Characterizing a Novel Enzyme Involved in Cardiolipin Remodelling
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Background: Cardiolipin (CL) is a specialized dimeric phospholipid comprising approximately 25% of total phospholipids in the inner mitochondrial membrane in eukaryotes, where it functions in energy metabolism, autophagy and apoptosis, and other important cellular processes. CL produced de-novo in the Kennedy pathway is considered nascent due to its non-specific acyl chain profile and is then remodeled to mature CL in the Lands pathway. CL plays several important roles within the mitochondria, including interacting with the electron transport complexes, maintaining the cristae architecture, mediating the apoptotic process, as well as acting in the initiation of mitophagy. Due to these reasons, the appropriate synthesis and remodelling of CL has implications for healthy physiological function. We have discovered a potential novel enzyme involved in CL metabolism, and the purpose of this thesis was to characterize the function of this enzyme in these processes. Major Aims and Approach: The major aims of this thesis were: 1) To investigate the in vitro effects of this enzyme on CL synthesis and 2) To characterize the role of this enzyme in normal physiology using mice deficient in this gene. Results: For our first aim, we discovered that overexpressing this enzyme in HEK-293 cells caused alterations in CL levels in vitro, while the partially purified enzyme displayed an ability to directly remodel monolysocardilipin using phosphatidylcholine as an acyl donor in vitro, which indicates a new and previously unreported function for this enzyme. My second aim had three objectives. The first was to assess alterations in CL content in tissues of mice deficient in this enzyme, versus Wt controls, and we discovered that gene ablated mice displayed tissue-specific alterations in CL content, and this was present in both male and female mice deficient in this enzyme. The second objective was to determine if there are differences in growth, food intake, and gross morphology between wildtype and gene ablated mice. Male gene-deficient mice exhibited differences in food intake, organ weights, and life span. These differences were not observed in female mice, though they followed similar trends despite not reaching statistical significance. The final objective of this thesis was to assess metabolic measures and exercise tolerance. Deficiency in this enzyme caused differences in oxygen consumption, carbon dioxide production, and energy expenditure in both sexes. Male gene-ablated mice also exhibited differences in rearing activity, and exercise tolerance. The differences in rearing activity and exercise tolerance were however not observed in female gene deficient mice. Conclusions: The findings of this thesis indicate a novel role for this enzyme in CL metabolism, including an ability to remodel CL in vitro. Characterization of gene-deficient mice also indicated significant phenotypic differences, especially in male mice, while gas chromatography analysis demonstrated major tissue-specific changes in CL content. These results provide insight into the function of this enzyme.
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Ashkan Hashemi (2023). Characterizing a Novel Enzyme Involved in Cardiolipin Remodelling. UWSpace. http://hdl.handle.net/10012/20137