Mitochondrial Metabolism and Redox Homeostasis in health and disease

Westbrook, Rebecca Louise ORCID: 0000-0003-0877-3959 (2023). Mitochondrial Metabolism and Redox Homeostasis in health and disease. University of Birmingham. Ph.D.

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Abstract

In order to maintain essential cellular metabolic pathways, the availability of required redox co-factors must remain favourable. For example, several reactions in the oxidative direction of the tricarboxylic acid (TCA) cycle, such as α-ketoglutarate conversion to succinyl-CoA, require nicotinamide adenine dinucleotide (NAD+) as an oxidising agent. The redox balance of the cell can be challenged by outside influences, such as the tumour microenvironment and oxygen availability. It can also be influenced by cell intrinsic factors, such as genetic loss or alteration of enzymes involved in redox-heavy pathways.
Hypoxia presents a significant challenge to redox homeostasis and leads to a marked rewiring of the metabolic network. This thesis will begin by exploring the metabolism of the non essential amino acid, proline, in cancer cells under low oxygen conditions. Proline metabolism is closely linked to redox state due to the requirement of NAD(P)H in multiple reactions. Use of stable isotope tracing reveals that the synthesis of proline from glutamine through pyrroline-5-carboxylate reductase 1 (PYCR1) is increased in hypoxia. The implications of loss of this isozyme, particularly the impact on the cellular redox balance, on the phenotype of breast cancer cells in 2D culture is then investigated. This reveals a reduced NAD+:NADH ratio, reduced oxidative incorporation of glutamine carbons into the TCA cycle and a slowed proliferative rate.
Leading on from these observations, a spheroid culture model is employed to more closely recapitulate the oxygen and nutrient gradients present in solid tumours. Again, stable isotope tracing suggests dysregulated redox balance with PYCR1 loss, leading to apoptosis and expression of hypoxic markers. Finally, this is explored in vivo, using a doxycyline-inducible shPYCR1 xenograft model. Under these conditions, chronic knockdown of PYCR1 results in slowed tumour growth, while acute knockdown results in increased hypoxia, apoptosis and necrosis.
Additionally, a suspected inborn error of metabolism is investigated, initially hypothesised to be caused by a mutation in decaprenyl phosphate synthase subunit 1 (PDSS1), a key step in the synthesis of coenzyme Q10. The mRNA and protein expression of this enzyme are not found to be altered, but a change in function cannot be ruled out. The metabolism of primary fibroblasts from this patient is investigated, and phenotype consistent with a dysregulated redox balance is discovered.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):