Browsing by Subject "Mitochondria"
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Item Biochemical and genetic studies of mitochondrial protein synthesis in Saccharomyces cerevisiae : characterization of the AEP3 and TRM5 gene products(2008-08) Lee, Changkeun, 1971-; Appling, Dean RamsayProtein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.Item Biochemical Pathways in Apoptosis(2005-05-03) Nijhawan, Deepak; Wang, XiaodongCaspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.Item Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae(2011-08-08) Garber Morales, Jessica H.An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.Item Defining a Novel Role for Hypoxia Inducible Factor-2 Alpha (HIF-2a)/EPAS1 : Maintenance of Mitochondrial and Redox Homeostasis(2005-12-20) Oktay, Yavuz; Garcia, Joseph A.The Epas1 gene encodes HIF-2alpha , a member of the Hypoxia Inducible Factor family of transcriptional regulators. The biological role for HIF-2alpha has been elusive due to embryonic lethality of the initial Epas1-/- mouse strains. Our lab reported the generation of the first viable Epas1-/- mice using a genetic breeding strategy. Adult Epas1-/- mice exhibit gross, histological, biochemical, and molecular evidence consistent with mitochondrial dysfunction. Similarities between Epas1 and Sod2 deficient strains suggest a biochemical etiology, increased oxidative stress, as well as a molecular etiology, decreased Sod2 gene expression, for the mitochondrial dysfunction in Epas1-/- mice. Consistent with this hypothesis, Sod2 gene expression is reduced in Epas1-/- mice whereas HIF-2a induces Sod2 gene promoter in transient transfection studies. Further studies revealed impaired mitochondrial respiration, sensitized mitochondrial permeability transition pore opening, increased electron transport chain activity and reduced mitochondrial aconitase activity. Given that it is the most sensitive enzymatic marker for oxidative stress, aconitase inhibition may explain impaired respiration. Also, redox balance in Epas1-/- liver is disturbed: the reduced cytoplasmic environment, and a relative oxidized environment for mitochondria from Epas1-/- liver implies a role for HIF-2a in maintenance of cellular redox balance. All these data suggest that HIF-2a is essential for maintenance of mitochondrial function, reactive oxygen species detoxification, and redox balance.Item Defining the role of Mtf1 and N-terminal domain of Rpo41 in transcription initiation and replication(2012-05) Chang, Hae Ryung; Yin, Yuhui Whitney; Hackert, Marvin L.; Jayaram, Makkuni; Johnson, Kenneth; Molineux, IanMitochondrion is an organelle found in the eukaryotic cell. It is responsible for essential metabolic processes as well as ATP production via oxidative phosphorylation (OXPHOS). The mitochondrion contains DNA that encodes for several subunits in the OXPHOS system as well as rRNA and tRNA for translation. It also has its own replication, transcription and translation machinery. Proper maintenance of the mitochondrial DNA is critical for the cell’s health. Saccharomyces cerevisiae mitochondrial transcription system has been a great model system for its ease of genetic manipulation as well as having conserved RNA polymerases across species. The polymerases are homologues to T7 RNA polymerase, but have longer N-terminal domain and require transcription factor(s). The reason for the extra domain as well as the need for an accessory factor is still unclear. This study reveals the role of Rpo41 N-terminal domain (NTD) as well as clarifies the role of Mtf1, the transcription factor, in transcription initiation. Rpo41 is the 153 kDa catalytic subunit, and Mtf1 is 40 kDa, the transcription factor of the yeast mitochondria. We have shown that Mtf1 is required for correct promoter sequence recognition as well as inhibition of incorrect initiation. Although it was thought that Rpo41 has intrinsic promoter recognition capability, we have shown that Rpo41 can initiate transcription on a pre-melted DNA, even if it is not the consensus promoter sequence. N-terminal truncation mutant studies showed that the NTD of Rpo41 is also required for correct transcription initiation. On linear duplex DNA, N-terminal truncation of 321 amino acids has little effect when Mtf1 is present. On pre-melted DNA, it shows opposite trend from the wild-type. 160 N-terminal amino acid residue truncation shows little activity, whereas Mtf1 increases activity, even on non-promoter initiation sites. We further investigated properties of Rpo41 in replication. A link between mitochondrial transcription and replication has been suggested before, where Rpo41 functions as the leading strand primase. Our studies show that Rpo41 can indeed function as the leading and lagging strand primase, and explains why Rpo41 is able to initiate transcription on non-promoter sites. N-terminal truncation resulted in loss of primase activity, which shows that NTD is required for replication.Item Fidelity of nucleotide incorporation by the human mitochondrial DNA polymerase(2005) Lee, Harold Ray; Johnson, Kenneth AllenThe human mitochondrial DNA polymerase (pol γ) is a nuclearly encoded enzyme, imported to the mitochondria, solely responsible for the replication of the mitochondrial genome. I have characterized the kinetics of nucleotide incorporation, determined the discrimination constants for misincorporation, and calculated the overall fidelity of this enzyme. Additionally, I have investigated the dependence of these parameters on the concentration of magnesium ion present in the reaction. There have been reports in the literature of pol γ having reverse transcriptase activity, and of attempts to determine a physiological role for this activity. Indeed, many steady-state kinetic assays of this enzyme reported in the literature are reverse transcriptase assays. I have characterized the kinetics of incorporation of the reverse transcriptase activity of polymerase γ, both in single turnover and processive polymerization assays. Additionally, I have characterized the activity of the 3′-5′ exonuclease domain on a DNA/RNA heteroduplex. For many years there has been research into the factors that contribute to polymerase discrimination. Base pair hydrogen bonding, base stacking, steric interactions, active site tightness, and factors unknown are all believed to play a role. However, recent attempts to investigate the contribution to discrimination afforded by base pair hydrogen bonding in Klenow fragment, using “shape mimic” nucleoside analogs have led to the supposition that base pairing plays little, if any, role in discrimination. In order to investigate the contribution of base pair hydrogen bonding to discrimination in pol γ, I have characterized the incorporation of natural nucleotides opposite the dT analog 2, 4-difluorotoluene deoxynucleoside (dF) and the dA analog 9- (1-aza-4-methyl-benzimidazolyl)-1′-β-2′-deoxyriboside (dQ). Additionally, the kinetic parameters of incorporation of dF opposite dT, and of dQ and 4-methylbenzimidazole (dZ) opposite dA have been determined. The rates of 3′-5′ exonuclease removal of natural nucleosides paired opposite dF and dQ have been determined as well. The mitochondrial polymerase is the sole enzyme responsible for replication of the mitochondrial genome. In the absence of accurate and efficient replication by polymerase γ, several clinical pathologies are observed including cardiac and neural myopathy, mitochondrial myopathy, anemia, and potentially fatal lactic acidosis. These symptoms are known effects of oxidative damage to the mitochondrial genome and of toxicity associated with the treatment of HIV infection with nucleotide reverse transcriptase inhibitors. Additionally, several diseases have been associated with mitochondrial genome mutation and depletion including Parkinson’s disease and Alzheimer’s disease. A full understanding of the fidelity of the mitochondrial DNA polymerase and the mechanisms by which this fidelity is insured will aid in the understanding of diseases associated with mitochondrial damage and in the design of drugs used to fight HIV, lacking the potentially fatal mitochondria based toxicities.Item Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae(2013-11-27) Cockrell, Allison LeighTransition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and toxicity mechanisms are employed for regulating and storing intracellular Fe. In Saccharomyces cerevisiae, vacuoles are responsible for sequestering, storing, and supplying Fe to the cytosol. Many of the proteins and regulatory pathways involved in Fe trafficking and storage in S. cerevisiae have been identified, but the forms of Fe which are involved in these processes have not been fully characterized. In these studies, biophysical and bioanalytical techniques were used to study intracellular Fe distributions in S. cerevisiae cells and organelles. Ultimately, Fe-containing species were biophysically characterized and absolute Fe concentrations in cells and organelles were quantified. The motivation for these studies stemmed from previous studies which revealed that the majority of the whole-cell Fe is a non-heme, high-spin (NHHS) form of Fe^(3+). This Fe is not localized to the mitochondria. The purpose of these studies was to determine if the vacuoles contained this NHHS Fe^(3+). A large-scale isolation procedure was developed to obtain purified vacuoles from S. cerevisiae and to investigate the Fe in these organelles. M?ssbauer and EPR analysis revealed that the primary form of Fe in vacuoles is a mononuclear, NHHS Fe^(3+) species. A second form of Fe was also observed as superparamagnetic ferric phosphate nanoparticles (NP). By investigating model compounds of Fe and polyphosphate we determined that a shift in vacuolar pH induces the conversion between NHHS Fe^(3+) and NP. These results showed that there are at least two forms of Fe in vacuoles, and that the ratio of these two forms is dependent upon the pH of these organelles. Biophysical analyses of whole cells also revealed the presence of low concentrations of a non-heme, high-spin Fe^(2+) species. The goal of these next projects was to determine if this NHHS Fe^(2+) species was localized to the cytosol. Genetic strains lacking or over-expressing the vacuolar Fe import protein Ccc1p were studied by M?ssbauer spectroscopy (?CCC1 and CCC1-up, respectively). ?CCC1 cells showed low vacuolar Fe (NHHS Fe3+ and NP), and increased NHHS Fe^(2+). We hypothesize that this NHHS Fe^(2+) is cytosolic Fe. We also propose that this NHHS Fe^(2+) is involved in the regulating intracellular Fe levels. CCC1-up cells accumulated more Fe than wild-type (WT) cells, and showed elevated levels of vacuolar Fe (NHHS Fe^(3+) and NP). These cells also accumulated high levels of NHHS Fe^(2+). The CCC1-up cells exhibited an adenine deficient phenotype, where the cells developed a red color during growth. With excess adenine the levels of NHHS Fe^(2+) declined, which indicated that this Fe accumulation was related to adenine deficiency. We conclude that adenine deficiency leads to the accumulation of a sequestered (possibly vacuolar) form of NHHS Fe^(2+). Overall, we have identified two separate pools of NHHS Fe^(2+) in ?CCC1 and CCC1-up cells. In ?CCC1 cells the NHHS Fe^(2+) pool is localized to the cytosol and is sensed by the cell. In CCC1-up cells the NHHS Fe^(2+) is sequestered from the Fe regulatory mechanism- possibly in the vacuoles. These data have helped us better understand the roles of vacuoles in Fe trafficking and the dynamics of vacuolar Fe trafficking.Item Mechanistic insights into the function of the mitochondrial uncoupling protein in Caenorhabditis elegans(2010-08) Pfeiffer, Matthew Edwin; Mills, Edward M.; Bratton, Shawn B.; Wright, Casey W.; Combs, Alan B.; Morgan, Philip G.The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics.Item Mitochondrial uncoupling links lipid catabolism to Akt inhibition and blockade of skin tumorigenesis(2014-08) Nowinski, Sara Marie; Mills, Edward MichaelIn order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling.Item Neurodegeneration caused by mitochondrial complex I dysfunction in the mouse retina(2005) Zhang, Xian; González-Lima, FranciscoItem New application of fluorescence correlation spectroscopy for early detection of apoptosis in cells(2013-08) Lyer, Di; Pappas, Dimitri; Thompson, Jonathan E.In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial proteins release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy, flow cytometry and microfluidic technology were employed to explore ultrasensitive and high throughput detection of apoptosis in cells using confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells and to develop high throughput cell screening using microfluidic technology, enabling new methods of rapid drug testing and dose-response analyses.Item Regulation of Cytochrome C Release in UV-Induced Apoptosis(2006-05-16) Traer, Elie; Mumby, MarcApoptosis, or programmed cell death, is vitally important for maintaining cellular homeostasis. When damaged cells do not appropriately enact the cell death program they have the potential to accumulate genetic mutations and become cancerous. Therefore, a mechanistic understanding of how cells decide to die would provide the foundation for drug discovery to specifically target cancer cells. Current genotoxic chemotherapeutics, and other sources such as ultraviolet (UV) radiation, damage DNA, leading to induction of apoptosis through the mitochondrial pathway. Cytochrome c is released from the mitochondria, binds dATP and Apaf-1, and together they form a structure called the apoptosome. The apoptosome then binds and activates caspase-9, which cleaves caspase-3 and other caspases resulting the morphological changes characteristic to apoptosis. To understand how UV causes apoptosis, an assay was developed to reproduce cytochrome c release in vitro. This assay revealed that UV radiation causes a rapid decrease of the anti-apoptotic protein Mcl-1 in HeLa cells. Reduction of Mcl-1 on the mitochondria causes in vitro release of cytochrome c from mitochondria three hours before in vivo release of cytochrome c is observed, i.e. the mitochondria are primed to release cytochrome c. Removal of Mcl-1 is required for UV-induced apoptosis, but it is not sufficient to induce apoptosis. This means that, in addition to mitochondrial priming, there is another required event, a second hit. It was reasonable to think that this required second hit might be bound specifically by Mcl-1. To that end, Mcl-1 was found to bind BimEL with far greater affinity than any other pro-apoptotic Bcl-2 family member. In addition, BimEL is dephosphorylated after UV in an ERK1/2-dependent manner. Despite perfectly fitting the profile of the second hit, BimEL and ERK1/2 phosphorylation do not have any affect on induction of caspases after UV. These results have helped gain insight into the regulation of cytochrome c release, which remains the most perplexing and important question in apoptosis.Item Regulation of DIAP1 function by Dropsophila Omi and the N-end rule pathway(2007-12) Malladi, Madhavi, 1976-; Bratton, Shawn B.The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.Item Regulation of Metabolic Processes by Micrornas and Class I Histone Deacetylases(2013-01-17) Carrer, Michele; Olson, Eric N., Ph.D.Obesity is a medical condition resulting from accumulation of excess body fat that affects more than 30% of the adult population in the U.S. Obesity-related pathological conditions include heart disease, stroke, type 2 diabetes and certain types of cancer. Despite the high incidence and the elevated social costs, the molecular basis of obesity and associated metabolic syndrome are still poorly understood. Yet, the need for novel therapeutic approaches for the treatment and prevention of obesity remains. In humans and animal model of disease, hallmarks of obesity include dysregulation of genes involved in mitochondrial function, lipid uptake and lipid storage. The dynamic and modifiable regulation of transcriptional pathways that control mitochondrial function and adipogenesis, as well as additional aspects of mammalian metabolism, will provide new approaches for pharmacological intervention in obesity. Thus, the modulation of epigenetic histone modifications and microRNA functions represents a potentially powerful approach for the treatment of metabolic disorders. We show that the Ppargc1b gene, which encodes the PGC-1? protein, also co-transcribes two microRNAs, miR-378 and miR-378*. Mice lacking miR-378/378* are resistant to high fat diet-induced obesity and display enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Taken together, our findings reveal that miR-378 and miR-378* function as integral components of the regulatory circuit formed by PGC-1beta and nuclear hormone receptors to control the overall oxidative capacity and energy homeostasis of insulin-target tissues. MiR-378/378* mutant mice do not display overt phenotypes under normal laboratory conditions, whereas their phenotypes become apparent under conditions of stress, in this case in response to excessive caloric intake. Thus, pharmacological modulation of miR-378/378* function might represent an effective approach in the treatment of obesity. In obese humans and mice, the unused caloric energy resulting from excessive net caloric intake is converted to triglycerides and stored in adipocytes for further usage. Lipid accumulation within adipocytes is under the control of a cascade of transcription factors that interact with histone acetyltransferases and deacetylases. We show that histone deacetylase inhibitors efficiently block adipocyte differentiation in vitro. Furthermore, through a loss-of-function approach, we provide evidence that histone deacetylases 1 and 2 play redundant and requisite roles in adipogenesis. In conclusion, we unveiled previously unrecognized roles for miR-378/378* in the control of mitochondrial metabolism and energy homeostasis, and for histone deacetylases in the control of adipocyte differentiation.Item Regulation of steroidogenesis in subclones of the Ma-10 mouse leydig tumor cell line(Texas Tech University, 1990-05) Kilgore, Michael Wayne.Item The role of the human mitochondrial polymerase in the toxicity of nucleoside analogs and aging(2004) Hanes, Jeremiah Wayne; Johnson, Kenneth AllenThe toxic side effects associated with the administration of nucleoside analogs used to treat HIV are correlated with the kinetics of incorporation by the human mitochondrial polymerase γ (Pol γ). The reconstitution of recombinant human enzyme has allowed for a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. One nucleoside analog in particular, zidovudine (AZT), exhibits unique kinetics of incorporation by Pol γ. Evidence is presented supporting a model in which the kinetics of incorporation of AZT differ from that of natural nucleotides and other nucleoside analogs, in that phosphoryl transfer is reversibly linked to binding by way of a slower than normal enzyme isomerization following phosphoryl transfer. The implication of the unique reaction kinetics on the toxicity associated with the use of AZT is also discussed. The most toxic nucleoside analog approved for treatment of HIV is zalcitabine (ddC). It was shown previously that the incorporation of ddC by Pol γ is relatively efficient and that the removal by the proofreading exonuclease was too slow to measure under the conditions used. Both efficient incorporation and slow removal are likely to contribute to the observed clinical toxicity. I revisited this phenomenon by measuring the rate of excision more accurately and under various conditions in order to better understand the mechanistic basis for the slow removal of ddC. In addition to a role in the toxicity of nucleoside analogs used to treat HIV, Pol γ may play an important part in the process of aging. The mitochondrial theory of aging states that electrons derived from the electron transport chain during normal respiration produce especially high levels of reactive oxygen species (ROS) in the mitochondria. These ROS can damage the mitochondrial genome, compromising its integrity. One of the most common products of oxidative damage to DNA is 8-oxodG. I have examined the kinetic parameters governing the replication of oxidatively damaged DNA by Pol γ and show that replication fidelity is reduced when incorporation is performed when 8-oxodG is the templating base. I provide evidence suggesting that oxidized free nucleotide (8-oxodGTP) may also be mutagenic.Item Studies of the cytochrome bc1 complex and its electon acceptors in purple photosynthetic bacteria(Texas Tech University, 2001-12) Li, JunThe cytochrome bc1 complex is a key component of electron transfer chains in mitochondria and in many aerobic and photosynthetic bacteria. The bc1 complexes from photosynthetic bacteria provide many advantages for understanding electron transfer through the complex, as they are structurally simpler than the mitochondrial complexes and fast electron transfer after photoactivation of the reaction center can be easily followed using absorbance changes, something that cannot be done with mitochondria. This dissertation focuses on three aspects related to the bc1 complexes: (1) The role of HiPIP and cytochrome cg, which are reduced by the bc1 complex, as alternative electron donors to the reaction center of Chromatium vinosum. Laser flash kinetics with intact Cm. vinosum cells showed that either HiPIP or cytochrome cg can be an efficient electron donor to the reaction center, depending on the media used to grow the cells. However, the preference for one electron donor over another does not arise from significant differences in protein abundance in the cells grown in the two different media. The mechanism of this "switch" remains to be elucidated. (2) Spectroscopic and oxidation-reduction properties of M183K and M183H variants of Rhodobacter capsulatus cytochrome c8. MCD and EPR spectra suggest that Rb. capsulatus cyt c\ is flexible and that one of the three histidines present outside the normal heme-binding domain can be recruited as an alternative to the methionine heme ligand found in the wild-type cytochrome. Titrations carried out in the oxidative direction differ markedly from those carried out in the reductive direction, mdicating the possible occurrence of redox-triggered conformational changes. (3) Interaction between Rb. capsulatus cytochrome bc1 and equine cytochrome c (a homolog for Rb. capsulatus cytochrome C2). Steady-state kinetic data, using site-specific cyt c1 mutants, showed that one acidic patch on the surface of cytochrome ci is unlikely to be involved in binding to cytochrome c. However, both steady-state kinetics and redox titration revealed that the phenylanaline at position 138 plays a critical role m maintaining a normal heme environment and suggest that this aromatic amino acid may participate in mediating electron transfer to and/or from the heme of cyt c1.Item Targeting mitochondria via methylene blue : implications in memory enhancement and neuroprotection(2015-05) Auchter, Allison Michelle; González-Lima, Francisco, 1955-; Monfils, Marie-H.; Schallert, Timothy; Haley, Andreana; Barea-Rodriguez, EdwinMemory—though seemingly simple in concept—is altogether a notoriously elusive and difficult process to understand. While some memories are fragile and changeable under certain circumstances, others are persistent and difficult to erase. This work is a comprehensive investigation into memories: how to change persistent ones, enhance complicated ones and protect delicate ones. The first objective of this work was to explore a strategy for manipulating fear memories, which are notoriously difficult to erase. This was done by testing different parameters for attenuation of fear using extinction as a strategy for interrupting fear memory reconsolidation. Briefly, by manipulating extinction parameters such that subjects were unable to predict the occurrence of fearful stimuli, we were able to maximize the degree of memory updating. Since memory processing is contingent upon specific neuronal activity, and neuronal activity is primarily fueled by products of mitochondrial respiration, the second and third objectives of this work focused largely on how manipulating mitochondrial activity enhances and protects memory processes respectively. This was done using mitochondrial enhancer methylene blue (USP grade). Methylene blue (MB) is a synthetic dye with a unique ability to cross the blood-brain barrier and diffuse into neuronal mitochondria. There, it serves as a redox electron cycler, increasing neurons’ capacity for mitochondrial respiration. The second objective of this work examined the effect of post-extinction administration of MB and its interaction with reconsolidation update mechanisms in the persistent attenuation of fear memories. Building on findings from the first objective, we found that administering MB after extinction helped subjects maintain the fear attenuation induced by extinction. The third objective explored the ability of MB to prevent the cognitive deficits arising from chronic cerebral hypoperfusion, a risk factor for mild cognitive impairment and Alzheimer’s disease. Finally, quantitative cytochrome oxidase histochemistry (a marker for mitochondrial activity) was used to map the effects of MB on brain mitochondria in the hypoperfusion model. Methylene blue attenuated some of the cognitive deficits that arose from chronic cerebral hypoperfusion, which was reflected in enhanced brain mitochondrial activity.Item The role of docosahexaenoic acid in mediating mitochondrial membrane lipid peroxidation and apoptosis in colonocytes.(Texas A&M University, 2005-11-01) Ng, Yee VoonColon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.Item Ultrastructural changes in synaptic and mitochondrial structure throughout postnatal development and long-term potentiation in rat hippocampus(2015-08) Smith, Heather Lynne; Harris, Kristen M.; Aldrich, Richard; Raab-Graham, Kimberly; Schallert, Timothy; Zemelman, BorisMitochondria, by providing the vast majority of ATP produced in neurons, fuel many steps of the synaptic vesicle cycle, but little is known about their role in synaptic vesicle clustering and mobilization during synaptic development and plasticity. Long-term potentiation (LTP), a cellular model for learning and memory, has a protein synthesis dependent late phase (L-LTP) that has been shown to recruit both pre- and postsynaptic mechanisms. In spite of their seeming importance in synaptic function, mitochondria are only found in roughly half of all mature presynaptic boutons, which implies that only a subset of boutons are capable of supporting increases in vesicle release. Preliminary exploration of CA1 boutons from perfusion- xed P15 rats, however, demonstrated that while smaller than boutons that contain mitochondria, boutons that are within three microns of mitochondria contain significantly more vesicles than those that are farther away from them, which raises the possibility that diffusion of ATP from nearby mitochondria could be sufficient to fuel LTP. To determine whether this was the case, I prepared 3D reconstructions from serial electron micrographs (3DEM) in order to quantify mitochondrial distance and vesicle counts in CA1 boutons following long-term potentiation in both P15 and adult Long-Evans rats. At both ages, mobilization of reserve pool vesicles following LTP required the presence of mitochondria. Mitochondria themselves also undergo structural changes following LTP. In adults, mitochondria become longer and less frequent along axons, implying that they undergo fusion. Although P15 mitochondria undergo no change in frequency, mitochondrial cristae become wider as mitochondrial matrix becomes more compact at both ages, which are both changes that correlate well with increased rates of respiration. To even further explore ultrastructural components required to support LTP, I used 3DEM to track changes in synaptic and subcellular structure from P8 to P12, during which animals undergo radical changes in their ability to support LTP. I found that spinogenesis may begin at P10 but increases sharply at P12, which coincides perfectly with the onset age of LTP. In conjunction with this increase in dendritic spines, axons built new single synaptic boutons from clusters of dense core and amorphous vesicles previously known to transport proteins and membrane to developing synapses. To fuel the creation of new boutons, mitochondrial division increased at P12, as evidenced by decreased mitochondrial size and increased mitochondrial frequency.