Submitted by LUÍS LENIN VICENTE PEREIRA null (luislenin@gmail.com) on 2017-03-02T15:07:34Z No. of bitstreams: 1 Tese Luis Lenin Vicente Pereira.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-08T13:17:27Z (GMT) No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) / Made available in DSpace on 2017-03-08T13:17:27Z (GMT). No. of bitstreams: 1 pereira_llv_dr_sjrp.pdf: 2894913 bytes, checksum: 01d77fed0e0eb6b5cfb7979e52829667 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A subordem Heteroptera possui sete infraordens com aproximadamente 80 famílias. A maioria ocorre em todos os continentes (exceto Antártica) e algumas ilhas. Além dos Heteroptera terrestres, há os aquáticos e semi-aquáticos que são amplamente distribuídos, e surpreendem por sua capacidade de habitar uma extraordinária variedade de ecossistemas, sendo encontrados em habitats de água doce e marinho, e variada faixa de altitude entre 0 e 4.700 m. Estudos sobre aspectos ultraestruturais da espermatogênese e, especificamente, a estrutura do espermatozoide em Heteroptera ainda são escassos, por este motivo o objetivo do presente estudo foi o de analisá-los, por meio de cortes semifinos corados com azul de toluidina ou impregnados por íons prata, e cortes ultra-finos analisados em microscopia eletrônica de transmissão, utilizando testículos de machos adultos das famílias Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae e Veliidae. Após a análise ultraestrutural da espermatogênese foi possível determinar que o padrão flagelar do axonema é de 9+9+2 para todas as espécies analisadas sendo, portanto, o padrão para essa subordem, as mitocôndrias durante a espermatogênese assumem diferentes morfologias, sendo que inicialmente as mitocôndrias se unem formando o complexo mitocondrial e, posteriormente, se divide em dois derivados mitocondriais que estão posicionados bilateralmente em relação ao axonema. Os derivados mitocondriais apresentaram tamanhos diferentes para as espécies B. amnigenus (Notonectidae) e R. c. crassifemur (Gerridae) e para as demais espécies o tamanho foi semelhante. As células germinativas possuem em seu citoplasma o acúmulo de um material denominado corpo cromatóide estando localizado próximo ao núcleo. Com relação ao comportamento nucleolar da espécie Martarega brasiliensis foi observado de um a quatro corpúsculos nucleolares em células de Prófase I comprovando uma grande atividade sintética das células nessa fase da divisão celular. Células em Metáfase I apresentaram regiões organizadoras nucleolares na região telomérica de um dos autossomos. Ainda, nessa espécie, foi possível observar, em Anáfase I, vários corpúsculos nucleolares persistindo até a fase de Telófase I. Todas as ultraestruturas descritas nas espécies analisadas foram semelhantes às descritas na literatura para Heteroptera, corroborando as características sinapomórficas dessa subordem sendo elas: a) a presença de duas pontes que ligam o material intertubular do axonema flagelar às cisternas achatadas que aderem aos lados internos dos derivados mitocondriais; b) padrão flagelar do axonema de 9+9+2 e c) ausência de corpos acessórios. / The suborder Heteroptera has seven infraorders with approximately 80 families. Most occur in all continents, except Antarctica and some islands. In addition to terrestrial Heteroptera, there are also widely distributed aquatic and semi-aquatic species. This suborder have adapted to live in an extraordinary variety of ecosystems as freshwater and marine habitats and at altitudes ranging from 0 m to 4,700 m. The research concerning the ultrastructural aspects of spermatogenesis is a large and growing field of study, however, in the case of Heteroptera, research is still scarce. For this reason, the aim of this study was to analyze the ultrastructures and spermatogenesis through semi-thin sections stained with toluidine blue or silver ions (Ag-NOR) and ultrathin sections examined in transmission electron microscopy, using testes of adult males ofthe following families: Belostomatidae, Gelastocoridae, Gerridae, Mesoveliidae, Notonectidae and Veliidae. After ultrastructural analysis of spermatogenesis, it was possible to determine that the flagellar pattern of the axoneme is 9+9+2 for all species, being therefore, the pattern for this suborder. As spermatogenesis progresses, the mitochondria begins to cluster and concentrate on only one side of the cell. Then, the mitochondria combine to form a single mitochondrial complex, which subsequently divides into two mitochondrial derivatives. They are positioned on opposite sides of the axoneme. The mitochondrial derivatives presented different sizes for the species B. amnigenus (Notonectidae) and R. c. crassifemur (Gerridae) and for the other species the size was similar. The germ cells have in their cytoplasm the accumulation of a material denominated the chromatoid body, being located near the nucleus. Regarding the nucleolar behavior, M. brasiliensis showed nucleus in prophase I composed by the nucleolus and nucleolar corpuscles that varied from one to four, emphasizing that this insect has great synthetic activity during meiosis. The analysis of cells in metaphase I, showed that M. brasiliensis presents nucleolar organizing region (NOR) in at least one autosome. Furthermore, was not observed the phenomenon of nucleolar persistence. All the ultrastructures described in the analyzed species were similar to those described in the literature for Heteroptera, corroborating the synapomorphic characteristics of this suborder, being them: a) two opposite bridges in the axoneme connect the flattened cisterns adherent to the internal side of each mitochondrial derivative to the intertubular material; b) flagellar pattern of the axoneme of 9+9+2; c) accessory bodies are absent all along the flagellum.

Mitochondria are a key organelle within human cells, with functions ranging from ATP synthesis to apoptosis. Changes in mitochondrial function are associated with many diseases, as well as 'natural' processes like ageing. Mitochondria have a unique evolutionary origin, as the result of an endosymbiotic relationship between a bacterium and an archaeal cell. Therefore, the phylogenetic history of the mitochondrial proteome is also unique within the total human proteome. A new description of the genes encoding the human mitochondrial proteome - IMPI (Integrated Mitochondrial Protein Index) 2017 - provided an opportunity for exploration of mitochondrial proteome history and the application of this knowledge to the understanding of gene function, disease and ageing. To facilitate the exploration of the mitochondrial proteome, I created a manually curated dataset of 190,097 predicted orthologues of the 1,550 IMPI 2017 human genes across 359 species, using reciprocal best hit analysis as the basis for orthologue prediction. I used this to explore gene history and the potential for phylogenetic profiling to predict the function of uncharacterised genes. This inspired the use of phylogenetic profiling within two phyla of animals, to link presence and absence of metabolic genes to the function of mitochondrial transporters. Potential transport substrates were predicted for two groups of uncharacterised mitochondrial carriers. I also used the dataset to identify features of genes associated with monogenetic disease, as well as differences between recessive and dominant disease genes. A similar orthologue identification method was used to explore the total sequenced viral proteome for potential orthologues of mitochondrial proteins. This showed that a range of mitochondrial proteins are shared with viruses, potentially facilitating the co-opting of mitochondrial function during viral infection of eukaryotic cells. I then used orthology to explore the conservation of residues linked to protein acetylation and identify a link with lifespan in warm-blooded vertebrates. In conclusion, I have used orthology to further the understanding of human mitochondrial proteome history and developed applications of this information. For example, phylogenetic features of disease genes are being used as part of a wider pipeline to predict mitochondrial disease genes. Furthermore, predicted substrates of the SLC25A14/30 mitochondrial carriers are being tested. My dataset provides further opportunities to explore the evolution and function of the mitochondrion.

La maladie de Parkinson (MP) est caractérisée par une dégénérescence des neurones dopaminergiques de la substance noire. Elle est le plus souvent sporadique mais des formes familiales monogéniques existent, notamment dues à des mutations de PARK2 et de PINK1. Ces gènes codent pour l'ubiquitine-protéine ligase cytosolique Parkine et la sérine/thréonine kinase mitochondriale PINK1, deux acteurs majeurs du contrôle de qualité mitochondrial. Ce travail étudie le rôle de leur interaction au niveau de la membrane mitochondriale externe dans la régulation de l'homéostasie mitochondriale.Nous avons montré que l'association de PINK1 et Parkine au complexe d'import mitochondrial TOM lors d'un stress mitochondrial permet l'import de la grande majorité des protéines adressées à la mitochondrie ; que déstabiliser ce complexe suffit à initier la mitophagie ; et que l'activation de Parkine par PINK1 facilite l'import de son substrat HSD17?10. Nous avons développé un biosenseur moléculaire inductible, permettant d'étudier la voie d'import classique des protéines à pré-séquence. Nous avons également montré, dans un modèle neuronal, qu'un stress mitochondrial, en présence de Parkine, induit une forte augmentation de l'expression de gènes clés de la biogenèse mitochondriale ; et que ces gènes sont up-régulés de façon basale dans les neurones PARK2-/-, indiquant une possible altération de la réponse aigüe au stress.Ces résultats approfondissent notre connaissance de la physiopathologie des formes autosomiques récessives de MP en soulignant l'importance de la voie PINK1/Parkine dans l'import et la biogenèse mitochondriaux. / Parkinson’s disease (PD) is linked to a specific loss of dopaminergic neurons of the substancia nigra. The disease is most often sporadic but familial monogenic forms exist, for example due to mutations in PARK2 or PINK1. Those genes encore the cytosolic ubiquitin-protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, both essential for mitochondrial quality control. This work studies the role of their interaction at the outer mitochondrial membrane in the regulation of mitochondrial homeostasis. We found that the association of PINK1 and Parkin to the mitochondrial import TOM complex during mitochondrial stress induces the import of most proteins targeted to mitochondria; that destabilizing this complex is sufficient to initiate mitophagy; and that Parkin activation by PINK1 facilitates the import of its substrate, HSD17β10. We developed an inducible BRET-based molecular biosensor to study the classical pre-sequence import pathway. We also found, in a neuronal model, that mitochondrial stress induced a strong increase in the expression of mitochondrial biogenesis key genes, in the presence of Parkin; and that these genes are basally up-regulated in PARK2-/- neurons, possibly reflecting an alteration of acute stress response. These results increase our understanding of the pathophysiology of autosomal recessive forms of PD, underlining the importance of the PINK1/Parkin pathway in mitochondrial import and biogenesis.

La dynamique mitochondriale est impliquée dans la maintenance et la fonction des mitochondries. Dans les graines sèches tout les processus cellulaires sont arrêtés du fait de la faible teneur en eau des tissues, et la transition développementale que représente la germination requiert la réactivation de la dynamique cellulaire. Une approche de bio-imagerie sur la plante modèle Arabidopsis a été utilisée afin d’étudier la réactivation des mitochondries nécessaire à la germination. La réactivation bioénergétique des mitochondries, mesurée par la présence du potentiel membranaire, intervient dès le début de l’hydratation des tissus. Cependant les mitochondries restent statiques et la dynamique mitochondriale ne reprend que plus tardivement. La réactivation des mitochondries provoque une réorganisation du chondriome impliquant la biogenèse de membranes et une fusion massive menant à la formation de structures réticulaires et périnucléaires, qui permet le mélange des nucléoïdes d’ADNmt précédemment isolés en unités discrètes. La mitophagie, un indicateur de la qualité mitochondriale, est réactivée de manière concomitante à la dynamique, alors qu’elle est réprimée durant la biogenèse des mitochondries. La fin de la germination coïncide avec la fragmentation du chondriome tubulaire, menant au doublement du nombre de mitochondrie et à une redistribution hétérogène des nucléoïdes dans le chondriome, générant une population de mitochondrie adaptée à la croissance des plantules. Cette thèse met en évidence l’imbrication des processus de dynamique mitochondriale, de biogenèse et de contrôle qualité des mitochondries requis pour la germination et pour la transition vers l’autotrophie. / Mitochondrial dynamics underpin their function and maintenance. In dry seeds, all cellular processes are in stasis due to a low water content. Thus, the developmental switch leading to germination necessarily involves a reactivation of cellular dynamics. In order tobetter understand the role played by mitochondrial dynamics during germination we used Arabidopsis as a model for a bioimaging approach to investigate the rapid reactivation of mitochondria that is required in order to provide ATP to support germination. Bioenergetic reactivation, visualised as the presence of a mitochondrial membrane potential, is almost immediate upon rehydration. However, the reactivation of mitochondrial dynamics only occurs after several hours of rehydration. The reactivation of mitochondrialbioenergetics and dynamics lead to a dramatic reorganisation of the chondriome involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. Mitophagy, an indicator of mitochondrial quality, is reactivated concomitant with a reactivation of mitochondrial dynamics, but is repressed at time of mitochondrial biogenesis. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondrialnumber and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth. This thesis provides strong evidence for the tight interweaving of mitochondrial dynamics, mitochondrialbiogenesis and mitochondrial quality control that is required to ensure effective germination and the transition to autotrophy.

<b>Français : </b> La mitochondrie est un organite dont les fonctions dépassent largement le rôle bioénergétique. De ce fait, il apparaît de plus en plus clairement qu’un grand nombre de pathologies sont liées à un dysfonctionnement mitochondrial. Au cours de ces dernières années l’existence d’une communication moléculaire rétrograde entre la mitochondrie non fonctionnelle et le noyau a été mise en évidence dans les cellules eucaryotes de mammifères. Les voies de signalisation moléculaire menant à l’expression différentielle de gènes nucléaires en réponse à un dysfonctionnement mitochondrial sont néanmoins encore peu connues. Dans ce domaine, l’utilisation de lignées cellulaire totalement (r0) ou partiellement (r-) déplétées en ADN mitochondrial (ADNmt) s’est révélée essentielle dans l’étude de la réponse cellulaire induite par un dysfonctionnement mitochondrial. L’objectif de ce travail était de mieux comprendre les mécanismes moléculaires impliqués dans la réponse cellulaire à un dysfonctionnement mitochondrial 1) en recherchant comment les cellules déplétées en ADNmt maintiennent un potentiel de membrane mitochondrial en étudiant les mécanismes impliqués dans le maintien de la biogenèse mitochondriale et 3) en caractérisant la sensibilité des cellules déplétées en ADNmt à l’apoptose. Au cours de la première partie de ce travail, nous avons mis en évidence le rôle de la protéine mtCLIC dans le maintien du Dym des cellules déplétées en ADNmt. Nous avons ainsi démontré que le gène codant cette protéine est surexprimé dans les cellules présentant un dysfonctionnement mitochondrial, et que l’activité de canal à chlore pouvait rendre compte du maintien du Dym dans ces cellules. Dans la deuxième partie de ce travail, nous avons caractérisé et comparé les populations mitochondriales des cellules parentales et déplétées en ADNmt (143B r0, ostéosarcome humain). L’activité de certains facteurs de transcription décrits pour jouer un rôle dans le processus de biogenèse mitochondriale a été recherchée ainsi que le niveau d’expression de certaines protéines marqueurs de la biogenèse mitochondriale. Le rôle de la voie calcium-CaMKIV-CREB dans le maintien de la biogenèse mitochondriale des cellules r0 a ainsi pu être mis en évidence. Nous avons également mis en évidence une diminution de l’activité d’importation de protéines chimériques matricielles dans les mitochondries des cellules déplétées en ADNmt. Cette diminution peut s’expliquer par la réduction du Dym et de la charge en ATP dans ces cellules mais n’est pas généralisable à l’ensemble des protéines mitochondriales. En effet, l’importation du cytochrome c est augmentée et celle de la sous-unité ß de la F1-ATPase est inchangée dans des cellules 143B r0. La dernière partie de ce travail a été consacrée à la caractérisation et à la comparaison de la réponse des cellules 143B et 143B r0 à un stimulus pro-apoptotique. Après avoir clairement établi que les cellules r0 présentent une sensibilité accrue à la staurosporine, nous avons recherché les mécanismes moléculaires pouvant expliquer cette réponse différentielle. Nous proposons que la sensibilité accrue des cellules r0 peut s’expliquer par la sous-expression constitutive des protéines anti-apoptotiques Bcl-2 et Bcl-XL. De plus, nous montrons également que les mécanismes impliqués pourraient faire intervenir la cathepsine B, libérée du lysosome par un mécanisme non encore élucidé. Nous montrons également que l’activation spécifique de l’autophagie dans les cellules 143B r0 en réponse à la staurosporine pourrait également contribuer à la plus grande sensibilité à l’apoptose des cellules présentant un dysfonctionnement mitochondrial. Les résultats obtenus au cours de ce travail ont permis d’identifier certains mécanismes d’adaptation mis en place dans des cellules de mammifères soumises à un stress énergétique chronique, et donc de mieux comprendre les implications d’un dysfonctionnement mitochondrial, une situation associée à ou responsable de nombreuses pathologies mitochondriales. <b>English : </b> Mitochondria are involved in numerous cell processes, such as ATP production, calcium homeostasis, fatty acid metabolism, heme synthesis, urea cycle, redox cell status, autophagy and apoptosis. Impairment of its bioenergetic activity is thus obviously associated with numerous pathologies. However, while various origins and symptoms have been described for mitochondrial diseases over the past 10 years, only very few retrograde signalling pathways (that could be defined as communication between impaired mitochondria and nucleus) have been identified. In addition, little is still known about the molecular mechanisms leading to differential gene expression in response to chronic or acute mitochondrial dysfunction. In that research field, the generation of cells totally (r0) or partially (r-) depleted in mtDNA has been very useful to study the response of cells to a chronic energetic stress. The major aim of this work was to get a better understanding of the molecular mechanisms involved in the retrograde communication between impaired mitochondria and the nucleus that participate to the maintenance of 1) the mitochondrial membrane potential (Dym), 2) the mitochondrial biogenesis and 3) the apoptotic response to staurosporine, an alkaloïd that inhibits numerous kinases. In the first part of this work, we highlighted the role of the protein mtCLIC/CLIC4 in the maintenance of the Dym in mtDNA-depleted cells. Using a “mRNA RT-PCR differential display” approach, we first identified that the gene was over-expressed in mtDNA-depleted cells. We also show that modifications of its abundance (over expresion and silencing by siRNA) were able to modify the Dym. Finally, we evidenced that mtCLIC allows the importation of chlorine into mitochondria of r-L929 (murine fibrosarcoma cells). In the second part of this work, we characterized and compared mitochondrial populations between 143B (osteosarcoma cell line) and 143B r0 cells. We monitored the activity status of several key transcription factors known to be involved in the control of mitochondrial biogenesis and we determined the expression level of several mitochondrial proteins used as common markers of mitochondrial biogenesis. We also clearly demonstrated the role for calcium-CaMKIV-CREB pathway in the maintenance of mitochondrial biogenesis in mtDNA-depleted cells. Indeed, we show that the over-expression of cytochrome c and the higher mitochondrial NAO (Nonyl Acridine Orange) staining (two indicators for a higher abundance of mitochondrial mass) observed in mtDNA-depleted cells could be reduced in r0 cells that over-express either a dominant negative forms of CREB or CaMKIV. Moreover, we show that the importation of matrix-targeted proteins is reduced in mtDNAdepleted cells, a feature that can be explained by the lower Dym and reduced ATP content in these cells. As several evidence were reported to link mitochondrial dysfunction and apoptosis in vivo, the last part of this work has been dedicated to the characterization of the apoptotic response of mtDNAdepleted cells to staurosporine. Indeed, the higher or lower sensitivity of mtDNA-depleted cells to apoptotic stimuli is still a debated question in the literature. We first show that r0 143B cells are hypersensitive to staurosporine-induced apoptosis, a phenomenon that could most likely be explained by the constitutive down-regulation of anti-apoptotic proteins such as Bcl-2 an Bcl-XL in r0 cells. Moreover, we show that the mechanisms of r0 cells response to staurosporine seems to be different from those triggered in parental cells. Indeed, we show that cathepsin B might play a role in staurosporine-induced mtDNA-depleted cell apoptosis, despite the activation of many caspases. Finally, we show that autopahgy is also triggered by staurosporine in r0 143B cells, an upstream event of caspase activation as 3-methyladenine (3-MA) strongly reduces caspase activation. In conclusion, our results bring new information in the understanding of mechanisms and cell signalling activated in mammalian cells facing a chronic energetic stress, and thus bring new insights into the cellular consequences of mitochondrial impairment, a feature found in numerous mitochondrial diseases and pathologies associated with aging.

<p>Mitochondria are the primary energy producers of the cell and play key roles in cellular signaling, apoptosis and reactive oxygen species (ROS) production. Mitochondria are the only organelles that contain their own genome which encodes for a small subset of electron transport chain (ETC) proteins as well as the necessary tRNAs and ribosomal subunits to translate these proteins. Over 300 pathogenic mitochondrial DNA (mtDNA) mutations have been shown to cause a number of mitochondrial diseases emphasizing the importance of mtDNA maintenance and integrity to human health. Additionally, mitochondrial dysfunction and mtDNA instability are linked to many wide-spread diseases associated with aging including cancer and neurodegeneration. Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mtDNA by important environmental genotoxins including polycyclic aromatic hydrocarbons, ultraviolet C radiation (UVC) and mycotoxins. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. Degradation of mitochondria and mtDNA is carried out by autophagy. Autophagy is protective against cell stress and apoptosis resulting from exposure to mitochondrial toxicants suggesting that it plays an important role in removal of unstable mitochondria that can serve as a source of ROS or initiate apoptotic cell death. Furthermore, dysfunctional mitochondria can be specifically targeted for degradation by the more specific process of mitophagy influenced in part by the processes of mitochondrial dynamics (i.e., fusion and fission). </p><p>The goals of this dissertation were to investigate the long-term fate of helix-distorting mtDNA damage and determine the significance of autophagy and mitochondrial dynamics in removal of and recovery from persistent mtDNA damage. Removal of irreparable mtDNA damage and the necessity of autophagy, mitophagy, fusion and fission genes in removal of this damage were examined using genetic approaches in adult <italic>Caenorhabditis elegans</italic>. In order to investigate the significance of autophagy, fusion and fission genes in recovery from mtDNA damage-induced mitochondrial dysfunction <italic>in vivo</italic>, an experimental method was developed to specifically induce persistent mtDNA damage and mitochondrial dysfunction without persistent nDNA damage in developing <italic>C. elegans</italic>. Additionally, the effect of persistent helix-distorting DNA damage on mitochondrial morphology, mitochondrial function and autophagy was investigated in <italic>C. elegans</italic> and in mammalian cell culture. The rate and specificity of mitochondrial degradation was further examined in cell culture using live-cell fluorescence microscopy and transmission electron microscopy. </p><p>Removal of UVC-induced mtDNA damage was detectable by 72 hours in <italic>C. elegans</italic> and mammalian cell culture, and required mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. UVC exposure induced autophagy with no detectable effect on mitochondrial morphology in both systems; mitochondrial function was inhibited in the <italic>C. elegans</italic> system but not in the cell culture system in which the degree of mtDNA damage induced was less. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the <italic>in vivo</italic> relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.</p> / Dissertation

In eukaryotes, folate-dependent one-carbon (1-C) metabolism is composed of two parallel pathways compartmentalized to either the cytoplasm or mitochondria. In each, 1-C units, carried on tetrahydrofolate (THF), are interconverted by four catalytic activities. Serine hydroxymethyltransferase transfers the 3-carbon of serine to THF forming 5,10-methylene-THF which is oxidized in 3 successive steps to formate via the intermediates, 5,10-methenyl-THF and 10-formyl-THF. Because of the redox potential in each compartment, 1-C flux is thought by most authors to be from formate to serine in the cytosol and in the opposite direction in mitochondria. Transport of serine, glycine and formate across the mitochondrial membranes creates a 1-C cycle. All eukaryotes characterized to date contain a cytoplasmic trifunctional C1-THF synthase possessing 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities which interconvert the catalytic intermediates between 5,10-methylene-THF and formate. However, despite the observation that adult rat liver mitochondria oxidize serine to formate, no known enzymatic activities correlate with those of cytoplasmic C1-THF synthase. In embryos, a bifunctional protein, containing 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase, accounts for two of these activities. But the 10-formyl-THF synthetase activity has no associated enzyme in mitochondria. Reported here is the discovery of a monofunctional homolog of C1-THF synthase in mammalian mitochondria. Characterization of the protein confirms mitochondrial localization and 10-formyl-THF synthetase activity. Likewise, the adult human transcript is present and differs in size and tissue distribution from cytosolic C1-THF synthase. In mouse embryos, the temporal expression of the mRNA starts out relatively low and increases as the embryos age. The spatial distribution of the transcript is ubiquitous but with areas of elevated expression corresponding to proliferative regions within the embryo. The temporal expression pattern of the protein and transcript correspond well. However, mitochondrial flux studies and immunoblotting data suggest that mitochondrial C1-THF synthase is not the rate-limiting enzyme in mitochondria, at least during the mid to later stages of embryogenesis. Additionally, studies modulating the expression of mitochondria 1-C proteins demonstrate the likelihood that most cytoplasmic 1-C units are mitochondrially derived. / text

The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. / text

The mitochondrion is an important therapeutic target due to its roles in energy metabolism, reactive oxygen species production, and activation of cell death. The connection of these cellular processes with diabetes, obesity, neurodegenerative disorders, and cancer makes this organelle a potential control point for treatment of these diseases. The advancement of mitochondrial medicine will be accelerated by the development of organelle-specific cellular transporters as well as by strategies to enhance the activity of mitochondrially-active drugs. Here, the discovery and development of a new class of mitochondria-specific agents, mitochondria-penetrating peptides (MPPs), is described. These peptides, exemplified by the sequences (FXY)3 and (FXY)4 where FX=cyclohexylalanine and Y=basic arginine or lysine residues, display hydrophobic and cationic residues critical for access to this organelle, and appear to accumulate within cells and mitochondria through membrane potential-dependent mechanisms. Subcellular localization of the peptides was determined by the interplay of hydrophobicity and positive charge, and necessary lipophilicity “thresholds” for access to the mitochondrion. MPPs can be engineered to have minimal mitochondrial disruptive activity and cytotoxicity through minimization of hydrophobicity and length. Furthermore, MPPs appear to accumulate predominantly in the mitochondrial matrix, a feature which places them within an exclusive class of mitochondria-specific transporters and may enable delivery applications for a number of therapeutically-relevant cargoes. Information gained from MPP studies on the physiochemical parameters that drive mitochondrial localization were applied to improve the activity of the anticancer peptide d-(KLAKLAK)2, an agent that activates apoptosis through mitochondrial disruption. Residue substitutions that increased peptide hydrophobicity, regardless of changes to secondary structure, enhanced mitochondrial localization, activity, and cytotoxicity induced by the peptide. In conclusion, these studies provide important guidelines for how to drive the subcellular localization and activity of peptides, and expand the possibilities for mitochondrially-targeted therapeutics.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.