MULTI-OMICS INVESTIGATION INTO THE ROLE OF CYCLOPHILIN D (CYPD) IN MITOCHONDRIA


Adegbite, Yetunde (2021) MULTI-OMICS INVESTIGATION INTO THE ROLE OF CYCLOPHILIN D (CYPD) IN MITOCHONDRIA. PhD thesis, University of Liverpool.

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Abstract

Cyclophilin D (CypD), a ~18KDa mitochondrial member of the peptidyl-prolyl cis-trans isomerase has been widely reported to be implicated as a regulator of the mitochondrial permeability transition pore (MPTP) dynamics; however, to date, its mechanism of action remains elusive. Being a peptidyl-prolyl isomerase, it is suggested that CypD functions alongside other mitochondrial chaperones to aid in the folding and refolding of nascent and unfolded matrix proteins, respectively. Indeed, less work has been directed towards the protein quality control of CypD, whereas many studies have investigated its role in the MPTP. As it is unlikely that the primary role of CypD in the mitochondria is regulating the MPTP, thus, this project aimed to explore and unravel other roles in the mitochondria. It was hypothesized that since the mitochondria are the major metabolic hub of the cytoplasm, understanding the basal metabolic changes associated with CypD deficiency both at acute and chronic levels would enhance the understanding of CypD and this may provide useful information for the characterisation of the MPTP. Three branches of OMICS; metabolomics, transcriptomics, and proteomics were utilised for the study in HAP1 cells. Metabolic changes were observed via NMR-based techniques in both animals (mouse models) and in vitro cell (HAP1) systems. Before this, preliminary optimisation of protocols for NMR-based metabolomics was performed and adapted for both the animal and cell-based systems. Transcriptomics and proteomics studies were achieved by next-generation RNA sequencing and liquid chromatography-mass spectrometry (LC-MS) respectively. The work herein revealed the dysregulation of mitochondrial proteostasis which is accompanied by the loss of the mitochondrial bioenergetic characteristics in CypD deficient cells and tissues. Thus, resulting in the activation of alternative pathways to offset the ensuing energy deficit. Asides from glucose, pyruvate, and some amino acids, other major metabolic markers associated with CypD deficiency include the cellular accumulation of succinate, polyamines, and 1 carbon donors which are potential modulators of the genomic landscape. Paramount changes at both transcript and protein levels were decreased mitochondrial electron transport chain components, PGC1a – the master regulator of mitochondrial biogenesis, and numerous mitochondrial translational machineries such as nuclear-encoded rRNA and tRNAs. Conversely, 9 out of the 13 mitochondrially generated mRNA were significantly elevated in both acute (KD) and chronic (KO) CypD deficiency, however, not at the protein level. More importantly, CypD KO was characterised with reductive stress phenotype, signified by elevated NAD(P)H, GSH, and relatively lower cellular ROS levels than the WT counterpart. This result emphasized the physiological importance of CypD in the regulation of both mitochondrial energy and protein homeostasis. Specifically, proteins involved in pyruvate oxidation and mitochondrial mRNA degradation may be directly regulated by CypD and CypD ablation may contribute to their dysregulation and subsequent disruption of the mitochondrial bioenergetics, hence the observed nuclear and cytoplasmic effects.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 16 Dec 2022 11:32
Last Modified: 18 Jan 2023 21:24
DOI: 10.17638/03143754
Supervisors:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3143754
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