Dissertations / Theses on the topic 'Mitochondrial apoptosis'

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed August 31, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 129-140).

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.

Le but de cette thèse est de montrer la réponse de la membrane mitochondriale au cours la régulation de l’apoptose en étudiant l’effet de domaines clés sur la dynamique membranaire et l’importance de la composition phospholipidiques des modèles utilisés. Le domaine BH4 est la partie spécifique anti-apoptotique de la famille Bcl-2. La première étape a été de synthétiser le peptide par voie chimique en utilisant la synthèse peptidique en phase solide. Un protocole décrivant les étapes de purification par chromatographie liquide et de caractérisation par spectroscopie de masse, garantissant une pureté indispensable pour des études biophysiques, a été établi. La modification de la structure secondaire du peptide interagissant avec des vésicules a été étudiée par spectroscopie infrarouge ainsi que par dichroïsme circulaire. Le peptide s’agrège à la surface et s’insère peu profondément dans la partie hydrophobe de la membrane. En utilisant la résonance magnétique nucléaire (RMN) et la calorimétrie, il a été montré que le peptide BH4 modifie l’organisation et la dynamique des liposomes mimant la surface mitochondriale. La deuxième étude a porté sur la première hélice de la protéine pro-apoptotique Bax (Bax-a1) qui a la propriété de diriger la protéine cytosolique vers la mitochondrie. Un protocole de synthèse et purification a été à nouveau établi. Le but de cette étude est de démontrer le rôle de l’interaction spécifique entre la cardiolipine, un phospholipide uniquement présent dans la mitochondrie et le peptide Bax-a1. Les études RMN ont montré que Bax-a1 n’interagissait uniquement que si la cardiolipine était présente, produisant un fort effet électrostatique piégeant le peptide à la surface de la membrane. Enfin, un nouveau protocole permettant d’étudier la réponse des lipides de mitochondries isolées toujours actives par RMN est présenté. Le but est de pouvoir directement observer les modifications subies par chaque phospholipide de la mitochondrie.
The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-a1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-a1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-a1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed June 2, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 190-218).

Mitochondria are essential organelles for life and death of the cell: they produce most of the cellular ATP (Danial et al., 2003), regulate cytosolic Ca2+ signalling (Rizzuto et al., 2000), and integrate and amplify different apoptotic stimuli (Green and Kroemer, 2004). Such a functional versatility is matched by a complex and dynamic morphology, both at the ultrastructural and at the cellular level (Griparic and van der Bliek). At the ultrastructural level, the mitochondrial cristae constitute a separate compartment connected to the thin intermembrane space by narrow tubular junctions (Frey and Mannella, 2000). In the cytosol, mitochondria are organized in a network of individual organelles that dynamically fuse and divide. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of "mitochondria-shaping" proteins, many of which are dynamin-related proteins initially identified by genetic screens in buddying yeast (Dimmer et al., 2002; Shaw and Nunnari, 2002). Dynamins are ubiquitous mechano-enzymes that hydrolyze GTP to regulate fusion, fission, tubulation and elongation of cellular membranes (McNiven et al., 2000). In mammalians, mitochondrial fission is controlled by a cytosolic dynamin related protein DRP-1 (Smirnova et al., 2001) that translocates to sites of mitochondrial fragmentation where it binds to FIS1, its adapter in the outer membrane (Yoon et al., 2003) (James et al., 2003). Fusion is controlled by mitofusin-1 (MFN1) and-2 (MFN2), two large GTPases of the outer mitochondrial membrane, orthologues of S. cerevisiae Fzo1p (Rapaport et al., 1998). OPA1, the mammalian homologue of S. cerevisiae Mgm1p, is the only dynamin-related protein of the inner mitochondrial membrane (Olichon et al., 2002). Loss-of-function or dominant-negative mutations in Opa1 are associated with autosomal dominant optic atrophy (DOA), the leading cause of inherited optic neuropathy, characterized by retinal ganglion cells degeneration followed by ascending atrophy of the optic nerve (Alexander et al., 2000; Delettre et al., 2000). The aim of my PhD has been to generate, use and analyze genetic models in order to unravel the biological function of OPA1 as well as its regulation. In order to dissect the biological function of OPA1, we undertook a combination of genetics and imaging to address its role in regulating mitochondrial fusion/fission equilibrium. Imaging of wild type mouse embryonic fibroblasts (MEFs) cotransfected with a mitochondrially targeted cyan fluorescent protein (mtCFP) showed mitochondria as individual organelles, rod or round-shaped, with an average length of 3±0.34 µm along their major axis. Morphometric analysis confirmed that only 23% of the analyzed cells displayed elongated mitochondria, i.e. cells with axial length >5 µm and roundness index <0.5 in more than 50% of mitochondria. Cotransfection of OPA1 with mtCFP induced visible changes in the shape of the mitochondrial reticulum. The rod-shaped mitochondria appeared now to be interconnected in a branched network. Morphometric analysis confirmed this mitochondria-shaping effect of OPA1, with more than 50% of the cells analyzed showing elongated mitochondria. Furthermore, we analyzed the effect of pathogenic mutations of OPA1 on its ability to elongate mitochondria. A missense mutation in the GTPase domain (K301A) that reduces the GTPase activity of more than 80%, as well as a truncative one in the coiled coil domain (R905stop), which eliminates the C-terminal coiled-coil domain required in protein-protein interactions, abolished the ability of OPA1 to elongate mitochondria, indicating that it requires a functional GTPase and coiled-coil domain. To address the effect of reduced OPA1 levels on mitochondrial morphology we turned to stable, plasmid-generated RNA interference (RNAi). In cell clones where OPA1 was ablated, mitochondria appeared globular and fragmented as opposed to the rod, elongated organelles of the control clones. Tubulation induced by OPA1 is not the results of simple juxtaposition of mitochondria, but it represents the steady state appearance of increased mitochondrial fusion events, as substantiated by assays of mitochondrial fusion in polykarions induced by PEG treatment. Expression of OPA1 significantly speeded up mixing of matricial content, whereas its downregulation reduced mitochondrial fusion. In yeast, the pro-fusion activity of Mgm1p, the orthologue of OPA1, depends on the outer membrane mitochondria-shaping protein Fzo1p. We therefore wished to ascertain whether this paradigm was maintained in higher eukaryotes. We turned to a genetic approach, testing the ability of overexpressed OPA1 to promote mitochondrial tubulation in MEFs deficient for either Mfn1 or Mfn2. Expression of OPA1 induced mitochondrial tabulation and fusion in wt and in Mfn2-/- but not in Mfn1-/- cells. This defect was complemented by re-introduction of MFN1 but not MFN2, unequivocally identifying outer membrane MFN1 as an essential functional partner of OPA1. Moreover, MFN1 was unable to promote mitochondrial elongation if OPA1 had been ablated. Thus, OPA1 and MFN1 appear to functionally depend one on each other. To address whether Mfn1-/- MEFs displayed any defect in the preparatory events of mitochondrial juxtaposition and docking, we performed 4D-imaging of mitochondria, i.e. time series of z-stacks of mitochondrial images. The total number of contacts between mitochondria was not affected by OPA1 overexpression or by MFN deficiency. OPA1 facilitated fusion following contacts between wt and Mfn2-/- but not Mfn1-/- mitochondria. Taken together, our results suggested that OPA1 requires MFN1 to fuse the membranes of two juxtaposed mitochondria and not to produce inter-mitochondrial contacts. Our genetic analysis provided the first evidence of a functional diversity between MFN1 and MFN2, suggesting a functional axis between OPA1 and MFN1 (Cipolat et al., 2004). The discovery that OPA1 is a pro-fusion protein raised the question of whether this protein participated in the regulation of apoptosis, during which fusion is impaired. We therefore decided to genetically dissect the role of OPA1 in fusion and apoptosis. We could demonstrate that OPA1 has an antiapoptotic activity, controlling the cristae remodelling pathway of apoptosis, independently of mitochondrial fusion. OPA1 did not interfere with the activation of the core mitochondrial apoptotic pathway of BAX and BAK activation. Yet OPA1 inhibited the release of cytochrome c by preventing the remodelling of the cristae and the intramitochondrial redistribution of cytochrome c. Inactivating mutations in the GTPase domain of OPA1 impaired its anti-apoptotic activity, enhancing susceptibility to apoptosis induced by stimuli that recruit the mitochondrial pathway. While our results contributed to clarify the biological function of OPA1, they left open a number of questions. In particular, if the pro-fusion activity of OPA1 was dispensable for the inhibition of apoptosis, how was this function controlled? In yeast Mgm1p is processed by the inner mitochondrial membrane rhomboid protease Rbd1/Pcp1 into a short active form, responsible for the effects of Mgm1p on mitochondrial morphology (Herlan et al., 2003; McQuibban et al., 2003). The mammalian orthologue of Rbd1p, PARL, could similarly play a role in the regulation of one of the two biological functions we ascribed to OPA1, i.e. its effect in mitochondrial fusion and its anti-apoptotic activity. In order to address this issue, we decided to analyze the phenotype of a mouse model of Parl deletion. Parl-/- mice were born with normal Mendelian frequency and developed normally up to 4 weeks. From then on, mice displayed severe growth retardation and progressive atrophy in multiple tissues, leading to cachexia and death. The atrophy of Parl-/- tymi, spleens and muscular tissues was caused by an increased apoptosis of double-positive (CD4+CD8+) thymic lymphocytes, splenic B lymphocytes (B220+) and myoblasts, respectively. We investigated to what extent mitochondrial dysfunction and morphology dysregulation contributed to this multisystemic atrophy. PARL was not required for normal mitochondrial function: Parl-/- mitochondria did not display primary respiratory defects or latent mitochondrial dysfunction in hepatocytes, MEFs, primary myocytes and myotubes. Mitochondrial dysfunction therefore did not explain Parl-/- muscular atrophy and multisystem failure. Moreover Parl was not required for maintenance of mitochondrial shape and fusion, even in tissues severely affected by Parl ablation like muscle, and Parl was dispensable for regulation of mitochondrial dynamics by OPA1. We therefore investigated whether PARL regulates mitochondrial apoptotic machinery by analyzing apoptosis in MEFs treated with different intrinsic mitochondria utilizing stimuli. Parl-/- MEFs were more sensitive to all the stimuli tested as compared to their wt counterparts. Reintroduction of a catalytically active PARL showed that the defect was specific. PARL exerted its antiapoptotic effect at the mitochondrial level, since cytochrome c release and mitochondrial dysfunction following treatment with an apoptotic stimulus occurred faster in Parl-/- fibroblasts than in their relative wt counterparts. PARL did not regulate activation of the core BAX, BAK dependent apoptotic pathway, but it was required to keep in check the cristae remodelling pathway and to prevent mobilization of the cristae stores of cytochrome c during apoptosis. Since these results pointed to a role for PARL in the cristae remodelling pathway, regulated by OPA1, we ought to understand whether OPA1 required PARL to regulate apoptosis. OPA1 protected wt but not Parl-/- MEFs from apoptosis; furthermore, expression of OPA1 in Parl-/- MEFs did not reduce cytochrome c release, or mitochondrial depolarization following intrinsic stimuli. When Opa1 was silenced by siRNA in Parl-/- cells, they were no longer rescued by re-expression of PARL, demonstrating that PARL is genetically positioned upstream of OPA1. This genetic interaction was confirmed at multiple levels, since PARL and OPA1 interacted in a yeast two-hybrid and co-immunoprecipitation assays. PARL participated in the production of a soluble, IMS located, "anti-apoptotic" form of OPA1. The catalytic activity of PARL was required for the efficient production of soluble OPA1 and the re-introduction of a form of OPA1 in the IMS rescued the pro-apoptotic phenotype of Parl-/- cells. Thus, this IMS form resulted pivotal in controlling the pathway of cristae remodelling and cytocrome c redistribution. IMS and integral IM OPA1 indeed were both found to participate in the assembly of OPA1-containing oligomers that are early targets during cristae remodelling and greatly reduced in Parl /- mitochondria. The reduced level of OPA1 oligomers could account for the faster remodelling and cytochrome c mobilization observed in the absence of PARL. OPA1 affects complex cellular functions other than apoptosis, as substantiated in overexpression studies showing a role for this protein in movement of leukocytes (Campello et al., 2006) and formation of dendritic spines (Li et al., 2004). Furthermore, Opa1 knockout mice demonstrated that OPA1 is required for embryonic development. Homozygous mutant mice die in uterus at 13.5 dpc, with first notable developmental delay at E8.5 (Alavi et al., 2007). We therefore reasoned that levels of OPA1 are likely to affect development and function of multiple organs, by regulating mitochondrial fusion or apoptosis. In the last part of this Thesis, we therefore decided to study whether ablation of OPA1 influences differentiation of embryonic stem (ES) cells in vitro using a hanging-drop differentiation system. To this end, we analyzed an ES cell line where Opa1 had been gene trapped (Opa1gt), resulting in an Opa1+/- genotype. We compared the differentiation potential into cardiomyocytes and neurons of this Opa1gt ES cell line to its relative wt ES cell line. Opa1gt ES cells displayed a decreased capacity to differentiate into beating cardiomyocytes, while they retained a normal neuronal differentiation potential. These preliminary results indicate that OPA1 is a good candidate to regulate differentiation of ES cells in vitro. We now aim at understanding the molecular mechanism by which levels of OPA1 influence differentiation into cardiomyocytes. In conclusion, the data presented in this Thesis demonstrate genetically distinct roles of the mitochondrial dynamin related protein OPA1 in the regulation of organellar shape and apoptosis. The individuation that the functional axis between OPA1 and MFN1 (that regulates mitochondrial fusion) and the regulatory IMM network comprised of the couple substrate-protease Parl-Opa1 could perhaps even control embryonic differentiation opens novel, unexpected avenues to investigate the role of mitochondria in life and death of the cell.

Bien que les statines forment la classe d'hypolipidémiants la plus utilisée, une toxicité musculaire a été reportée, pouvant ainsi provoquer l’apparition d’une myopathie. Dans la première partie, nous avons montré chez l’Homme et l’animal que les statines inhibent directement la chaine respiratoire mitochondriale, et induisent la production de radicaux libres dérivés de l’oxygène (RLO), qui active les voies apoptotiques dans les muscles glycolytiques, alors que les muscles oxydatifs ne sont pas atteints. Nous avons ensuite montré in vitro que le stress réducteur peut engendrer une oxydation mitochondriale, pouvant conduire à une activation de la voie de biogenèse mitochondriale. De plus l’augmentation du contenu mitochondrial induite a permis de protéger les cellules contre l’apoptose induite par les statines. Enfin, nous avons montré in vivo que l’induction des voies de biogenèse mitochondriale est nécessaire à la tolérance des statines dans les muscles oxydatifs. En conclusion, le phénotype mitochondrial, tant au niveau quantitatif que qualitatif, semble être un facteur clé dans l’apparition de la myopathie aux statines
Although statins are the most prescribed class of lipid-lowering agents, adverse muscular toxicity has been reported, which can lead to the appearance of a myopathy. In the first part, we showed in Humans and animals that statins inhibit directly the mitochondrial respiratory chain, and induce the production of reactive oxygen species (ROS), that trigger apoptotic pathways in glycolytic skeletal muscles, whereas oxidative muscles are not impaired. We then showed in vitro that reductive stress can provoke mitochondrial oxidation, that could lead to an activation of mitochondrial biogenesis pathways. Moreover, the consequent increase in mitochondrial content enabled to protect cells against statin-induced apoptosis. Finally, we showed in vivo that the induction of mitochondrial biogenesis is necessary for statin tolerance in oxidative skeletal muscles. In conclusion, mitochondrial phenotype, both quantitatively and qualitatively, seems to be a key factor in the appearance of statin myopathy

Mitochondria exist in a dynamic network regulated by the opposing processes of mitochondrial fusion and fission. Regulation of mitochondrial morphology is critical for metabolism, quality control and cell survival, among other cellular processes. Large GTPases are responsible for shaping the mitochondrial network. Mitofusins 1 and 2 and Opa1 regulate outer and inner mitochondrial membrane fusion, respectively. Conversely, Drp1 is recruited to mitochondria to carry out fission. Although many proteins have been implicated in these processes, there are still many unknowns. We sought to identify novel regulators of mitochondrial morphology and conducted a genome-wide RNAi screen to identify candidate genes. We identified Reactive Oxygen species Modulator 1 (ROMO1) as a novel regulator of mitochondrial fusion and cristae integrity. In the absence of ROMO1, the mitochondrial network fragments and cristae are lost. These defects lead to impaired mitochondrial respiration and sensitization to cytochrome c release and downstream apoptosis. ROMO1 is regulated by mitochondrial REDOX at 4 cysteine residues that couple REDOX signaling to mitochondrial morphology. We have characterized ROMO1 as an interactor with the MINOS complex, required for cristae junction maintenance, and the inner mitochondrial membrane fusion GTPase OPA1. Through these interactions ROMO1 couples cristae junction security to mitochondrial fusion.

Mitochondria are essential organelles for the life of the cells since it is the major source of ATP, key molecule for many endoergonic reaction. Recently it has been demonstrated that mitochondrial play a key role in many other cellular processes like Ca2+ signaling and programmed cell death. Following an apoptotic insult mitochondria release cytochrome c and other proteins required in the cytosol for the activation of the effector caspases required for cell demise. What is remarkable about cytochrome c release is that is fast, complete and usually is not associated with mitochondrial swelling. Thanks to the advances in 3D electron microscopy it has been demonstrated that cristae are not just invagination of the inner mitochondrial membrane (IMM) as previously depicted by Palade (Palade, 1952) but rather distinct compartments of it, separated from the inter membrane space (IMS) by tubular narrow cristae junctions. The majority of cytochrome c and the other respiratory chain components are restricted in this compartment. To reach a complete cytochrome c release in the absence of mitochondrial swelling mitochondria remodel their internal structure: individual cristae fuse and tubular narrow cristae junctions widen; this process, defined cristae remodeling is associated with the mobilization of cytochrome c towards the IMS for its subsequent release across the outer mitochondrial membrane (OMM) (Scorrano et al., 2002). The molecular mechanism beyond this dynamic process is not well understood and in the laboratory where I did my doctoral Thesis it has been hypothesized that OPA1, the only dynamin related protein of the IMM (Alexander et al., 2000; Delettre et al., 2000) could control cristae remodeling. Dynamin related proteins are regulators of mitochondrial morphology promoting mitochondrial fusion and fission. To this family belong Mitofusins (MFN) 1 and 2 in the OMM and OPA1 that resides in the IMM. OPA1 is a large GTPase anchored in the IMM, facing the IMS (Olichon et al., 2002; Satoh et al., 2003); it has been shown that in yeast, its orhologue Mgm1p is required for fusion competent mitochondria by the cooperation with a protein of the same family on the OMM called Fzo1p. In our laboratory it has been demonstrated that in mammalian cells OPA1 promotes mitochondrial fusion through one of the two mammaliam orthologue of Fzo1p called MFN1. In 2000 two distinct laboratories demonstrated that mutations in OPA1 gene are the cause of dominant optic atrophy (ADOA), the leading case of inherited blindness in human, characterized by selective death of retinal ganglion cell (RGC) (Alexander et al., 2000; Delettre et al., 2000). The fact the mutation in a mitochondrial protein involved in mitochondrial morphology caused cell death opened a new scenario that corroborates the central position of mitochondria in regulating apoptotic signaling. The aim of my thesis was to analyze the role of OPA1 in mitochondria-dependent apoptosis. We started with a brute force approach by overexpressing OPA1 in murine embryonic fibroblasts (MEFs) and measuring cells viability in response to intrinsic apoptotic stimuli that specifically trigger apoptosis through the mitochondrial pathway. Overexpression of wt OPA1 but not of mutant in the GTPase domain (OPA1K301A) or a truncated mutant in the coiled coil domain (OPAR905*) is able to prevent from apoptosis induced by hydrogen peroxide, staurosporine, etoposide and overexpression of tBID, a BH3 only protein of the Bcl-2 family that promotes cristae remodeling. To confirm that OPA1 antiapoptotic activity was exerted at the mitochondrial level we analyzed two aspects of the mitochondrial dysfunction: cytochrome c release and mitochondrial depolarization. To this aim we overexpressed a mitochondrially targeted red fluorescent protein (mtRFP) as marker of the mitochondrial network and then we immunodecorated cytochrome c with a FITC-conjugated secondary antibody. OPA1 overexpression prevented cytochrome c release in response to intrinsic stimuli while its inactive mutant OPAK301A aggravated cytochrome c release kinetic. We then analyzed another aspect of the mitochondrial dysfunction: mitochondrial depolarization, taking advantage of the potentiometric probe tetramethylrhodamine-methyl ester (TMRM) which mitochondrial fluorescence is proportional to mitochondrial potential. Overexpression of OPA1, but not of its inactive K301A mutant, prevented mitochondrial depolarization induced by intrinsic stimuli, confirming that OPA may prevent from apoptosis at the mitochondrial level by reducing cytochrome c release and mitochondrial depolarization. How can a dynamin related protein prevent from apoptosis? We asked this because when our study was ongoing an intriguing hypotesys emerged: during apoptosis mitochondrial network undergoes irreversible massive fragmentation; this event and apoptotic cristae remodeling are required for complete cytochrome c release. In principle, OPA1 could prevent apoptosis at both of these levels either counteracting mitochondrial fragmentation thanks to its pro-fusion activity or by the regulation of cristae remodeling. To understand at which of these levels OPA1 was exerting its antiapototic activity, we started a genetic approach, overexpressing OPA1 in Mfn1-/-, where OPA1 pro-fusion activity was prejudiced. Overexpression of OPA1 in these cells prevented from apoptosis induced by intrinsic stimuli; in view of the fact that a residual pro-fusion activity of OPA1 could be mediated by the presence of MFN2 we repeated the same experiments in cells in which both mitofusins were ablated (DMF). Also in this conditions OPA1 prevented from apoptosis at the mitochondrial level, slowing down cytochrome c release kinetic. OPA1 has an antiapoptotica function that is independent of its pro-fusion activity on the mitochondrial network. At this point we asked whether OPA1 may have a role on apoptotic cristae remodeling. We generated stable cell lines that stably overexpressed OPA1 and its K301A mutant both in wt and in Mfn1-/- cells and a cell line depleted of OPA1 by short hairpin RNA interference (shOPA1RNAi). We then isolated mitochondria and measured cytochrome c release induced by recombinant caspase 8 cleaved BID (cBID) using a specific ELISA immunoassay. Stable overexpression of OPA1 is able to prevent cytochrome c relase independently of MFN1 while its downregulation dramatically increases its release. Using a specific assay we observed that OPA1 is also able to prevent cytochrome c mobilization from the cristae independently of MFN. These results were confirmed by the fact that overexpression of the OPA1K301A mutant increased cytochrome c mobilization that was almost complete when OPA1 levels were depleted by RNAi. A thorough morphometric analysis of isolated mitochondria from these cell lines, associated with 3D reconstruction of electron microscopy tomography, showed that OPA1 controls cristae morphology and prevents cristae junction widening in response to cBID. To better understand the molecular mechanism through which OPA1 controls cristae remodeling and cristae junctions diameter we based our hypothesis on the possible analogy with vesciculation processes regulated by cytosolic dynamin, where GTPase activity of it mediated mechanoenzimatic constriction of the vesicle collar. Despite this analogy, we should mention that OPA1, unlike dynamin, is located on the inner side of the membrane to be constricted and not on the outside as dynamin complicating the model. First, we analyzed biochemical characteristic of OPA1: gel filtration studies showed that OPA1 is eluted at very high molecular weight fractions (>600 KDa) and in response to cBID incubation it is retrieved in low molecular weight fractions. Parallel studies in our laboratory demonstrated that OPA1 is processed by a rhomboid protease, PARL, into a short form found soluble in the IMS that is responsible for the antiapototic but not of the pro-fusion activity of OPA1. We therefore reasoned that OPA1 could organize into high molecular weight complexes made up at least by the PARL generated soluble form and the membrane bound form of OPA1. To confirm this hypothesis we crosslinked this complex and confirmed the presence of a high molecular weight immunoreactive band for OPA1 that disappear following the mechanical expansion of the cristae induced by osmotic swelling. These crosslinker-stabilized oligomers contain both the soluble and the membrane bound forms of OPA1 as demonstrated by their immunoreactivity for properly tagged and co-expressed forms. The OPA1-containing oligomers is targeted by cBID in a time dependent manner and OPA1 overexpression stabilizes these complexes. We can conclude that OPA1 controls cytochrome c mobilization and cristae remodeling that occurs during apoptosis. This function of OPA1 is independent of MFNs and is correlated to the formation of high molecular weight complexes. The data collected so far on OPA1 antiapoptotic function open a new scenario. First we need to investigate on the molecular composition of these complexes in normal and apoptotic conditions. To this aim we started a biochemical study on OPA1-containing complexes in mitochondria isolated from different genetic background in normal and apoptotic conditions. The proteomic analysis of the proteins eventually found in complex with OPA1 will allow us to comprehend the function and regulation of OPA1 oligomers before and after cell death induction. OPA1 appears as a crucial protein in the apoptotic process; as a confirmation of this, it has been found that OPA1 is highly overexpressed in some lung cancer (Dean Fennel, personal communication); we then asked whether OPA1 could be a target for the development of new drugs that enhance apoptosis in tumor cells. To this aim, we started a collaboration with Stefano Moro from the Department of Medicinal Chemistry of the University of Padova, to generate a library of candidate inhibitors of OPA1 performing a virtual screening of compounds targeted to the GTPase pocket of OPA1 obtained following an homology modeling on the Dyctiostelium Discoideum GTPase domain of Dynamin A. In conclusion, the data presented in this doctoral thesis show that mitochondrial protein OPA1 participates in the regulation of cytochrome c mobilization and cristae remodeling during apoptosis. We demonstrated that OPA1 organizes into high molecular weight complexes which disruption correlates with cristae junction widening. This function is distinct from its role in mitochondrial morphology and this suggest a bifurcation and specialization of OPA1 function during evolution.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
The retinoblastoma protein (pRB) tumor suppressor is deregulated in the vast majority of human tumors. pRB is a well-established transcriptional co-regulator that influences many fundamental cellular processes. It has been most well characterized in its ability to block cell proliferation by inhibiting the E2F family of transcription factors. Importantly, pRB also plays a pivotal role in apoptosis. This function has been extensively characterized in the context of genotoxic stress. Specifically, these studies have revealed that pRB can act in both an anti-apoptotic manner by inducing cell cycle arrest, and a pro-apoptotic manner by transcriptionally co-activating proapoptotic genes. Here, we show that pRB can also promote TNF[alpha]-induced apoptosis. Moreover, this investigation led us to uncover a novel, non-transcriptional and non-nuclear role of pRB in the induction of apoptosis. Specifically, we found that pRB can enhance TNFainduced apoptosis even in the presence of an inhibitor of translation, and that a fraction of endogenous pRB is localized at the mitochondria both in the absence and presence of treatment with apoptotic stimuli. Further characterization revealed that pRB can directly bind to and activate BAX, resulting in mitochondrial outer membrane permeabilization and apoptosis. Importantly, targeting ectopically expressed pRB specifically to the mitochondria generated a separation-of-function mutant deficient for pRB's classic, nuclear roles. Remarkably, we found that this mito-tagged pRB mutant can promote apoptosis in response to many apoptotic stimuli, arguing that mitochondrial pRB is a general mediator of apoptosis. Moreover, expression of this mito-pRB mutant in vivo was sufficient to suppress tumorigenesis. Taken together, our data uncover a role for pRB in the direct activation of mitochondrial apoptosis. To our knowledge, this is the first characterization of a non-nuclear and transcription-independent function for pRB. Moreover, most human tumors are wild-type for pRB, but contain alterations that result in constitutive phosphorylation of pRB. While this functionally inactivates pRB's cell cycle function, we show that pRB's mitochondrial role is unaffected. This raises the possibility that this novel pro-apoptotic pRB mechanism can be exploited for chemotherapeutic treatment.
by Keren Ita Hilgendorf.
Ph.D.

The mitochondrion is a complex, double membrane organelle that serves several important cellular functions including ATP synthesis, Ca ²⁺ buffering, and ROS homeostasis. Although classic mitochondrial diagrams depict the mitochondrion as a simple oval or “bean” shaped organelle, the mitochondria can form extensive tubular networks or numerous small spheres in response to various cellular environments through two opposing processes, mitochondrial fission and fusion. Deregulation of mitochondrial dynamics has been implicated in a wide range of diseases, including Parkinson’s disease, heart disease and cancer. While significant emphasis for the last 15 years has been placed on the identification of the protein machinery responsible regulating mitochondrial morphology, it remains less clear how mitochondrial morphology affects various cellular functions and cellular fate outcomes. This thesis summarizes our findings on how mitochondrial morphology regulates cellular fate in the context of mitotic cell division and apoptosis. Using live cell microscopy and image analysis software we characterized mitochondrial dynamics with single cell resolution. We found that loss of key components of the mitochondrial fission machinery promotes a defect in cell cycle progression, characterized by an inability for cells to exit G2/M. Prolonged periods of mitochondrial fusion induced potent cell death, suggesting a novel mechanism to target the replicative potential of cancer cells. We also found that mitochondrial fission and fusion can alter the kinetics of cell death following apoptotic stimuli by inducing mitochondrial fusion prior to the commitment step in apoptosis, mitochondrial membrane permeabilization. This thesis summarizes our work in trying to elucidate how the structure of the mitochondria influences both mitochondrial and cellular fate.

Les dommages tissulaires associés à l'ischémie/reperfusion ont été largement étudiés. Plusieurs études ont montré que des dysfonctionnements mitochondriaux sont responsables de la survenue de ces dommages, et que la transition de perméabilité pourrait y être impliquée. Cette transition de perméabilité est médiée par l'ouverture du pore de transition de perméabilité (PTP). Cette ouverture du PTP pourrait survenir pendant la phase d'ischémie ou pendant la phase de reperfusion. L'objectif de ce travail était de visualiser l'ouverture du PTP dans des conditions d'ischémie/reperfusion sur cellules intactes (HMEC-1 et INS-1) et d'étudier son implication dans ce phénomène. Nous avons pu visualiser pour la première fois l'ouverture du PTP par le suivi des dommages qu'elle engendre (sortie du NADH et la chute du ΔΨ) induits par une ischémie/reperfusion. Nous avons constaté que l'activation du PTP a lieu pendant la phase d'ischémie tant dans les cellules HMEC-1 que dans les cellules INS-1. Cette induction a été prévenue dans les deux modèles cellulaires par la cyclosporine A. Nos résultats suggèrent également que le complexe I pourrait être impliqué dans la prévention de la chute du ΔΨ et de la sortie du NADH. Nous avons aussi montré que la capacité de rétention calcique des cellules perméabilisées diminue à l'ischémie et que cette diminution disparait après 60 minutes de reperfusion. Ainsi, la visualisation de l'ouverture du PTP dans un modèle d'ischémie/reperfusion constituerait une piste intéressante qui apporterait plus de certitude quant à l'implication du PTP dans ce phénomène. De plus, l'étude du phénomène d'ischémie in vitro, apporterait plus de réponses quant à l'implication des modifications du fonctionnement cellulaire dans les dommages tissulaires.

Menschliche Mitochondrien enthalten etwa 1500 bis 2000 Proteine. Die meisten dieser Proteine werden im Zellkern kodiert und im Zytoplasma synthetisiert, und müssen daher über eine spezielle Maschinerie in die Mitochondrien transportiert werden. Obwohl mittlerweile viele Details über die Wirkungsweise dieser Proteinschleusen bekannt sind, wurden einige wichtige Aspekte des Proteinimports noch nicht ausreichend untersucht. Zum einen ist nicht bekannt, ob die einzelnen Importkomplexe einen Einfluss auf die mitochondrienvermittelte Apoptose haben. Weiterhin ist offen, welche genaue Rolle der Mitochondrienimport in der Pathogenese von Neisseria gonorrhoeae spielt. Außerdem ist unklar, ob Faktoren des Importapparates für die Aufrechterhaltung der mitochondrialen Morphologie notwendig sind. Um diese Fragestellungen zu untersuchen, wurden im Rahmen der vorliegenden Arbeit permanente Zelllinien hergestellt, in denen die Expression einzelner am Mitochondrienimport beteiligter Proteine mittels RNA-Interferenz (RNAi) inhibiert werden kann. Mithilfe dieser Zelllinien wurde getestet, ob die proapoptotischen Proteine Bax und Bak die Importmaschinerie benötigen, um in die äußere Mitochondrienmembran zu gelangen. Die Präsenz der beiden proapoptotischen Proteine in Mitochondrien während der Apoptose ist sehr entscheidend, da Bax und Bak in den Mitochondrien oligomerisieren und damit weitere Schritte der Apoptose einleiten. Im Widerspruch zu früheren Publikationen konnte hier gezeigt werden, dass die Translokation von Bax und Bak in die äußere Mitochondrienmembran unabhängig von Proteinimportfaktoren erfolgt. Der zweite Teil dieser Arbeit beschäftigt sich mit dem Einfluss mitochondrialer Importproteine auf die Pathogenese von Neisseria gonorrhoeae. Das Neisserienprotein PorB transloziert während der Infektion in die Mitochondrien der Wirtszelle und induziert Apoptose. Aufgrund der strukturellen Ähnlichkeit von PorB zu bestimmten Proteinen der äußeren Mitochondrienmembran wurde bisher angenommen, dass PorB diesen endogenen Proteinen auf ihrem Importweg in die äußere Mitochondrienmembran folgt. Überraschenderweise wurde im Rahmen dieser Arbeit entdeckt, dass PorB nicht von allen Komplexen der Importmaschinerie in den Mitochondrien erkannt wird. Infolgedessen transloziert es in die innere Mitochondrienmembran und wirkt dadurch toxisch auf die Wirtszelle. In einem weiteren Projekt wurde untersucht, welche Rolle die Proteinimportkomplexe der äußeren mitochondrialen Membran in der Aufrechterhaltung der Mitochondrienmorphologie spielen. Unter Verwendung der beschriebenen Zelllinien wurde entdeckt, dass in Abwesenheit des SAM (sorting and assembly) Importkomplexes die Struktur der inneren Mitochondrienmembran derangiert ist. Es wurden zudem Hinweise darauf gefunden, dass die Ursache für diesen Befund in einer Unterbrechung von Kontaktstellen zwischen den beiden Mitochondrienmembranen liegen könnte, für deren Aufrechterhaltung möglicherweise der SAM-Komplex verantwortlich ist. Die in dieser Arbeit vorgestellten Ergebnisse erlauben neue Einblicke in verschiedene Aspekte des Proteinimports in Mitochondrien. Zudem wurde mit der Entwicklung der stabilen Zelllinien ein neues Model geschaffen, anhand dessen in Zukunft weitere Detail des mitochondrialen Proteinimports erforscht werden können.
Human mitochondria comprise about 1500 to 2000 proteins. While only 13 proteins are encoded by the mitochondrial DNA the vast majority of mitochondrial proteins is encoded in the nucleus, synthesized in the cytosol, and translocated into mitochondria by a special protein import machinery. Although many details are now known about its function several important aspects of protein import in mitochondria were not unraveled yet. To begin with, the influence of the different mitochondrial import complexes on apoptosis is not known. Further, the exact role of the protein import machineries in mitochondria in the pathogenesis of Neisseria gonorrhoeae has not been clarified yet. Moreover, the question whether factors involved in protein import are required for the maintenance of the mitochondrial morphology is still unsolved. In order to address these open issues, permanent cell lines were generated within the frame of the present thesis in which the expression of single proteins implicated in mitochondrial import can be inhibited via RNA interference (RNAi). Using these cell lines, it was investigated whether the proapoptotic proteins Bax and Bak require the import machinery in order to gain access to the outer mitochondrial membrane. The presence of both proapoptotic proteins in mitochondria is essential during apoptosis as Bax and Bak oligomerize in the outer mitochondrial membrane leading to the execution of apoptosis. In contrast to earlier publications, results presented here prove that the translocation of Bax and Bak into the outer mitochondrial membrane occurs independent of its import machineries. The second part of this thesis explores the influence of mitochondrial import proteins on the pathogenesis of Neisseria gonorrhoeae. The neisserial protein PorB translocates into the mitochondria of host cells during infection and induces apoptosis. Because of structural similarities of PorB to a certain class of proteins in the outer mitochondrial membrane, it was assumed that PorB would follow the import pathway of these endogenous proteins into the outer mitochondrial membrane. Surprisingly, it was found within the present study that PorB is not recognized by all complexes implicated in this import pathway. As a consequence, it translocates into the inner mitochondrial membrane to exert its toxic effect on the host cell. In a further project, the role of import complexes of the outer mitochondrial membrane in the maintenance of the mitochondrial morphology was investigated. Using the described cell lines, it was found that in the absence of the SAM (sorting and assembly) import device, the structure of the inner mitochondrial membrane was disrupted. Further, evidence was found that the reason for this phenotype could be an interruption of contact sites between the two mitochondrial membranes, whose preservation possibly requires the SAM complex. The results presented here allow new insights into different aspects of mitochondrial protein import. Further, with the development of the stable cell lines a new model was generated that will allow future investigations on details about mitochondrial protein import.

Thesis (Ph. D.) -- University of Maryland, College Park, 2007.
Thesis research directed by: Biology. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Les microtubules sont une des cibles les plus établies dans le traitement contre le cancer. Les taxanes, paclitaxel et docetaxel, stabilisent les microtubules et ont montré une activité clinique significative mais des facteurs tels que le développement d’une résistance à ces agents limite leur utilisation clinique. Les épothilones sont des macrolides, et ont une activité in vitro et in vivo sur différents types cellulaires résistants aux taxanes. Plusieurs épothilones dont l'ixabepilone, le patupilone, BMS-310705, KOS-862, KOS-1584, et ZK-EPO sont en développement clinique. Le patupilone (épothilone B, EPO906) est un composé très actif et est actuellement en essais cliniques de phase III pour les cancers péritonéaux et ovariens. Le patupilone a la propriété de facilement passer la barrière hématoméningée et n'est pas substrat pour la P-glycoprotéine. Il a également montré des effets antitumoraux dans le système nerveux central (CNS) dans les modèles animaux. En raison de ces avantages, nous avons réalisé pour la première fois une étude in vitro de cette drogue sur les cellules de neuroblastome (SK-N-SH) afin d’en étudier le mécanisme d’action qui s’avère plus efficace que le paclitaxel (IC50 = 1. 8 nM comparé à 100 nM). La perméabilisation de la membrane mitochondriale, les changements morphologiques et la libération du cytochrome c après traitement au patupilone sont des phénomènes précoces aboutissant à l'apoptose. Le patupilone augmente la génération des espèces réactives de l'oxygène (ROS) de façon précoce et spécifique des mitochondries. Nous avons également pu montrer une accumulation des protéines pro-apoptotiques Bim et p53 au niveau des mitochondries à 6 heures et 12 heures de traitement respectivement. En revanche, les niveaux de Bax et de Bcl-2 mitochondriaux n'ont pas été changés pendant le traitement. Le balayage de ROS ou délétion partielle de l'ADN mitochondrial a protégé de manière significative les cellules reflétant l'importance cruciale des ROS mitochondriaux pour l'activité du patupilone. L'accumulation des protéines pro-apoptotiques aux mitochondries, des changements morphologiques des mitochondries et le relargage du cytochrome c ont été nettement diminués quand les ROS mitochondriales ont été empêchées. Nos résultats montrent donc l'importance cruciale des ROS produits par les mitochondries pendant le traitement au patupilone pour le déclenchement de la voie intrinsèque en induisant l'accumulation de Bim aux mitochondries. Ces résultats pourraient expliquer l'activité supérieure du patupilone sur des cellules tumorales comparé au paclitaxel. En outre, le rôle important des mitochondries pour le déclenchement et l'exécution des signaux apoptotiques, déclenchés par le patupilone, est démêlé par nos données. Ces résultats suggèrent fortement de poursuivre la recherche clinique sur le patupilone contre le neuroblastome.

DMPK is a serine/threonine protein kinase that was initially proposed to be the cause of the most frequent adult muscular dystrophy, myotonic dystrophy 1 (DM1). Recently, it has been shown that DMPK is not the primary determinant of the DM1, but its deletion causes late onset myopathy and cardiac abnormalities in knock-out mice. Evidence present in the literature suggests a mitochondrial localization of high MW DMPK isoforms in muscle and cardiac tissue. However, to date, there is not a single association of mitochondria-anchored isoforms with the respective function of the organelle in the affected tissues. Therefore, we have examined the role of mitochondria-anchored isoform A, either by stably expressing it in cells lacking endogenous protein, or by stably silencing the endogenous one. DMPK significantly decreased levels of mitochondrial superoxide and consequently increased cell survival in prolonged serum and glucose depletion, both in SAOS-2 and rhabdomyosarcoma cells. At the molecular level we have found DMPK to interact with HK II and Src, increasing the HK II association to mitochondria. Detachment of HK II from mitochondria abolished differences in superoxide levels, while a HK II inhibitor 5-TG protected cells from death by stabilizing HK II on the OMM and by decreasing mitochondrial ROS in the absence of DMPK. Src activity was also important for HK II maintenance on OMM since its inhibition sensitized only DMPK-expressing cells to detachment of HK II. These data attribute an antiapoptotic role to DMPK due to an unprecedented link to HK II and its protective effect against mitochondrial ROS.
DMPK è la serina/treonina protein kinasi, la quale è stata inizialmente proposta come la causa della più frequente distrofia muscolare negli adulti, la distrofia miotonica del tipo 1 (DM1). Recentemente si è visto che la DMPK non è la causa principale della DM1, ma la sua delezione causa miopatia ad insorgenza tardiva e anomalie cardiache nei topi knock-out. I dati presenti in letteratura attribuiscono la localizzazione mitocondriale alle isoforme ad alto peso molecolare nel muscolo e nel tessuto cardiaco. Comunque, finora non vi sono stati studi volti ad associare il ruolo delle isoforme mitocondriali della DMPK alla funzione dell’organulo nei tessuti in questione. Perciò, abbiamo deciso di esaminare il ruolo dall’isoforma A associata ai mitocondri, sia esprimendola stabilmente nelle cellule prive della DMPK endogena, sia silenziando stabilmente quella endogena. DMPK ha significativamente diminuito i livelli del superossido mitocondriale e, di conseguenza, ha aumentato la sopravvivenza delle cellule SAOS-2 e rabdomiosarcoma in deplezione di siero e glucosio. A livello molecolare, abbiamo trovato che la DMPK interagisce con HK II e Src aumentando l’associazione dell’HK II ai mitocondri. Il distacco dell’HK II dai mitocondri ha cancellato le differenze nei livelli di superossido, mentre l’inibitore dell’HK II 5-TG ha protetto le cellule dalla morte stabilizzando l’HK II sulla membrana mitocondriale esterna e diminuendo i livelli di ROS mitocondriali in assenza della DMPK. Src aveva la funzione di mantenere HK II sulla membrana mitocondriale esterna, in quanto la sua inibizione ha sensibilizzato le cellule al distacco dell’HK II solo se esprimevano la DMPK. Questo studio attribuisce un ruolo anti-apoptotico alla DMPK grazie all’interazione con HK II e la sua funzione protettiva contro i ROS di origine mitocondriale.

Le gène rb est le premier suppresseur de tumeur découvert chez l’homme. Il prévient l’apparition de tumeurs notamment en régulant négativement le cycle cellulaire. Le rôle de pRb dans le contrôle de l’apoptose est plus complexe et les mécanismes moléculaires contrôlés par ce facteur de transcription ne sont pas complétement élucidés. Il existe un homologue de rb chez la drosophile : rbf1. J’ai contribué à caractériser les évènements mitochondriaux induits au cours de l’activation de l’apoptose par Rbf1 dans le disque imaginal d'aile, un tissu en prolifération de la larve de drosophile. Dans cette voie d’apoptose, la protéine Debcl, seule membre pro-apoptotique de la famille Bcl-2 chez la drosophile, est activée et induit le recrutement et l’oligomérisation de Drp1, protéine effectrice principale de la fission mitochondriale. C’est ainsi qu’est déclenchée la fragmentation mitochondriale et l’accumulation d’espèces activées de l’oxygène (EAOs) mitochondriales. Ces deux évènements participent à la transmission du signal apoptotique. J’ai par ailleurs pu mettre en évidence l’implication de facteurs participant au maintien du contrôle qualité mitochondriale. Celui-ci s’assure de l’intégrité des mitochondries et, le cas échéant, déclenche la digestion des éléments défaillants par mitophagie. Enfin, j’ai contribué à l’étude des liens entre la traduction et l’apoptose induite par Rbf1. Dans cette étude, nous montrons que la poly-A binding protein (PABP) peut supprimer le phénotype d’encoche induit par Rbf1 chez l’adulte alors que la mort cellulaire induite au cours du stade larvaire n’est pas inhibée mais augmentée. Ces résultats nous ont poussé à étudier les mécanismes de compensation induits par l’appareil traductionnel, ce qui nous a permis de montrer qu'une modulation de la traduction pourrait permettre de compenser la perte de tissu consécutive à l'apoptose induite par Rbf1 sans impliquer une inhibition de l'apoptose
The gene rb is the first tumor suppressor discovered in humans. Its prevents the appearance of tumors by regulating negatively the cell cycle. The role of pRb in apoptosis is more complex and the molecular mechanisms triggered by this transcription factor are not completely elucidated. There is a rb homologue in drosophila: rbf1. I participated in the characterization of mitochondrial events induced during activation of apoptosis by Rbf1 in a proliferating tissue of this model organism, the wing disc. In this apoptosis pathway, the Debcl protein, the only drosophila pro-apoptotic member of the Bcl-2 family, is activated and induces recruitment and oligomerization of Drp1, the main effector of mitochondrial fission. This triggers the mitochondrial fragmentation and the accumulation of mitochondrial reactive oxygen species (ROS). Both events participate to the transmission of the apoptotic signal. I have also been able to highlight the implication of factors involved in maintaining mitochondrial quality control which ensures the integrity of the mitochondria and, if necessary, triggers the degradation of damaged elements by mitophagy. Finally, I have contributed to the study of the links between translation and apoptosis induced by Rbf1. In this study, we show that the Poly-A Binding Protein (PABP) can suppress the Rbf1-induced notch phenotype in adults while cell death induced during larval stage was not inhibited but increased. These results prompted us to study the compensation mechanisms induced by the translational apparatus, which allowed us to show that a mRNA translation-related mechanism could counteract the loss of tissue resulting from Rbf1-induced apoptosis independently of apoptosis inhibition

The GTPase activity of OPA1, a dynamin-related mitochondrial protein upregulated in several tumors, controls cristae remodeling, cytochrome c release and apoptosis. To pharmacologically target OPA1 in cancer, we setup and iterated a high-throughput screening of a diversity based chemical library of 10,000 drug-like small molecules for recombinant purified OPA1 GTPase activity inhibition, identifying 8 candidates that were confirmed in a secondary screen. The most promising hit (MYLS22) was highly specific, as it could bind to recombinant OPA1 GTPase and did not inhibit recombinant Dynamin 1 GTPase activity. MYLS22 was not mitochondriotoxic, but it increased OPA1 oligomers disassembly and cytochrome c release in response to the proapoptotic stimulus BID in purified mitochondria and to hydrogen peroxide in cells, where MYLS22 caused the expected mitochondrial fragmentation. MYLS22 also phenocopied the inhibition of breast cancer cells migration caused by OPA1 silencing. Thus, we identified a first-in-kind OPA1 inhibitor with potential anti-cancer properties.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Breast cancer is the most frequently diagnosed cancer among Canadian women. Despite the use of advanced therapeutics, breast cancer remains the second leading cause of cancer death among Canadian women. Therefore, the development of novel and effective therapeutics to treat breast cancer remains an important goal. Defective or inhibited apoptosis is a major causative factor in the development and progression of cancer. Zinc is considered an apoptotic regulator. Further, previous work has also shown that there is an association between zinc depletion-induced apoptosis and an elevated intracellular Ca²⁺ level in human breast cancer MDA-MB-231 cells. Ca²⁺ is a known mediator of the mitochondrial apoptotic pathway. The overall objective of my thesis research was to investigate the role of intracellular Ca²⁺ and its involvement of the mitochondria in zinc depletion-induced apoptosis in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were cultured in DMEM containing FBS (10%) followed by depletion of intracellular zinc using N,N,N’,N’-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN; 20 µM) with or without the presence of intracellular Ca²⁺ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 10 or 20 µM). Apoptosis was assessed by caspase-9 and -3 activities using corresponding fluorogenic substrates and the proportion of cells with fragmented DNA using PI-staining flow cytometry assay. Intracellular Ca²⁺ was assessed using Fura-2 assay. Mitochondrial membrane potential was assessed by DiOC₆-staining flow cytometry assay. Cytochrome c release was detected by Western blot. Addition of TPEN resulted in an increase of caspase-9 and -3 activities, an increase in the proportion of cells with fragmented DNA, and a prolonged increase in intracellular Ca²⁺ level. TPEN treatment also reduced mitochondrial membrane potential and induced cytochrome c release. Zinc replenishment (10 – 40 µM) prevented TPEN-induced apoptosis. Intracellular Ca²⁺ chelation with BAPTA-AM suppressed TPEN-induced apoptosis, mitochondrial membrane potential loss, and cytochrome c release. Collectively these results showed that zinc depletion-induced apoptosis was mediated through the Ca²⁺-dependent mitochondrial apoptotic pathway in human breast cancer MDA-MB-231 cells.

Chemoresistance is a highly significant problem affecting a diverse array of cancers at all clinical stages. In an attempt to identify molecular mechanisms leading to chemoresistance, we performed a RNAi screen against all known and putative kinases and phosphatases in the human genome. The knockdown of one of these genes, MK-STYX, resulted in potent chemoresistance in response to a diverse array of chemotherapeutic agents. As many of these drugs function through the induction of the apoptotic program, we hypothesized that the RNAi-mediated knockdown of MK-STYX blocks the cellular response to chemotherapeutic-induced apoptosis.

To investigate this hypothesis, we determined the ability of both control and MK-STYX knockdown cells to undergo apoptosis after exposure to an array of cell death inducing agents with different mechanisms of action. The results of these experiments demonstrated that MK-STYX knockdown protects against intrinsic, but not extrinsic apoptotic stimuli. These data were recapitulated with knockdown of the pro-apoptotic genes caspase-9 and Bax/Bak, suggesting that MK-STYX may modulate the regulation of one of these key apoptotic regulatory nodes. We demonstrated that the loss of MK-STYX blocks cytochrome c release, placing the apoptotic deficiency at the level of Bax/Bak-mediated mitochondrial outer membrane permeabilization, or MOMP. MK-STYX was found to localize to the mitochondria, but is neither released from the mitochondria upon apoptotic stress nor localized proximal to the machinery currently known to control MOMP. These results are summarized in Chapter 2.

In an effort to more fully define molecular mechanism of MK-STYX, we performed an unbiased TAP-tagging experiment to identify its interaction partners. The most significant and unique protein identified was the mitochondrial phosphatase PTPMT1. Interestingly, MK-STYX is a catalytically inactive dual specificity phosphatase, and catalytically inactive phosphatases have a precedent for regulating the activity and/or localization of active phosphatases. Because of this potential phosphatase regulatory mechanism, as well as similar localization patterns of both genes, we chose to further explore the interaction between PTPMT1 and MK-STYX.

Due to the robust survival phenotype seen in MK-STYX knockdown cells when treated with chemotherapeutic, we predicted that the knockdown of PTPMT1 may have a similar phenotype. Surprisingly, we found that PTPMT1 knockdown causes a Bax/Bak dependent cell death, suggesting that MK-STYX and PTPMT1 may functionally oppose one another in the mitochondria. Experiments in which both enzymes are downregulated show that PTPMT1 is epistatic to MK-STYX, as cells are resensitized to chemotherapeutic agents and cytochrome c release under these conditions. Interestingly, PTPMT1 was recently shown to be an important enzyme in the cardiolipin biosynthetic pathway, positively regulating the synthesis of this mitochondrial lipid. The genetic interaction provided by the robust changes in viability seen when these enzymes are downregulated suggests that MK-STYX may function to dampen PTPMT1 enzymatic activity. This allows us to hypothesize that the loss of MK-STYX results in increased cardiolipin biosynthesis, leading to altered mitochondrial membrane composition and subsequently, an altered apoptotic response. These results are summarized in Chapters 3 and 4.

We further hypothesize that the upregulation of cardiolipin levels directly inhibits the ability of Bax/Bak to permeabilize the outer mitochondrial membrane, effectively blocking the induction of mitochondrial apoptosis. These data suggest a novel mechanism by which dysregulated cardiolipin can facilitate chemoresistance, and suggest that this pathway could be exploited by recurrent cancers to evade therapies.

I mitocondri sono strutture intracellulari in grado di decodificare una varietà di stimoli extracellulari, che vanno dalla produzione di energia alla morte cellulare. Quando l'omeostasi del Ca2+ mitocondriale è compromessa, possono verificarsi condizioni patologiche molto diverse, dal cancro alle malattie neurodegenerative, a seconda del tipo di cellula e del pathway molecolare coinvolto. Durante il mio dottorato, ho cercato di fare luce sul meccanismo molecolare attraverso il quale PTRH2, una proteina mitocondriale, regola la sopravvivenza e la morte cellulare sia in neuroblastoma che nelle malattie neurodegenerative. PTRH2 è un protettore dell'apoptosi indotta da stress nelle cellule aderenti. L'attività pro-survival di PTRH2 è stata attribuita ai suoi effetti sui mitocondri, tuttavia il meccanismo d’azione resta ancora sconosciuto. I pazienti con una mutazione germinale omozigote PTRH2 (PTRH2DF) sviluppano una malattia neurologica multisistemica infantile caratterizzata da microcefalia postnatale, atrofia cerebellare progressiva e malattia neurodegenerativa (IMNPED). Le cellule con bassi livelli di espressione di PTRH2 o quelle da pazienti con PTRH2DF mostrano una maggiore sensibilità all'apoptosi indotta da stress, mentre le cellule con alti livelli di espressione di PTRH2 sono resistenti alla morte indotta da stimoli apoptotici. In questo progetto, ho dimostrato che un'elevata espressione di PTRH2 nei tumori dei pazienti con neuroblastoma (NB) è correlata a prognosi sfavorevole, con sviluppo di metastasi allo stadio 4, livelli di Ca2+mitocondriale bassi e resistenza al trattamento chemioterapico. Al contrario, cellule di NB che esprimono poco o mancano di PTRH2 mostrano un elevato Ca2+ mitocondriale e una risposta apoptotica migliorata al trattamento chemioterapico. In questo lavoro, dimostriamo che PTRH2 modula, interagendo con la deubiquitinasi Trabid, l'espressione proteica di mt-ND5, subunità del complesso I mitocondriale. Pertanto, PTRH2 stabilizza l'attività di mt-ND5 modulando i flussi di Ca2+ e i livelli di ATP nei mitocondri e proteggendo le cellule dallo stress indotto apoptosi. Proponiamo che PTRH2 sia un gatekeeper mitocondriale, per cui la perdita di PTRH2 provoca sovraccarico di Ca2+ mitocondriale e morte cellulare che porta a sviluppo di malattie neurodegenerative. Al contrario, un'elevata espressione di PTRH2 induce un basso assorbimento mitocondriale di Ca2+ promuovendo la resistenza alla morte cellulare e allo sviluppo del tumore. I nostri dati forniscono un razionale meccanicistico per la capacità del PTRH2 di regolare l'apoptosi indotta dai mitocondri e forniscono un nuovo punto di partenza per future indagini volte a identificare nuove terapie per e contro la morte cellulare mediata dai mitocondri.
Mitochondria are structures within the cells able to decode a variety of extracellular stimuli into greatly different intracellular actions, ranging from energy production to cell death. When mitochondrial Ca2+ homeostasis is compromised, very different pathological conditions can occur, from cancer to neurodegenerative diseases, depending on the cell type and pathway involved. During my Phd, I tried to shed light on the molecular mechanism by which PTRH2, a mitochondrial protein, regulates cell-survival and death both in cancer and in neurodegenerative disease. PTRH2 is a protector of stress-induced apoptosis in adherent cells. The pro-survival activity of PTRH2 has been attributed to its effects at the mitochondria by an unknown mechanism. Patients with an inherited homozygous germ line PTRH2 mutation (PTRH2DF) develop infantile multisystem neurologic disease characterized by postnatal microcephaly, progressive cerebellar atrophy and neurodegenerative disease (IMNPED). Cells with reduced PTRH2 levels or PTRH2DF patient cells exhibit increased sensitivity to stress-induced apoptosis whereas cells with high PTRH2 levels are resistant. In this project, I demonstrated that high PTRH2 expression in Neuroblastoma (NB) patient tumors correlates with poor prognosis, stage 4 metastasis, low mitochondrial Ca2+ and resistance to treatment. NB cells with reduced PTRH2 and PTRH2DF cells display high mitochondrial Ca2+ and enhanced apoptotic response to treatment. In this work, we demonstrate that PTRH2 modulate, by interacting with deubiquitinase Trabid, protein expression of mt-ND5 of mitochondrial complex I. Thus, PTRH2 stabilizes MT-ND5 activity modulating Ca2+ fluxes and ATP levels in mitochondria and protecting cells from stress-induced apoptosis. We propose that PTRH2 is a mitochondrial gatekeeper whereby loss of PTRH2 results in mitochondrial Ca2+ overload and cell death leading to neurodegenerative disease. High PTRH2 expression induces low mitochondrial Ca2+ uptake promoting resistance to cell death and tumor development. Our data provide a mechanistic rationale for the ability of PTRH2 to regulate mitochondrial-induced apoptosis and provide a new starting point for future investigations aimed at identifying new therapies for and against mitochondrial-mediated cell death.

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Faculty of Medicine, Division of Cancer Sciences and Molecular Pathology, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

The Bcl-2 family of proteins regulates the mitochondrial apoptotic pathway and consists of both pro- and anti-apoptotic members. Bax-cc is present in the cytosol of healthy cells. Apoptotic stimuli induce the translocation of Bax-oc to the mitochondria leading to the permeabilisation of the outer mitochondrial membrane (OMM) and the release of downstream pro-apoptotic proteins from the intermembrane space (IMS). The mechanism by which Bax permeabilises the OMM remains unclear. Recent evidence suggests Bax alone might be sufficient to permeabilise the OMM. However, other models indicate the involvement of the mitochondrial permeability transition pore complex or the voltage-dependent anion channel (VDAC) present in the OMM. In this study Bax and C-terminally truncated Bax were expressed as GST-fusion proteins and were immobilised on agarose-GSH. The binding of mitochondrial membrane proteins that might be involved in the Bax mediated release of proteins from the IMS was investigated. The results showed that VDAC and the adenine nucleotide translocase (ANT) were retained by GST-Bax. Exogenous cyclophilin D (Cyp D) was added in the presence of VDAC and ANT and was also retained indicating that Bax interacts with the components of the permeability transition pore complex. The anti-apoptotic Bcl-2 protein Bcl-XL was expressed with a hexahistidine tag at its N-terminus. This was used to investigate its effects on Bax interaction with VDAC and ANT. The ANT ligands atractyloside and bongkrekic acid which promote and inhibit apoptosis respectively were shown to change the relative amounts of VDAC and ANT that bind GST-Bax. Apoptotic cell death has been identified in cardiomyocytes subjected to ischaemia. In this investigation cardiomyocytes transfected with GFP-Bax were treated with cyanide to simulate ischaemia and GFP-Bax translocation was observed using fluorescence microscopy. GFP-Bax co-immunoprecipitated with VDAC and ANT after translocation to mitochondria but not with Cyp D. The implications of these findings are discussed in this thesis.

The apoptotic function of Apoptosis-inducing factor (AIF) is well documented in theliterature, but its physiological role in the mitochondrion is less certain. Using a smallinterfering RNA (siRNA) strategy, we studied whether modulation of AIF expression incultured cells influenced the production of reactive oxygen species (ROS). We foundthat siAIF-transfected cells had reduced AIF protein levels and this was paralleled by asignificant increase in ROS. We tested the generality of this response by using twodifferent human cell lines, the hepatoma cell line Hep3B and cervix carcinoma lineHeLa, and also by employing a mouse ES AIF-KO cell line. The increased ROS weremitochondrial in origin as a similar silencing strategy in cells devoid of a functioningmitochondrial electron transport chain (ETC) did not result in a ROS-increase. Theaugmented ROS levels were sufficient to activate Hypoxia-inducible factor 1α (HIF-1α),a ROS-sensitive transcription factor, and this effect could be reversed usingantioxidants, both the broad-range general antioxidant (N-acetyl cysteine) and aspecific mitochondrial-targeted antioxidant (MitoQ), proving the implication of ROS inthe HIF-1α stabilization. We also studied another two redox-sensitive transcriptionfactor and thus observed up-regulation in the expression of Nuclear factor (erythroidderived2)-like 2 (Nrf2), however without major changes in Nuclear factor-kappa B(NF-κB) levels. Examination of the cellular oxygen consumption rate revealed that AIFdepletedcells had a major impairment of respiration, at Complex I in the ETC. Westernblot analysis also showed a loss of Complex I 39 and 20 kDa subunits. Studies usingthe antioxidants mentioned above, revealed that the respiratory competence could beregained in AIF-silenced cells. However, neither of the antioxidant treatments we usedcould recover Complex I assembly. Studies of the energetic state of siAIF cells showedthat despite a 30% decrease in the overall intact cell respiration, these cells maintainnormal basal levels of ATP, presumably due to a higher glycolytic capacity and a lowerproliferation rate. Moreover, we analyzed the expression of another redox-activeprotein, thioredoxin, by Western blot and found that the mitochondrial isoform, Trx2,was significantly decreased when AIF was silenced. Preliminary co-immunoprecipitationanalyses and proteomic studies failed to show any direct correlation between AIF andTrx2 at the protein level.Our results lead us to the conclusion that the defect in respiration in siAIF cells isdownstream of Complex I protein loss and is presumably due to ROS-mediateddamage to the ETC. This suggests an integral mitochondrial function of AIF, as a redoxmodifier and chaperone-like molecule, necessary for Complex I assembly. Additionalstudies are required to define the detailed mechanism of the AIF enzymatic activity inthe mitochondrion and to establish its binding partners.
La función proapoptótica del Factor Inductor de Apoptosis (AIF) está biendocumentada, sin embargo su papel fisiológico en la mitocondria es menos conocido.Empleando la metodología de interferencia por ARN, estudiamos si la modulación de laexpresión proteica de AIF en cultivo celular modifica la producción celular de especiesreactivas de oxígeno (ROS). Observamos que el silenciamiento de AIF estaba seguidopor un incremento significativo en los niveles de las ROS. Estas ROS fueronmitocondriales de origen, puesto que el silenciamiento de AIF en células que carecende la cadena de transporte electrónico funcional (ETC) en la mitocondria no llevó a unincremento de ROS. Este incremento fue suficiente para activar el Factor inducible porhipoxia (HIF-1α), efecto que se puede revertir usando los antioxidantes, N-AcetilCisteina y MitoQ, demostrando así la implicación de los ROS en la estabilización deHIF1-α. Los análisis del consumo de oxigeno celular mostraron que las células de AIFsilenciado sufren una disminución en la respiración celular, al nivel del Complejo I de laETC, acompañada por una disminución significativa en la expresión de sus subunidades39 y la 20kDa. Tratamientos con los antioxidantes previamente nombrados mostraronque la tasa de respiración se puede recuperar, no siendo así con la expresión delComplejo I de la ETC. Estudios del estado energético de las células siAIF mostraronque a pesar de la disminución de 30% en la tasa de la respiración celular, estas célulasmantienen niveles normales de ATP, como resultado de un incremento en la capacidadglucolítica y una reducción en la tasa de proliferación. Posteriormente, analizamos laexpresión de la proteína tioredoxina y observamos una disminución significativa en laisoforma mitocondrial, la tioredoxina 2 (Trx2), aunque los análisis preliminares de coinmunoprecipitacióny proteómica no mostraron la existencia de una correlacióndirecta entre las proteínas AIF y Trx2.Concluyendo, nuestros resultados sugieren que el defecto de la respiración celular esposterior al defecto en el Complejo I, probablemente como consecuencia al daño de laETC por ROS. Esta observación apunta a un papel integrador de AIF en la mitocondria,como modulador del estatus redox y necesario para el ensamblaje del Complejo I.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality. The majority of patients present with advanced disease where platinum based systemic therapy forms the mainstay of treatment but is ) associated with high failure rates. Defective apoptosis underpins drug resistance. Mitochondrial apoptosis is regulated by the BCL-2 family proteins, BAX and BAK. Loss of these proteins confers multidrug apoptosis resistance, however their requirement for the action of cisplatin is unclear. Altered expression of BAX and BAK is frequent in NSCLC. Results presented show that BAX and BAK gene expression is positively correlated suggesting BAX and BAK double negativity is a common phenotype in NSClC. BAXBAK double negativity was modelled in the H460 and H1299 NSCLC cell lines. Unexpectedly, in H460 shBAXBAK models we demonstrate redundancy of core apoptosis signalling in relation to cisplatin through its ability to activate the death receptor pathway. This pathway has de novo block in H1299 cells such that H1299 shBAXBAK cells fail to induce apoptosis in response to cisplatin. This pathway is abrogated in cisplatin ) resistance, whereas sensitivity to death receptor agonism is conserved. Synthetic lethality, through targeting BRCA1/2 mutated tumours with PARP inhibitors, is a promising therapeutic strategy. Decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. Results presented demonstrate that silencing of BRCA 1 expression sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block. Furthermore, we demonstrate that BRCA 1 inhibition cannot override platinum resistance, but can still sensitize to PARP inhibition. 11-19% of NSCLC patients have absent BRCA1 protein expression. This molecular subgroup could be effectively targeted by PARP inhibitors. As a result of this work, the PIN study (PARP inhibitors in NSCLC) has been funded to assess the effectiveness of this therapy.

Breast Cancer is one of the major causes of mortality among women in the world. During normal tissue development, cell growth is controlled by a mechanism of cell death called apoptosis. However, during cancer, the balance between cell division & apoptosis is altered, leading to cell survival, cell proliferation and tumour formation. The nuclear receptor Farnesoid X Receptor (FXR) is expressed in human breast cancer tissue and the breast cancer cell lines MCF-7 and MDA-MB-468. In these cells, activators of FXR caused apoptosis, but the exact mechanism of how FXR regulates apoptosis in breast cancer is unknown. The aim of this research is to elucidate the mechanism of FXR-mediated apoptosis using human breast cancer cell lines MCF-7 and MDA-MB-231. Cell viability after treatment with FXR agonists chenodeoxycholic acid (CDCA) & GW4064 was measured by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The effect of staurosporine was also examined as a positive control for induction of apoptosis. All three chemicals significantly decreased the viability of MCF-7 and MDA-MB-231 cells in a concentration dependent manner, with IC50 values determined as 35µM, 245µM and 6µM for GW4064, CDCA and staurosporine, respectively for MCF-7 cells and 22µM, 440µM and 4µM for GW4064, CDCA and staurosporine, respectively for MDA-MB-231 cells. The difference in efficacy of the two FXR agonists corresponds to differing potency as GW4064 is a high affinity synthetic ligand for FXR, whereas CDCA is an endogenous bile acid that has much lower affinity for FXR. FXR expression was confirmed in both MCF-7 and MDA-MB-231 cell lines by Western blotting. Poly-ADP ribose polymerase (PARP) cleavage is the classical marker for apoptosis. Cleaved PARP, indicative of active caspase-3, was observed in MCF-7 and MDA-MB-231 cells exposed to staurosporine (positive control for apoptosis) or the FXR agonists GW4064 and CDCA. Luminescent analysis for caspases: 3/7, 9 and 8 were measured for MCF-7 and MDA-MB-231 cells treated with staurosporine (positive control for apoptosis), FXR agonists GW4064 and CDCA. Further research focused more specifically on the mechanism of apoptosis. Proteins involved in apoptotic pathway such as BAX, Bcl-2, and cytochrome c were studied through Western blotting performed on MCF-7 and MDA-MB-231 cell lines treated with staurosporine and FXR agonists. Reactive oxygen species were studied on MCF-7 and MDA-MB-231 cell lines treated with FXR agonists, measured using ROS luminescence assay. The results showed that FXR agonists GW4064 and CDCA induced intrinsic apoptotic pathway via ROS activation in both MCF-7 and MDA-MB-231 cell lines. Chemotherapy has been extensively used to treat patients with breast cancer. Tamoxifen (Tam) has been in use for over 30 years to treat patients. However, due to its toxicity and resistance, there is constant need for new therapeutic alternatives. In this study, the interaction between tam and FXR agonists was studied using MTT and clonogenic assay. The results showed a synergistic interaction of GW4064 with tam in both MCF-7 and MDA-MB-231 cell line. However, the mechanism needs further investigation. In summary, FXR agonists induce mitochondrial dependent apoptosis in breast cancer cells. Also GW4064 synergises with tam and reduces cell proliferation in MCF-7 and MDA-MB-231 irrespective of their estrogen status.

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Mitochondrial injury and apoptosis promote organ failure after ischemic acute kidney injury (AKI), a common cause of morbidity and mortality. In these studies, we propose that hexokinase (HK), mitofusin 2 (MFN2) and Bax, key mitochondrial associated proteins, modulate apoptotic cell death and organ function after ischemia. In the kidney, HKI and HKII isoforms both possess mitochondrial localization sequences. In vivo ischemia reduced murine proximal tubule HKII content and caused mitochondrial HKII dissociation. In cultured renal epithelial cells, expression of HKI or II significantly improved survival after ATP depletion, an in vitro model of ischemia, without preventing Bax activation or reducing mitochondrial fragmentation, a determinant of organelle sensitivity to injury. HKII over-expression increased mitochondrial associated HKII during stress and decreased mitochondrial Bax accumulation, a major cause of outer membrane permeabilization and apoptosis, suggesting that HK improves renal cell survival by antagonizing Sax-mediated injury. Deficiency of MFN2, a pro-fusion protein, caused mitochondrial fragmentation in primary proximal tubule cells without altering baseline or maximal oxygen consumption rate, or cell apoptosis. However, MFN2 deficiency significantly increased mitochondrial Bax accumulation and exacerbated mitochondrial outer membrane injury after stress. In the mouse, whole kidney MFN2 knockout caused severe mitochondrial fragmentation in renal epithelial cells. However, despite a small (20%) decrease in nephron number compared to littermate controls, newborn knockouts exhibited normal tubular and organ function. Surprisingly, proximal tubule specific MFN2 knockouts were also protected from renal ischemia. Although histologic injury scores as well as levels of apoptosis and necrosis, were similar, renal function and animal survival were significantly higher in proximal tubule specific MFN2 knockout mice at 24 and 48 hours post-ischemia. Interestingly, cortical oxidant stress was halved while cortical proliferation was nearly 4 times higher in proximal tubule knockouts compared to control, suggesting that MFN2 deficiency promotes organ recovery and survival after ischemia by enhancing proximal epithelial cell growth. While HK and MFN2 modulate Bax-mediated mitochondrial injury and apoptosis, "off-target" effects of MFN2 on renal cell proliferation ameliorate ischemia-reperfusion injury. These studies highlight the role of Bax-mediated mitochondrial injury in ischemic organ failure and suggest new targets for both attenuating injury and promoting organ recovery.

Strap is a DNA damage responsive p53 cofactor, reflecting its control by the DNA damage signalling pathway. This study identified Strap at the mitochondria where it is damage regulated and can augment p53-dependent cytochrome c release leading to apoptosis. Moreover, p53 and Strap facilitate each other’s localisation from the mitochondria to the nucleus during the DNA damage response. Two ATM/ATR phosphorylation consensus sites in Strap were identified by mass spectrometry and phosphorylation of all the ATM/ATR consensus sites resulted in mitochondrial localisation of Strap during DNA damage. Targeting Strap to the mitochondria depletes cellular ATP when cells favour energy production through oxidative phosphorylation and sensitises cells to p53-dependent damage-induced apoptosis. These results thus imply that Strap co-ordinates different arms of the p53 response, and might be responsible for integrating its mitochondrial and nuclear functions.

Ce travail a porté sur une nouvelle protéine impliquée dans l’apoptose, la mitogaligine, et plus particulièrement sur le fragment interne [31-53] responsable de son adressage à la mitochondrie. L’objectif général du projet est de comprendre au niveau atomique son mécanisme d’action sur les membranes mitochondriales. Le fragment d’adressage est à lui seul cytotoxique. C’est pourquoi j’ai concentré l’essentiel de mon travail de thèse sur son étude. Nous avons défini sa toxicité sur des cellules humaines et montré qu’il perturbait l’intégrité membranaire, excluant certaines protéines de la mitochondrie. Ce phénomène concorde avec le relargage de cytochrome c, à l’origine du déclenchement de l’apoptose par la voie mitochondriale. Pour mieux comprendre le mode d’action du fragment d’adressage et le rôle joué par la cardiolipine, lipide caractéristique des membranes mitochondriales, j’ai étudié par différentes techniques biophysiques complémentaires l’effet du milieu membranaire sur la structuration du peptide et l’effet du peptide sur la membrane. Le peptide a une très forte affinité (13nM) pour des membranes contenant de la cardiolipine. Il se place à plat sur la membrane, s’enfouissant dans l’interface, sans induire d’organisation particulière des lipides. De plus, nous avons mis en évidence que le peptide était capable d’induire une courbure positive de la membrane, ce qui va interférer avec de nombreux processus vitaux pour la cellule. Enfin, pour réaliser les études structurales et fonctionnelles de la protéine entière, j’ai participé aux premières étapes de production de mitogaligine, qui s’est avéré très délicate aussi bien par voie d’expression que par synthèse chimique
This work is about a new protein of apoptosis, mitogaligin, and more particularly about the internal fragment [31-53] responsible for its mitochondrial targeting. General aim of the project is to understand at the atomic scale its mechanism of action on mitochondrial membranes. The addressing fragment is cytotoxic by itself. That is the reason why I focused the main part of my work on this peptide. We defined its toxicity on human cells and showed that it was capable of disrupting the membrane integrity, excluding some proteins from mitochondrion. This phenomenon agrees with the release of cytochrom c, which induces apoptosis by the mitochondrial pathway. In order to better understand the mode of action of the addressing fragment and the role played by Cardiolipin, a specific lipid of mitochondrial membranes, I studied by various and complementary biophysics techniques the effect of membrane environments on the peptide structuration and the effect of the peptide on the membrane. The peptide has a very high affinity (13nM) for cardiolipin-containing membranes. It takes place parallel to the membrane, standing at the interface, without leading to a particular lipids organization. Furthermore, we highlighted that the peptide was capable of inducing a positive curvature of the membrane, what is going to interfere with numerous vital processes for the cell. Finally, to realize the structural and functional studies of the whole protein, I was involved in the first stages of mitogaligin’s production, which has proved to be very tricky either by recombinant pathway or by chemical synthesis

The upregulation of apoptosis is a hallmark of several neurodegenerative disorders including ischemic stroke. In neurons, as in other cell types, Bax and tBid are critical regulators of the intrinsic pathway upstream of mitochondrial outer membrane permeabilization (MOMP) and caspase activation. The characterization of the molecular events that occur during the early stages is therefore extremely important from a therapeutic standpoint. Here I show that two independent genetic pilot screens looking for novel regulators of Bax activation identified a common hit in the E3 ubiquitin ligase Trim17. Knockdown of Trim17 was found to protect against tBid-induced death in primary cortical neurons and allowed for the maintenance of mitochondrial function and oxidative phosphorylation under this apoptotic stress. The RING-domain of Trim17 was found to interact with Opa1 in mouse brain extracts. Furthermore, Opa1 co-immunoprecipitated with exogenously expressed full-length Trim17 from HEK293 cells. Knockdown of Trim17 in neurons increased Opa1 protein levels under steady-state conditions. These results suggest that Trim17 regulates Bax-dependent apoptosis in neurons via the modulation of Opa1 levels.

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

Gain- and loss-of-function studies of the BCL-2 family of proteins have shown that they can impact chemotherapeutic sensitivity. However, cells contain myriad anti-apoptotic and pro-apoptotic BCL-2 family members making it difficult to predict cell fate decisions based on the initial conditions of these proteins. BH3 profiling is a tool that measures mitochondrial priming, the readiness of a cell to die through the intrinsic (or mitochondrial) apoptotic pathway. Priming is due to the cumulative effect of the BCL-2 family of proteins that act as the gate keepers of the mitochondrial apoptotic pathway. Priming is measured by determining the sensitivity of mitochondria to perturbation by peptides derived from the BH3 domains of pro-apoptotic proteins. Using BH3 profiling, we now have a functional readout that can quantify priming and assess its contribution to drug sensitivity. Here we show that priming affects the sensitivity of acute myeloid leukemia (AML) cell lines to various standard chemotherapeutics, especially topoisomerase II inhibitors. Priming predicts clinical response to conventional induction chemotherapy as well as the long term maintenance of remission in AML patients. Interestingly, the priming of normal hematopoietic stem cells (HSCs) sits at the boundary line between the priming of cured and refractory patient AML. This HSC priming likely defines the therapeutic index since AML that are lower primed than HSCs are often refractory and cannot be cured without transplantation. Additionally, our BH3 profiles revealed that AML cells are more sensitive to BCL-2 antagonism than normal HSCs, which are primarily dependent on MCL-1. Indeed, we were able to kill primary refractory AML cells in vitro with the BCL-2 antagonist ABT-737 at doses that left HSCs unharmed. Cumulatively, these findings show that priming is a major mechanistic determinant of AML response in vitro and in the clinic to standard induction chemotherapy. With the ability to predict outcome, BH3 profiling may offer physicians and patients a promising tool for treatment decision-making.

Colon 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.

Grb10 is a member of the Grb7 family of adaptor proteins that also includes Grb7 and Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of Grb10 to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as well as many non-receptor molecules such as MEK1, Raf-1, and Nedd4. Grb10 has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-1 and the mitochondrial membrane.
In this report the pattern of transient Grb10 translocation following IGF-I cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new Grb10 binding partners.

Apoptosis is a crucial process in multicellular organisms in sculpting them, especially during embryogenesis. In addition, apoptosis is responsible for the clearance of harmful or damaged cells which can otherwise be detrimental to the organism. The Bcl-2 family proteins are key players in the regulation of the intrinsic pathway of the apoptotic machinery. This family consists of three subfamilies with B-cell CLL/lymphoma 2 (Bcl-2) protein itself representing anti-apoptotic members, the Bcl-2-associated X protein (Bax), and pro-apoptotic BH3-only signaling proteins. The interplay between pro- and anti-apoptotic proteins on the mitochondrial membranes is central to the balance between the life and death decision of whether the membrane should be permeabilized or not. The cytosolic Bax protein can upon cellular stress translocate to the mitochondrial membrane where it can either carry out its action of forming homo-oligomers that cause outer membrane permeabilization or be inhibited there by the anti-apoptotic membrane protein Bcl-2. Upon mitochondrial outer membrane permeabilization (MOMP) apoptogenic factors leak out from the intermembrane space (IMS) of the mitochondria, leading to caspase activation and ultimately cell death. A common stress signal initiating apoptosis is an increased formation of reactive oxygen species (ROS in the mitochondria, who can cause oxidative damage to lipid membranes. This membrane damage presumably influences the lipid landscape and the membrane features and hence the interactions of the Bcl-2 family proteins with each other and the mitochondrial outer membrane (MOM). To investigate the significance of membrane oxidation on the behavior of the Bcl-2 family proteins, especially Bax, synthetically produced oxidized phospholipids (OxPls) were incorporated in MOM-mimicking vesicles. Differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectroscopy revealed a major perturbation in membrane organization in the presence of OxPls. These changes in membrane properties increase the affinity of Bax to its target membrane and enable its partial penetration and formation of pores, as fluorescence leakage assays confirmed. However, in the absence of BH3-only proteins these pores are not sufficiently large for the release of apopototic factors such as cytochrome C (CytC). To understand the inhibition of Bax by the full-length Bcl-2 protein, suitable detergent solubilizing conditions were carefully chosen to enable the measurement of their direct binding to each other outside the membrane, by an antimycin A2 fluorescence assay. The observed protein-protein interaction was confirmed by surface plasmon resonance (SPR). An established protocol for the reconstitution of Bcl-2 into stable proteoliposomes now paves the way for structural studies of this key protein, in its membrane environment near physiological conditions; information essential for understanding its function, on a molecular level, and its potential as a cancer drug target.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress. Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex. Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences