalling which may be relevant to cellular health and disease, in particular, neurodegenerative disease, the PERK-protein interactome was examined using mass spectrometry. This led to the identification of the mitochondrial protein, ATAD3A, as a PERK kinase insert loop interactor. Using biochemical and biophysical techniques, a common binding site within the kinase insert loop of PERK was identified for eIF2α and ATAD3A. To further investigate the dynamics of these interactions bioluminescence resonance energy transfer (BRET) based biosensors were developed, enabling the interaction between PERK and eIF2α to be monitored in real-time in live cells. Mutating the kinase insert loop supported the in vitro binding assays by also indicating the presence of two eIF2α binding sites within PERK’s loop. Furthermore, ATAD3A can outcompete eIF2α for occupancy of this site thus reducing phosphorylation of eIF2α and dampening PERK downstream signalling. The potential future use of these sensors in high through-put drug screening assays to identify compounds that specifically disrupt the interaction between PERK and eIF2α is discussed. Finally, to understand the implications of PERK-ATAD3A interaction on cellular protein synthesis rates, the SunTag-PP7 system was used to monitor live mRNA translation through real-time fluorescent labelling of a reporter mRNA and newly translated proteins. This revealed that during ER stress, protein synthesis local to the mitochondria is relatively resistant to translational shutdown compared to the ER or cytosol. Further, ATAD3A overexpression has minimal further protective effects on protein synthesis rates local to mitochondria but a greater impact upon that at the ER. This data suggests a mechanism for the protection of local protein synthesis rates during UPR-induced ER stress, addressing perhaps how cells manage translational repression in cellular compartments beyond the ER and broadening the understanding of PERK signalling and subcellular translational control. Further, the PERK-ATAD3A interaction and its effect on eIF2α signalling offers a new therapeutic target for the modulation of dysregulated PERK signalling that occurs during disease.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Brar, Karinder

Advisor dc:contributor.advisor

• Balmus, Gabriel

• biology

Licence

• https://www.repository.cam.ac.uk/bitstreams/31eb194d-6d1d-4196-9b56-15094c3f29bb/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.108433

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/368102