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No description available.

No description available.

Mitochondrial proteins are presently believed to reside and function only within the mitochondria, under normal physiological conditions. However, in recent years many examples have come to light where proteins originally identified on the basis of extramitochondrial functions, upon characterization were found to be bonafide mitochondrial proteins. This raises important questions concerning the cellular functions of mitochondrial proteins. To investigate this phenomenon of mitochondrial proteins being present at extramitochondrial locations, we have examined by means of immunoelectron microscopy, the subcellular localization in normal rat tissues of a number of well characterized mitochondrial proteins which are encoded either by the nucleus (chaperonin 10 (Cpn10) and mitochondrial aspartate aminotransferase (mAspAT)), or by mitochondrial DNA (Cytochrome c oxidase subunits I and II (COX I and II)), using highly specific antibodies. Both Cp10 and mAspAT have already been shown to be involved in extramitochondrial functions. Cpn10 has been shown to be identical to early pregnancy factor, which is an immunosuppressant and growth factor found in maternal serum. mAspAT is identical to fatty acid binding protein isolated from plasma membranes of several cell types, involved in the transport of long chain free fatty acids. Immunofluorescent labeling of BS-C-1 African monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In all tissues examined, the antibodies showed strong labeling of mitochondria, as was expected. In several tissues, the binding was seen exclusively within mitochondria. However, in a variety of tissues such as pancreas, anterior pituitary and kidney, these antibodies showed strong and specific labeling of one or more of the following compartments- pancreatic zymogen granules, growth hormone granules, secretory granules in islet cells, red blood cells, condensing vacuoles in kidney and on the cell surface of different regions of the kidney. All of the observed labeling with these antibodies, both within mitochondria and in other compartments, was abolished upon omitting the primary antibodies or upon adsorption of the Cpn10 antibody (in the case of Cpn10) with purified recombinant protein. The extramitochondrial localization of these proteins, particularly those encoded by mitochondrial DNA provides strong evidence that these proteins have reached these sites by exiting from mitochondria. These observations have important implications concerning the roles of mitochondria and mitochondrial resident proteins in different mitochondrial diseases. Also, the function of these proteins and the precise mechanism by which they reach these extramitochondrial sites is of great interest. / Thesis / Master of Science (MSc)

Thesis / Master of Science (MS)

Mitochondrial DNA (mtDNA) aberrations have been detected in a large proportion of tissue samples isolated from human tumours, although the functional significance of these mutations is yet to be determined. Proponents of the selective advantage mtDNA defect acquisition model believe these mutations arise spontaneously, but propagate because they afford some benefit to overall neoplastic cell proliferation. The most tantalizing of these growth advantage properties is an elevation of electron transport chain (ETC) generated reactive oxygen species (ROS), which are already associated with some pathogenic mtDNA defects. However, others theorize that these mutations may arise spontaneously and expand through the ambivalent process of genetic drift alone. The 2008 human ovarian cancer cell line and its cis-platinum(II)-diammine-dichloride (CDDP) resistant C13* cell variant have divergent phenotypes when mitochondrial morphology, membrane potential (ΔΨₘ), ROS production, and rate of oxygen consumption are considered. Furthermore, the increased ΔΨₘ observed in C13* cells has been implicated in its CDDP resistant characteristic. In order to determine if mtDNA mutations were responsible for these phenotypic variations, the complete mitochondrial genomes of both cell lines were directly sequenced. Two novel mtDNA mutations were identified within cytochrome c oxidase subunit II (COXII) and the d-loop of 2008 and C13* cells respectively, however the functional significance of these defects were not obvious. To expunge the effects of nuclear DNA (nDNA) cybrids were created by transferring the mtDNA of 2008 and C13* cells to a common 143B TK⁻ nDNA background, creating 2008cyb and C13cyb cells. C13*cyb cells did not demonstrate CDDP resistance, decrease in rate of oxygen consumption or increase in ΔΨₘ when compared to 2008cyb cells. However, C13*cyb cells did retain mitochondrial morphological properties as well as increased Rh123 sensitivity and ROS production. This data would imply that mtDNA mutations are responsible for a proportion of mitochondrial-associated changes in the context of carcinogenesis. / Thesis / Master of Science (MS)

The regulation of gene expression at the level of transcription is an important mechanism for maintaining homeostasis. The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear hormone receptor superfamily that is involved in transcriptionally modulating such pathways as lipid and fatty acid metabolism. This receptor binds to enhancer elements, peroxisome proliferator-responsive elements (PPREs), upstream of a variety of target genes including those involved in β-oxidation of fatty acids, in the peroxisome and mitochondria, and ketogenesis. One such element has been identified upstream of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase (mtHMG-CoAS) gene. This enzyme has been shown to be one of the key regulatory points of ketogenesis. To learn how PPARα mediated transcriptional regulation occurs, this receptor was used as bait in a yeast dihybrid screen and was found to interact with human mtHMG-CoAS. Reproduction of this interaction 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 was performed by solid phase capture assays, using GST fusion proteins, and by co-immunoprecipitations. It was also ascertained that the synthase enzyme interacts with the retinoid X receptor α (RXRα). The hamster cytoplasmic form of the enzyme was chosen as a control and showed no binding capabilities to either nuclear hormone receptor. Interestingly, the mitochondrial enzyme contains a motif, LXXLL that has previously been shown to be important for binding between a transcriptional coactivator and a receptor. A site-directed mutant of the mitochondrial synthase sequence from LASLL to LASVL was made. The mutant showed a reduced ability to interact with both nuclear receptors. Consequently, the LXXLL motif is responsible, at least in part, for the interaction between mtHMG-CoAS and both PPARα and RXRα. The cytoplasmic synthase does not contain the motif; its corresponding sequence is LASVL. The effect ofthe mitochondrial synthase within a cell was then determined by transient transfection assays. It was discovered that on the HMG PPRE the mitochondrial synthase potentiated PPARα mediated transactivation while on the AOx PPRE the enzyme inhibited it. Thus, mtHMG-CoAS modulates PPARα activity in a PPRE dependent manner. The LXXVL mitochondrial mutant inhibited PPARα transactivation on both the HMG and AOx PPRE. Therefore, the mutant acts as a dominant-negative inhibitor of α mediated activity. The cytoplasmic control enzyme had no effect on either PPRE. To determine if the mitochondrial enzyme could be detected within the nucleus where it appeared to be modulating PPARα mediated transcription, localization studies were performed with the use of immunofluorescence. Immunofluorescence was done by utilizing hemagglutinin (HA) epitope tagged fusions of the mitochondrial, mutant and cytoplasmic enzymes. When the HA tag was placed at the carboxyl terminus of mtHMG-CoAS, the enzyme was localized to the mitochondria with no apparent nuclear staining. This is consistent with previous localization studies done with the rat mtHMG-CoAS. Also, the mutant enzyme, with the HA tag at the carboxyl terminus, was only detected in the mitochondria. However, under certain conditions, when PPARα and the mitochondrial synthase were co-transfected, the mitochondrial enzyme was detected within both the mitochondria and the nucleus. The mutant, on the other hand, when co-transfected with PPARα was found to remain non-nuclear. The HA tagged cytoplasmic control enzyme was also non-nuclear. Therefore, mtHMG-CoAS can be detected within the nucleus, it binds, due to at least in part a LXXLL motif, to nuclear receptors, and it is capable of modulating transcription in a PPRE dependent manner. Ketogenesis becomes an important mechanism for fuel production during starvation, prolonged exercise and diabetes. Perhaps, during extreme circumstances such as starvation, mtHMG-CoAS is involved in autoregulation which allows for an amplification of the transcription of its own gene. Also, mtHMG-CoAS appears to inhibit the transcription of the AOx enzyme which is involved in peroxisomal fatty acid β-oxidation. Again, perhaps under extreme conditions, the β-oxidation of fatty acids is concentrated within the mitochondria which allows for the production of acetyl-CoA that can subsequently be converted to ketone bodies that can then be utilized for fuel. / Thesis / Master of Science (MS)

[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.

Indiana University-Purdue University Indianapolis (IUPUI) / The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.

LD2668 .T4 1966 S971 / Master of Science