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Apoptosis is essential for cellular homeostasis and is also a pathologic feature of various diseases. The balance between Bcl-2 family proteins determines whether a cell will live or die. Bax, a member of the BCL-2 family proteins, is a pro-apoptotic protein that exists in both a soluble, cytoplasmic form and a membrane-bound form. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the mitochondria, initiating apoptotic events. However, little is known about whether Bax is involved in the regulation of mitochondrial function under non-apoptotic conditions, and how Bax binds to mitochondria to exert its activity. Here, we investigate the role of Bax in the regulation of mitochondrial function under non-apoptotic conditions and explore the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli. Using Bax-containing and Bax-deficient (Bax⁻/⁻) HCT-116 cells, we examined Bax cellular localization and its effects on mitochondria bioenergetics, and also tested whether over-expression of full-length Bax in Bax⁻/⁻ cells would recover mitochondrial metabolic activity. To determine the effects of Bax localization upon mitochondrial function, we measured citrate synthase activity and ATP generation. We showed that Bax localized to the outer and inner mitochondrial membranes in non-apoptotic cells, enabling the activity of citrate synthase and the generation of ATP. Loss of Bax led to impairment of respiring mitochondria morphology and reduced oxidative capacity, all of which was restored by expression of full-length or C-terminal-deleted Bax. These findings indicate that under non-apoptotic conditions, the constitutive expression of Bax is necessary for mitochondrial bioenergetics. To determine the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli, we previously performed in silico-mutagenesis and predicted that Lysines 189/190, in the C-terminal [alpha]9 helix, could regulate Bax binding to mitochondria. We demonstrated here that these lysines are the structural elements responsible for controlling how Bax interacts with the mitochondrial membrane. Expression of full-length Bax led to mitochondrial translocation and apoptosis, whereas deletion of the [alpha]9 helix resulted in cytosolic retention and dramatically reduced cell death. Mutation of the two lysine residues changed how Bax bound to mitochondrial membranes. We replicated the results achieved with full-length Bax by attaching the [alpha]9 helix of Bax to GFP or to a regulatory element, the degradation domain (DD), and induced apoptosis upon expression in cells. We demonstrated that the [alpha]9 helix alone promoted the mitochondrial translocation of Bax and increased apoptosis. These results indicate that the C-terminal [alpha]9 helix could be further studied for use in cancer therapies. Overall, we have demonstrated that the constitutive expression of the inactive form of Bax in non-apoptotic cells is necessary for mitochondrial bioenergetics, and have identified the C-terminal [alpha]9 helix of Bax as the effector domain of apoptotic function.

Over the past 20 years, our collaborative research team at the Ottawa Hospital and the University of Ottawa have extensively investigated molecular and metabolic differences between individuals with obesity in the highest (DS) and lowest (DR) quintiles for rate of weight loss following 6 weeks of caloric restriction. Research on these cohorts of individuals with extreme phenotypes in diet-response has revealed that DS individuals have several skeletal muscle metabolic advantages, including increased proportions of type I oxidative fibres, increased mitochondrial proton leak, enhanced fatty acid metabolism, and a greater antioxidant capacity. Regular physical exercise provides a vast array of beneficial effects to metabolic health, including increases in skeletal muscle mitochondrial bioenergetic capacity and muscle cross-sectional area, leading to the hypothesis that exercise may be particularly beneficial to women with diet-resistant obesity. The overall aim of this thesis was to determine whether six weeks of exercise training improves skeletal muscle mitochondrial function and attenuates chronic low-grade inflammation in women with obesity previously identified as diet-sensitive (DS) and diet-resistant (DR). Here, we demonstrate that exercise training improves body composition, enhances cellular maximal respiration, and increases mitochondrial length preferentially in DR women. Contrary to our hypothesis, exercise training increased skeletal muscle IKK-NFκB inflammatory signaling to a greater extent in DR individuals, despite improvements in systemic cytokine concentrations. In response to an inflammatory challenge, LPS-treated primary myotubes derived from DR and DS skeletal muscle responded similarly and respiratory capacity was preserved. Taken together, these findings suggest that exercise can be especially beneficial as part of a treatment plan for DR individuals, and that DS and DR skeletal muscle have systemic and mechanistic differences in inflammatory responses.

Bax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells, MitoTracker staining patterns differed, suggesting the existence of Bax -dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in Bax -deficient cells, while glycolysis was increased. These results suggest that cells lacking Bax have a deficiency in the ability to generate ATP through cellular respiration, supported by detection of reduced citrate synthase activity in Bax -deficient cells. Expression of either full length or C-terminal truncated Bax in Bax -deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of Bax was not dependent upon its C-terminal helix. Expression of BCL-2 in Bax-containing cells resulted in a subsequent loss of ATP measured, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. Bax is composed of nine alpha-helices. While three of these helices have features of a trans-membrane region, the contribution of each domain to the apoptotic or non-apoptotic functions of Bax remains unknown. To examine this, we focused on the C-terminal alpha-9 helix, an amphipathic domain with putative membrane binding iv properties and discovered that it has an inherent membrane-binding and cytotoxic capacity. A peptide based on the last twenty amino acids (CT20p) of the alpha-9 helix was synthesized and proved a potent inducer of cell death independent of any apoptotic stimuli. The solubility of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs), and these CT20p-NPs caused the death of colon and breast cancer cells in vitro and induced tumor regression in vivo, using a murine breast cancer tumor model. CT20p caused increased mitochondrial membrane potential followed by cell death via membrane rupture, without the characteristic membrane asymmetry associated with apoptosis. Hence, while CT20p is based on Bax, its innate cytotoxic activity is unlike the parent protein and could be a powerful anti-cancer agent that bypasses drug resistance, can be encapsulated in tumor-targeted nanoparticles and has potential application in combination therapies to activate multiple death pathways in cancer cells. While previous work revealed novel aspects of the biology of Bax that were unrecognized, new structural information is needed to fully elucidate the complexity of Bax’s function. One approach is to use computational modeling to assess the solved structure of Bax and provide insight into the structural components involved in the activity of the protein. Use of molecular dynamics simulators such as GROMACS, as well as other computational tools provides a powerful means by which to test the feasibility of certain modifications in defined parameters. Such work revealed that the removal of the C-terminal alpha-9 helix of Bax, which normally resides within a hydrophobic pocket, significantly destabilized the protein, perhaps explaining how the protein transitions from soluble to membrane-bound form and maintain energy v production via aerobic respiration or, conversely, how the C-terminus helix conveys cytotoxicity. Collectively, this work reveals that Bax is more than an inducer of cell death but has complex activities yet to be determined.

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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23247号 / 農博第2454号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5337(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23954号 / 農博第2503号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5389(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23955号 / 農博第2504号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5390(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Huntington’s disease is an autosomal dominantly inherited neurodegenerative disorder characterized by degeneration of striatal and cortical neurons. The neurons in these regions are particularly energy-demanding and need to maintain high levels of oxidative phosphorylation to support cellular activities. Reactive oxygen species are generated as a byproduct of oxidative phosphorylation and can damage DNA and other biomolecules if not properly metabolized. In HD, there is elevated oxidative DNA damage and impaired DNA damage repair, likely due to impaired function of the mutant huntingtin protein in base excision repair (BER). Previous studies have shown that mutant huntingtin is hypo-phosphorylated at serines 13 and 16 in the N17 domain, and that restoring phosphorylation can reestablish normal protein function and is protective in HD. In this thesis, we show that a metabolite of the DNA damage product N6- furfuryladenine (N6FFA), kinetin triphoshate (KTP) increases N17 phosphorylation through casein kinase 2 (CK2) by acting as an ATP analog, with protective effects in cell and animal models of disease. We additionally show N6FFA increases the activity of CK2 on other substrates, specifically p53. We hypothesize that in times of ATP stress CK2 can utilize KTP as an alternate energy source, promoting DNA repair and cell viability. In HD, inefficient BER inhibits generation of KTP and promotes hypo- phosphorylation of CK2 substrates, which can be overcome by exogenous addition of N6FFA. Additionally, we show that another DNA-responsive kinase, PKCζ, can also phosphorylated N17, potentially priming this domain for CK2 phosphorylation. Finally, we propose that the protective effects of N6FFA may be via a two-pronged pathway, involving both CK2 and the mitochondrial quality control kinase, PINK1. Thus, this thesis presents a novel mechanism where a product of DNA damage acts as a phosphate source for critical kinases in DNA repair and mitochondrial maintenance in conditions where ATP levels are low. / Thesis / Doctor of Philosophy (PhD)