RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in mitochondria of several maize races. The extent to which this molecule associates in vivo with proteins was investigated by both rate zonal and CsCl equilibrium density gradient centrifugations of clarified lysate of mitochondria from maize plants with the S-type cytoplasm. A soluble endogenous complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in in vitro conditions (in mitochondrial lysate), was identified. The complex had a density of 1.49 g/cm$ sp3$ but a surprisingly low sedimentation coefficient, only slightly larger than the naked RNA b. Only a minor fraction of RNA b molecules were bound in the complex; the majority of RNA b sedimented as naked RNA molecules. Complexes synthesizing other, less abundant, maize RNA plasmids were not identified. However, in vitro synthesis of all RNA plasmid species in mitochondrial lysate was resistant to heparin, suggesting that in all cases preformed RdRp-RNA template complexes, capable of elongating in vivo preinitiated RNA plasmid strands, were present.

Mitochondria contain their own genetic system, encoding key subunits of the oxidative phosphorylation system. These subunits are expressed by an organelle-specific gene expression machinery. This work revealed a number of fundamental aspects of mitochondrial gene expression and provides evidence that this process is organized in a unique and organelle-specific manner which likely evolved to optimize protein synthesis and assembly in mitochondria. Most importantly, improving the experimental handling of ribosomes we could show that mitochondrial ribosomes are organized in large assemblies that we termed MIOREX complexes. Ribosomes present in these complexes organize gene expression by recruiting multiple factors required for post-transcriptional steps. In addition, we could reveal mechanisms by which ribosome-interactor complexes modulate and coordinate the expression and assembly of the respiratory chain subunits. For example we showed that the Cbp3-Cbp6 complex binds to the ribosome in proximity to the tunnel exit to coordinate synthesis and assembly of cytochrome b. This location perfectly positions Cbp3-Cbp6 for direct binding to newly synthesized cytochrome b and permits Cbp3-Cbp6 to establish a feedback loop that allows modulation of cytochrome b synthesis in response to assembly efficiency. Likewise the interaction of the membrane-anchor proteins Mba1 and Mdm38 with the tunnel exit region enables them to participate in the translation of the two intron-encoding genes COX1 and COB in addition to their role in membrane insertion.  In summary, work presented in this thesis shows that mitochondrial gene expression is a highly organized and regulated process. The concepts and technical innovations will facilitate the elucidation of many additional and important aspects and therefore contribute to the general understanding of how proteins are synthesized in mitochondria. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

The human genome project has allowed for the rapid identification of a large number of protein families based on similarities in their genetic sequences. In the present study, I report the functional characterization of a 44 kDa protein that functions in cardiolipin synthesis and remodeling. Although it is present in most tissues, it is abundant in multiple brain regions including olfactory bulbs, hippocampus, cerebellum, cortex, and brain stem, and is detectable in both primary neurons and glial cells. In assays performed in vitro, this protein significantly increased the incorporation of [14C]oleoyl-CoA into phosphatidylinositol and CL using either lysophosphatidylinositol, or monolysocardiolipin or dilysocardiolipin as acyl acceptors, respectively. This protein did not display significant acyltransferase activity with a number of other lysophospholipid acyl acceptors. Overexpressing this enzyme in HEK-293 cells increased total CL content, but did not significantly affect levels of other glycerophospholipids. Analysis of the fatty acyl profile of CL from cells overexpressing this protein indicated increased total saturated fatty acids, particularly stearate, palmitate, and myristate, and increased levels of n-3 polyunsaturated fatty acids α-linoleic acid (18:3n-3), eicosatrienoic acid (20:3n-3), and eicosapentanoic acid (20:5n-3). In accordance with its observed role in CL remodeling, subcellular localization of this protein was predominately mitochondrial. This protein is also regulated during embryogenesis, and in varying metabolic states.

A subset of mitochondrial group II introns of flowering plants has, over evolutionary time, lost characteristic features and employs unconventional splicing pathways. Given the potential impact of cold treatment on RNA folding, as well as on enzymatic activity and import of nuclear-encoded splicing machinery, I have examined the physical excised forms of aberrant introns from wheat embryos subjected to 4oC. My findings suggest a shift in biochemistry with cold treatment to novel splicing pathways that generate heterogeneous in vivo circularized forms for nad1 intron 2, nad2 intron 1 and the cox2 intron, in contrast to predominantly linear excised intron forms at room temperature. Interestingly, the highly degenerate nad1 intron 1, which due to DNA rearrangement has been broken into two halves that interact for splicing in trans, is excised exclusively by first-step hydrolysis at room temperature and under cold treatment. In this case, splicing culminates in two distinct linear half introns that appears correlated with an unusual 5’ terminal insert. This represents the first in vivo demonstration of hydrolytic trans-splicing. Based on northern analysis, cold treatment was further associated with reduced splicing efficiency for all introns surveyed. Moreover, study of precursor transcripts of the nad1a-intron 1a locus suggests the efficiency of end-maturation, including processing of the cotranscribed tRNA-Pro gene, is also reduced in the cold. My findings demonstrate a temperature-sensitivity of transcript maturation, particularly for RNA splicing, providing new insight into the impact of cold growth conditions on plant mitochondrial gene expression.

One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

There are ~12 supergroups of complex-celled organisms (eukaryotes), but relationships among them (including the root) remain elusive. For Paper I, I developed a dataset of 37 eukaryotic proteins of bacterial origin (euBac), representing the conservative protein core of the proto-mitochondrion. This gives a relatively short distance between ingroup (eukaryotes) and outgroup (mitochondrial progenitor), which is important for accurate rooting. The resulting phylogeny reconstructs three eukaryote megagroups and places one, Discoba (Excavata), as sister group to the other two (neozoa). This rejects the reigning “Unikont-Bikont” root and highlights the evolutionary importance of Excavata. For Paper II, I developed a 150-gene dataset to test relationships in supergroup SAR (Stramenopila, Alveolata, Rhizaria). Analyses of all 150-genes give different trees with different methods, but also reveal artifactual signal due to extremely long rhizarian branches and illegitimate sequences due to horizontal gene transfer (HGT) or contamination. Removing these artifacts leads to strong consistent support for Rhizaria+Alveolata. This breaks up the core of the chromalveolate hypothesis (Stramenopila+Alveolata), adding support to theories of multiple secondary endosymbiosis of chloroplasts. For Paper III, I studied the evolution of cox15, which encodes the essential mitochondrial protein Heme A synthase (HAS). HAS is nuclear encoded (nc-cox15) in all aerobic eukaryotes except Andalucia godoyi (Jakobida, Excavata), which encodes it in mitochondrial DNA (mtDNA) (mt-cox15). Thus the jakobid gene was postulated to represent the ancestral gene, which gave rise to nc-cox15 by endosymbiotic gene transfer. However, our phylogenetic and structure analyses demonstrate an independent origin of mt-cox15, providing the first strong evidence of bacteria to mtDNA HGT. Rickettsiales or SAR11 often appear as sister group to modern mitochondria. However these bacteria and mitochondria also have independently evolved AT-rich genomes. For Paper IV, I assembled a dataset of 55 mitochondrial proteins of clear α-proteobacterial origin (including 30 euBacs). Phylogenies from these data support mitochondria+Rickettsiales but disagree on the placement of SAR11. Reducing amino-acid compositional heterogeneity (resulting from AT-bias) stabilizes SAR11 but moves mitochondria to the base of α-proteobacteria. Signal heterogeneity supporting other alternative hypotheses is also detected using real and simulated data. This suggests a complex scenario for the origin of mitochondria.

Tikslas: Ištirti dviskiaučių ginkmedžių lapų tinktūros (GT) poveikį žiurkės širdies mitochondrijų funkcijoms mechanizmą. Uždaviniai: 1) Ištirti GT poveikį žiurkės širdies mitochondrijų vidinės membranos laidumui. 2) Ištirti GT poveikį žiurkės širdies mitochondrijų kvėpavimui po girdymo GT. 3) Įvertinti laisvųjų deguonies junginių susidarymą žiurkės širdies mitochondrijose po girdymo GT. Tyrimo metodai: Eksperimentiniame darbe buvo tiriamas dviskiaučių ginkmedžių lapų tinktūros (GT) poveikis žiurkės širdies mitochondrijų funkcijoms. Tyrimams naudojami Wistar veislės žiurkių suaugę patinėliai. Eksperimentinis gyvūnėlis užmigdomas anglies dioksido dujomis ir nutraukiamas stuburo kanalas kaklo srityje (leidimo, atlikti laboratorinius bandymus su gyvūnais, Nr.0006). GT poveikio žiurkės širdies mitochondrijų funkcijų tyrimui buvo pasirinkti du eksperimentiniai modeliai: 1) Tyrimai in vitro. Žiurkės širdies mitochondrijos izoliuojamos diferencinio centrifugavimo būdu, GT poveikis žiurkės širdies mitochondrijų oksidacinio fosforilinimo sistemai buvo tiriamas naudojant spektrofotometrinį bei poliarografinį metodus; 2) Tyrimai in vivo. Wistar veislės žiurkių patinėliai 7 dienas buvo girdomi GT (54 ar 108 μl/parai). Po to izoliuojamos širdies mitochondrijos ir vertinamas GT poveikis širdies mitochondrijų kvėpavimo parametrams bei H2O2 susidarymui širdies mitochondrijose. Rezultatai: Tyrimuose in vitro, nustatėme, kad GT didina mitochondrijų vidinės membranos laidumą dėl protonoforinių... [toliau žr. visą tekstą] / Aim: To investigate the mechanism of the effect of Ginkgo leaves tincture (GT) on mitochondrias functions. The objectives: 1) To investigate the impact of GT on permeability properties of mitochondrial inner membrane; 2) To investigate GT effect on rat hart mitochondrial respiration after oral treatment with GT; 3) To evaluate reactive oxygen species production in rat harts mitochondrias after oral GT administration. Methods: In this research work we investigated the impact of Ginkgo leaves tincture (GT) on rat hart mitochondrial functions. Male adult Wistar rats were used. The experimental animal was taken to sleep by carbon dioxide gas and its spinal canal had been broken in the neck zone (number of permission of laboratory testing on animals, No. 0006). We chose two experimental models of research on GT effects on rat heart mitochondrial functions: 1) In vitro studies. Rat heart mitochondrias were isolated by differential centrifugation, the impact of GT on rat heart mitochondrial oxidative phosphorilation system was evaluated by spectrophotometric and oxygraphic methods. 2) In vivo studies. Male adult Wistar rats were given GT (54 or 108 μl/day) for 7 days. After that mitochondrias were isolated and GT effects on mitochondial respiration and H2O2 production were evaluated. Results: In vitro studies have shown that GT increases permeability of mitochondrial inner membrane, due to higher permeability of protons. This effect is related to impact on ADP/ATP translocator and... [to full text]

Mitochondria play essential roles in numerous cellular processes, including oxidative phosphorylation and apoptotic initiation. As a result, organellar dysfunction has been implicated in several pathologies such as cancer, diabetes and neurodegenerative diseases. The opportunity to deliver compounds to probe or treat these conditions would be highly beneficial but accessing this organelle is challenging. Prior work investigated the physicochemical properties required for mitochondrial targeting and yielded mitochondria-penetrating peptides (MPPs). MPPs possess hydrophobic and cationic character and exhibit efficient cellular uptake and mitochondrial localization. In this proof-of-principle study, MPPs were harnessed to re-route an anti-leukemia agent, chlorambucil (Cbl), from the nucleus to mitochondria. This DNA alkylating agent was selected for its rapid kinetics and facile conjugation to an MPP. In addition, because mitochondria possess their own genome, the target of this drug would also be present in the organelle. Conjugation of an MPP to Cbl (mt-Cbl) confirmed that the drug was re-routed to the mitochondria and an increase in potency was observed in several cell lines and patient samples. This gain in activity was due to the increased accessibility of the mitochondrial genome, its lack of introns and its limited repair capacity. However, despite this enhanced toxicity, a therapeutic window continued to be maintained due to the elevated mitochondrial membrane potential in cancer cells. The re-routing of Cbl also resulted in evasion of several drug resistance mechanisms. Damage directly within the organelle was sufficient to initiate apoptosis even in cell lines with disabled apoptotic triggering. In addition, mitochondrial sequestration protected mt-Cbl from drug inactivation mechanisms. Lastly, mt-Cbl inhibited Pgp efflux by unexpectedly interacting with the pumps and inhibiting activity for a short period of time. The anti-cancer activity of mt-Cbl was also assessed in vivo in xenograft models of leukemia. The conjugate was stable in mouse plasma and displayed an improved pharmacokinetic profile. In addition, mt-Cbl successfully delayed tumor growth in two xenograft models and continued to alkylate mitochondrial DNA in vivo. These studies demonstrate that MPPs can be harnessed to re-route drugs to this organelle. Mitochondrial re-targeting could be a novel method of re-purposing FDA-approved drugs to enhance activity and evade resistance.

Mitochondria play essential roles in numerous cellular processes, including oxidative phosphorylation and apoptotic initiation. As a result, organellar dysfunction has been implicated in several pathologies such as cancer, diabetes and neurodegenerative diseases. The opportunity to deliver compounds to probe or treat these conditions would be highly beneficial but accessing this organelle is challenging. Prior work investigated the physicochemical properties required for mitochondrial targeting and yielded mitochondria-penetrating peptides (MPPs). MPPs possess hydrophobic and cationic character and exhibit efficient cellular uptake and mitochondrial localization. In this proof-of-principle study, MPPs were harnessed to re-route an anti-leukemia agent, chlorambucil (Cbl), from the nucleus to mitochondria. This DNA alkylating agent was selected for its rapid kinetics and facile conjugation to an MPP. In addition, because mitochondria possess their own genome, the target of this drug would also be present in the organelle. Conjugation of an MPP to Cbl (mt-Cbl) confirmed that the drug was re-routed to the mitochondria and an increase in potency was observed in several cell lines and patient samples. This gain in activity was due to the increased accessibility of the mitochondrial genome, its lack of introns and its limited repair capacity. However, despite this enhanced toxicity, a therapeutic window continued to be maintained due to the elevated mitochondrial membrane potential in cancer cells. The re-routing of Cbl also resulted in evasion of several drug resistance mechanisms. Damage directly within the organelle was sufficient to initiate apoptosis even in cell lines with disabled apoptotic triggering. In addition, mitochondrial sequestration protected mt-Cbl from drug inactivation mechanisms. Lastly, mt-Cbl inhibited Pgp efflux by unexpectedly interacting with the pumps and inhibiting activity for a short period of time. The anti-cancer activity of mt-Cbl was also assessed in vivo in xenograft models of leukemia. The conjugate was stable in mouse plasma and displayed an improved pharmacokinetic profile. In addition, mt-Cbl successfully delayed tumor growth in two xenograft models and continued to alkylate mitochondrial DNA in vivo. These studies demonstrate that MPPs can be harnessed to re-route drugs to this organelle. Mitochondrial re-targeting could be a novel method of re-purposing FDA-approved drugs to enhance activity and evade resistance.