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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Tartu, 2004. / Includes reprint of 5 previously published articles. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 119-137).

Plusieurs études ont découvert et renforcé l'implication de la dynamique mitochondriale dans le cancer. J'ai découvert un rôle inattendu des protéines kinases de la famille PKD dans la fission mitochondriale. La perte de l'activité PKD a conduit à un blocage de la fission et a entraîné une élongation significative des mitochondries par fusion continue. D'un point de vue mécanique, nous avons montré que les protéines PKD régulent la dynamique mitochondriale en activant le facteur de fission mitochondrial (MFF) par phosphorylation de plusieurs sites. MFF agit comme un récepteur principal de la GTPase DRP1, qui resserre les mitochondries, et il est essentiel à une bonne division mitochondriale. Les trois membres de la famille PKD peuvent phosphoryler MFF. La phosphorylation de MFF est médiée par PKD et la fragmentation mitochondriale se produit pendant la mitose. Comme démontré dans études sur les phosphoprotéomes, la phosphorylation du MFF est augmentée dans les cancers très mitotiques. Ainsi, l'axe de signalisation PKD-MFF régulant la dynamique mitochondriale en mitose pourrait devenir une voie thérapeutique attrayante pour le traitement du cancer. / Over the last two decades, multiple studies have uncovered and strengthen the implication of mitochondrial dynamics in cancer. During my thesis, I discovered an unanticipated role for the PKD kinase family in mitochondrial fission. Loss of PKD activity led to blockade of mitochondrial fission and resulted in a significant elongation of mitochondria by unopposed fusion. Mechanistically, we showed that PKDs regulated mitochondrial dynamics by activating the mitochondrial fission factor (MFF) through phosphorylation of multiple sites. MFF acts as a main receptor for the large GTPase DRP1, which constricts mitochondria, and it is critical for proper mitochondrial division. All three PKD family members could phosphorylate MFF. PKD-mediated MFF phosphorylation and mitochondrial fragmentation occurred specifically during mitosis. As MFF phosphorylation was found to be significantly upregulated in highly mitotic cancers, which was evidenced in several global phosphoproteome studies, the discovered PKD-MFF signaling axis regulating mitochondrial dynamics in mitosis could become an attractive therapeutic avenue for cancer treatment.

La mitochondrie est une organelle essentielle possédant son propre ADN circulaire. Cet ADN peut présenter des mutations et/ou des délétions, consécutives à l’exposition à différents types de dommages ou en raison de protéines mutées. Ces mutations ou délétions sont impliquées dans de nombreuses pathologies, dont les cancers, et le vieillissement. Leur apparition peut survenir notamment lors de la réplication ou de la réparation. A ce jour, la réplication et la réparation mitochondriales ne sont pas encore bien élucidées. L’objectif de ce projet est donc de mieux en appréhender les mécanismes et de mieux comprendre l’émergence d’anomalies en nous intéressant plus particulièrement à une délétion appelée « Common Deletion ». Ce travail reposait sur l’hypothèse que cette délétion put résulter d’une mauvaise réparation de cassure(s) double-brin et/ou d’une erreur durant la réplication de l’ADN mitochondrial. L’analyse de ces résultats révèle que la formation de la « Common Deletion » ne nécessite qu’une seule cassure double-brin proche des séquences répétées entourant cette dernière et implique les protéines de la réplication de l’ADN mitochondrial. Ainsi, ce travail permet de mieux saisir les mécanismes de réplication et de réparation assurant la stabilité de l’ADN mitochondrial. Un second projet a été de proposer un modèle d’étude in vitro des topoisomérases en utilisant des minicercles d’ADN permettant la visualisation du complexe covalent, étape clef de la réaction de relaxation de ces enzymes. / Mitochondria is an essential organelle with its own circular DNA. This DNA may exhibit mutations and/or deletions, as a result of exposure to different types of damage or due to mutated proteins. These mutations or deletions are involved in many pathologies, including cancers, and aging. They may occur during replication or repair. For now, mitochondrial replication and repair have not yet been fully elucidated. The objective of this project is therefore to better understand the mechanisms and the emergence of anomalies by focusing on a deletion called "Common Deletion". This work was based on the assumption that this deletion could result from poor repair of double-strand break(s) and/or error during mitochondrial DNA replication. Analysis of these results reveals that the formation of the "Common Deletion" requires only a single double-strand break close to the repeated sequences surrounding the latter and involves the proteins of mitochondrial DNA replication. Thus, this work makes it possible to better understand the mechanisms of replication and repair ensuring the stability of mitochondrial DNA. A second project was to propose an in vitro model for topoisomerases using DNA minicircles allowing visualization of the covalent complex, a key step in the relaxation reaction of these enzymes.

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-09-12T14:10:40Z No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:07Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-13T14:25:18Z (GMT) No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) / Made available in DSpace on 2016-09-13T14:25:25Z (GMT). No. of bitstreams: 1 DissCHM.pdf: 2847929 bytes, checksum: db6163924f9983d42120de5673f3df0a (MD5) Previous issue date: 2015-11-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Mitochondrial dysfunctions caused by mutations in the mitochondrial DNA (mtDNA) represent an important group of human pathologies. However, it is not possible to predict with accuracy the risk of a woman with mutant mtDNA to transmit her pathology to her descendants. This is mainly due to out limited understanding of the molecular basis of mitochondrial inheritance. Since development of a technology that enabled derivation of induced pluripotent stem cells (iPSCs) from in vitro culture of somatic cells, iPSCs have become an interesting model to study mitochondrial inheritance. Derivation of iPSCs from patients with pathogenic mtDNA mutations has revealed that the mutant load decreases through in vitro culture of iPSCs, suggesting the existence of a specific mechanism that eliminates mutant mtDNA in the germ line. Thus, the aim of this work was to use iPSCs derived from patients with mitochondrial disorders to investigate the existence of a mechanism that eliminates mtDNA molecules with pathogenic mutations. In this way, we used heteroplasmic fibroblasts harboring a point mutation A3243G in mtDNA causing mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS); heteroplasmic fibroblasts harboring a deletion in mtDNA causing Kearn-Sayre Syndrome (KSS) and homoplasmic fibroblasts containing only wild-type mtDNA (Control). The KSS lineage derivation resulted in iPSCs with low levels of mutant mtDNA (<0,1%), and the elimination of mutant molecules during the culture. The MELAS derivation resulted in iPSCs with high levels of mutant mtDNA (> 98%), and indication of mutant molecules elimination as well. However, unexpectedly, there was no reduction of mtDNA content in iPSCs compared to fibroblasts in all lineages. On contrary, mtDNA copy number increased in MELAS and KSS iPSCs, perhaps due to the high levels of mutations in the cells. No effect of Rapamycin (mitophagy inductor) treatment was detected on the yield of colony formation in MELAS iPSCs. Additionally, Rapamycin did not affect the mutation levels in MELAS iPSCS compared to untreated iPSCs. Finally, gene expression analysis of MELAS iPSCs provided evidences of an autophagic mechanism directed towards the mitochondrion. / Disfunções mitocondriais causadas por mutações no DNA mitocondrial (mtDNA) representam um importante grupo de patologias humanas. No entanto, não é possível predizer com acurácia o risco de uma mulher acometida por uma mutação no mtDNA transmitir a patologia para seus descendentes. Isso se deve, em parte, ao desconhecimento dos mecanismos moleculares que controlam a herança mitocondrial. Com o desenvolvimento de metodologias que possibilitam a derivação de células pluripotentes induzidas (iPSCs) a partir de células somáticas cultivadas in vitro, as iPSCs se tornaram um interessante modelo para o estudo da herança mitocondrial. A derivação de iPSCs de pacientes com mutações patogênicas no mtDNA tem revelado que a porcentagem de moléculas mutantes diminui ao longo do cultivo, sugerindo a existência na linhagem germinativa de mecanismos específicos para eliminação de mtDNAs mutantes. Portanto, o presente trabalho investigou em iPSCs derivadas de pacientes com desordens mitocondriais a existência de um mecanismo celular que elimina as moléculas de mtDNA com mutações patogênicas. Para tanto, foram utilizados fibroblastos heteroplásmicos portadores da mutação pontual A3243G no mtDNA causadora de encefalomiopatia mitocondrial, acidose lática e episódios tipo acidente vascular cerebral (MELAS); fibroblastos heteroplásmicos portadores de uma deleção de 4,9 kb no mtDNA causadora da Síndrome de Kearns-Sayre (KSS) e fibroblastos Controle, contendo apenas mtDNA selvagem. A derivação de linhagens portadoras de KSS resultou em iPSCs com baixos níveis de mtDNA mutante (< 0,1%), e na eliminação de moléculas mutantes ao longo do cultivo. A derivação de linhagens portadoras de MELAS resultou em iPSCs com alta taxa de mutação (> 98%), também com indícios de diminuição da quantidade de moléculas mutantes ao longo do cultivo. No entanto, ao contrário do esperado, não houve diminuição da quantidade de cópias de mtDNA nas iPSCs em relação aos fibroblastos em todas as linhagens (Controle, KSS e MELAS), sendo que as iPSCs de MELAS e KSS apresentaram um aumento significativo na quantidade de cópias de mtDNA, provavelmente devido a efeitos causados pela mutação no mtDNA. Ao analisar o efeito do tratamento com Rapamicina (indutor de mitofagia) durante a derivação de MELAS não observamos aumento na eficiência de formação de colônias, além de o tratamento não afetar a quantidade de mtDNA mutante, resultando em iPSCs com níveis de mutação similares aos encontrados nas iPSC MELAS não tratadas com o rapamicina. Por fim, resultados de expressão gênica das iPSCs do grupo MELAS revelaram indícios de mecanismos autofágicos direcionados a mitocôndria provavelmente devido ao efeitos causados pela a alta taxa da mutação. / 2013/13869-5

Abstract Mitochondrial DNA (mtDNA) is a maternally inherited 16.6 kbp circular genome that codes for 13 subunits of the mitochondrial respiratory chain, 2 rRNAs and 22 tRNAs. The mutation rate in mtDNA is high and therefore, mutations have accumulated sequentially to lineages that have diverged tens of thousands of years ago. The neutral theory predicts that a proportion of these variations may be slightly deleterious, associated with diseases and selected against, but the issue is still controversial. This study reports an analysis of selection against mutations in mtDNA. First, the population prevalence of one of the most pathogenic mtDNA mutations, the common MELAS mutation (3243A>G), was determined in a population-based screening setting in Northern Ostrobothnia, and the reproductive capacity, or genetic fitness, of women with the mutation was estimated in order to measure for the first time the degree of host-level selection against this highly pathogenic mutation. The frequency of 3243A>G was high, as the minimum estimate for the prevalence was 10.2/100,000, and this together with the geographical distribution of maternal ancestors of the mutation carriers suggested that nuclear genes may be involved in the population history of the mutation. Surprisingly, the genetic fitness of mutation carriers was not reduced, suggesting that the average host-level selection against carriers is not strong. Second, all available complete human mtDNA sequences worldwide (N=847) were collected into a database and analysed for evidence to support the hypothesis concerning slightly deleterious mutations and selective constraints imposed by lineage-specific interactions. 465 distinct missense and 6 nonsense mutations were identified. 48% of the amino acid replacements changed the polarity, 44% hydropathy, 32% aliphaticity, 26% size, 13% aromaticity, and 8% charge. Nonconservative amino acid replacements were found to be more common among the evolutionarily recent mutations than among the older ones, and mutations that have arisen more than once during human evolution showed different properties from the remaining ones. The major continent-specific mtDNA lineages were analysed in terms of nucleotide diversity indices, neutrality tests and nonsynonymous/synonymous rate ratios, and patterns suggesting selective constraints possibly due to lineage-specific interactions were identified. Moreover, a general correlation between nucleotide position and nucleotide polymorphism was identified in the mtDNA. The results are compatible with the assumption that selection has a marked role in human mtDNA evolution and that selective constraints may vary between populations, so that the pathogenic potential of a given mutation may depend markedly on the presence of other, interacting mutations.