Search

Mitochondria are dynamic organelles that exchange a multiplicity of signals with other cell compartments, in order to finely adjust key biological routines to the fluctuating metabolic needs of the cell. During neoplastic transformation, cells must provide an adequate supply of the anabolic building blocks required to meet a relentless proliferation pressure. This can occur in conditions of inconstant blood perfusion leading to variations in oxygen and nutrient levels. Mitochondria afford the bioenergetic plasticity that allows tumor cells to adapt and thrive in this ever changing and often unfavorable environment. Here we analyse how mitochondria orchestrate the profound metabolic rewiring required for neoplastic growth.

Drosophila Bet1 and Slh, needed for vesicle trafficking at the Golgi, are required also for mitochondrial quality control. When they are deficient, a signature of bioenergetic dysfunction and intramitochondrial proteotoxic stress results and cells enter a senescencelike state. Slh-deficient cells lose coassociation of mitochondria and lysosomes with Bet1., The Drosophila gene products Bet1, Slh, and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 cooperate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential, and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, cofractionated with highly purified mitochondria, while live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This three-way association was disrupted in cells knocked down for Slh, although colocalized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 was required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24, or Syntaxin6. Our findings implicate vesicle sorting at the cis-Golgi in mitochondrial quality control.

Mutations in neurofibromin, a Ras GTPase-activating protein, lead to the tumor predisposition syndrome neurofibromatosis type 1. Here, we report that cells lacking neurofibromin exhibit enhanced glycolysis and decreased respiration in a Ras/ERK-dependent way. In the mitochondrial matrix of neurofibromin-deficient cells, a fraction of active ERK1/2 associates with succinate dehydrogenase (SDH) and TRAP1, a chaperone that promotes the accumulation of the oncometabolite succinate by inhibiting SDH. ERK1/2 enhances both formation of this multimeric complex and SDH inhibition. ERK1/2 kinase activity is favored by the interaction with TRAP1, and TRAP1 is, in turn, phosphorylated in an ERK1/2-dependent way. TRAP1 silencing or mutagenesis at the serine residues targeted by ERK1/2 abrogates tumorigenicity, a phenotype that is reverted by addition of a cell-permeable succinate analog. Our findings reveal that Ras/ERK signaling controls the metabolic changes orchestrated by TRAP1 that have a key role in tumor growth and are a promising target for anti-neoplastic strategies.

SummaryProliferating cells require coordinated gene expression between the nucleus and mitochondria in order to divide, ensuring sufficient organelle number in daughter cells [1]. However, the machinery and mechanisms whereby proliferating cells monitor mitochondria and coordinate organelle biosynthesis remain poorly understood. Antibiotics inhibiting mitochondrial translation have emerged as therapeutics for human cancers because they block cell proliferation [2, 3]. These proliferative defects were attributable to modest decreases in mitochondrial respiration [3, 4], even though tumors are mainly glycolytic [5] and mitochondrial respiratory chain function appears to play a minor role in cell proliferation in vivo [6]. Here we challenge this interpretation by demonstrating that one class of antiproliferative antibiotic induces stalled mitochondrial ribosomes, which triggers a mitochondrial ribosome and RNA decay pathway. Rescue of the stalled mitochondrial ribosomes initiates a retrograde signaling response to block cell proliferation and occurs prior to any loss of mitochondrial respiration. The loss of respiratory chain function is simply a downstream effect of impaired mitochondrial translation and not the antiproliferative signal. This mitochondrial ribosome quality-control pathway is actively monitored in cells and constitutes an important organelle checkpoint for cell division.

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an "emergency shutdown" system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.

Made available in DSpace on 2018-11-26T16:18:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-17 Academy of Finland (CoE grant) European Research Council EU (Marie Curie International Incoming Fellowship) Tampere University Hospital Medical Research Fund Sigrid Juselius Foundation The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression. Tampere Univ, BioMediTech, FI-33014 Tampere, Finland Tampere Univ, Tampere Univ Hosp, FI-33014 Tampere, Finland Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 Jaboticabal, SP, Brazil Univ Helsinki, Inst Biotechnol, FI-00014 Helsinki, Finland INSERM, UMR 1141, F-75019 Paris, France Univ Paris 07, Fac Med Denis Diderot, Hop Robert Debre, F-75019 Paris, France Univ Estadual Paulista, Fac Ciencias Agr & Vet, Dept Tecnol, BR-14884900 Jaboticabal, SP, Brazil Academy of Finland (CoE grant): 272376 European Research Council: 232738 EU (Marie Curie International Incoming Fellowship): 328988

In developing rat brain cytochrome c oxidase subunit IV (COXIV) expression is also regulated at post-transcriptional level and two 3′UTR-COXIV RNA-binding factors have been identified. Here, we report the enrichment and identification of the factors from just born rat brains by affinity chromatography of biotinylated 3′UTR-COXIV RNA–protein complexes on streptavidin-conjugated paramagnetic particles. We successfully isolated two main proteins of about 86 and 42 kDa, whose sequences were highly attributable to Hsp90 and Actin. The purified proteins maintain RNA-binding ability and specificity for COXIV messenger and, interacting with the 3′UTR, then could negatively modulate mRNA translation. We also studied the content of Hsp90 and Actin during postnatal brain development and demonstrated that in just born rat brain, when the COXIV protein appears at low level, Hsp90 was not phosphorylated. Vice versa in the adult tissue, when COXIV accumulates, Hsp90 appeared phosphorylated in serine therefore it could be unable to bind COXIV messenger, suggesting that the phosphorylation event could provoke the loss of Hsp90 binding to mRNA. We hypothesize a new post-transcriptional mechanism regulating a messenger encoded by nuclear genome for a mitochondrial protein and that Hsp90 and Actin, could represent key players in COXIV translation.

The heavy metal cadmium (Cd) a pollutant associated with several modern industrial processes, is absorbed in significant quantities from cigarette smoke, water, food and air contaminations. It is known to have numerous undesirable effects on health in both experimental animals and humans, targeting kidney, liver and vascular system. The molecular mechanism accounting for most of the biological effects of Cd are not well-understood and the toxicity targets are largely unidentified. The present review focuses on important recent advances about the effects of cadmium on mitochondria of mammalian cells. Mitochondria are the proverbial powerhouses of the cell, running the fundamental biochemical processes that produce energy from nutrients using oxygen. They are among the key intracellular targets for different stressors including Cd. This review provides new additional informations on the cellular and molecular aspects of the interaction between Cd and cells, emphasizing alterations of mitochondria as important events in Cd cytotoxicity, thus representing an important basis for understanding the mechanisms of cadmium effect on the cells.

Cytochrome c oxidase (COX) complex is built up with both nucleus- and mitochondrion-encoded subunits. Biogenesis and assembly of the complex thus requires fine cross-talk between the two compartments. In order to shed light on the regulation of nuclear–mitochondrial interactions, we studied the expression of COXIII (mitochondrion-encoded) and COXIV (nucleus-encoded) in adult rat tissues and rat developing brain. We found that the levels of COXIV protein and mRNA are not linearly related, thus suggesting a post-transcriptional mode of regulation. In agreement with this observation, we report the presence of a protein that specifically binds to the 3′-untranslated region of COXIV mRNA. This factor, that forms with RNA a complex of about 60 kDa, is present both in the cytoplasm and mitochondria, where its concentration decreases throughout development with inverse correlation with COXIV accumulation. Interestingly, using an antibody raised in our laboratory, we found that, in developing rat brain, COXIII does not localize exclusively to mitochondria, but is also present in the cytosol, where it could exert a yet unknown regulatory role.

Cadmium–breast epithelial cell interactions were studied by analyzing some mitochondria-related aspects of stress response. We treated immortalized non-tumor breast cells with 5 or 50 μM CdCl2 for 24 or 96 h demonstrating that the exposure did not cause a significant mitochondrial proliferation, while it induced a significant increase in the respiratory activity and mitochondrial polarization. In addition, we found that hsp60 was up-regulated while hsp70 and COXII and COXIV were down-regulated. The mRNA for hsp70 remained constant and only the inducible form of the 70-kDa heat shock protein was over expressed. The mRNAs for COXII and COXIV remained constant after 24 h and increased after longer incubations while the respective proteins decreased. These findings provide additional information on the cellular and molecular aspects of the interaction between Cd and epithelial cells, and on alterations of mitochondria as early events in Cd cytotoxicity.

It was reported that cadmium is able to exert a cytotoxic effect on tumor MDA-MB231 cells, which shows signs of "non-classical" apoptosis and is characterized by drastic changes in gene expression pattern. In this study, we have extended our knowledge of metal-breast cancer cell interactions by analyzing some mitochondria-related aspects of the stress response to CdCl(2) at either 5 or 50 microM 24- or 96-h exposure, by cytochemical, conventional PCR and Northern/Western blot techniques. We demonstrated that (i) no modification of the mitochondrial mass was detectable due to CdCl(2) exposure; (ii) the respiration activity appeared to be increased after 96-h exposures, while the production of reactive oxygen species was significantly induced, as well; (iii) hsp60, hsp70, COXII and COXIV expressions were dependent on the duration of Cd exposure; (iv) a different hsp60 protein distribution was observed in mitochondrial and post-mitochondrial extracts and (v) 96-h exposure induced the over-expression of hsc/hsp70 proteins and, conversely, the down-regulation of cytochrome oxidase subunits II and IV. These observations, in addition to providing more information on the cellular and molecular aspects of the interaction between CdCl(2) and MDA-MB231 breast tumor cells, contribute to the comprehension of the intracellular molecular mechanisms implicated in the regulation of some mitochondrial proteins.

The biogenesis of mitochondria depends on the coordinated expression of nuclear and mitochondrial genomes. Consequently, the control of mitochondrial biogenesis and function depends on extremely complex processes requiring a variety of well orchestrated regulatory mechanisms. It is clear that the interplay of transcription factors and coactivators contributes to the expression of both nuclear and mitochondrial respiratory genes. In addition, the regulation of mitochondria biogenesis depends on proteins that, interacting with messenger RNAs for mitochondrial proteins, influence their metabolism and expression. Moreover, a tight regulation of the import and final assembly of mitochondrial protein is essential to endow mitochondria with functional complexes. These studies represent the basis for understanding the mechanisms involved in the nucleus–mitochondrion communication, a cross-talk essential for the cell.

Introduction Diabetes Associated Protein in Insulin-sensitive Tissues (DAPIT) is a subunit of mitochondrial ATP synthase and has also been found to associate with the vacuolar H+-ATPase. Its expression is particularly high in cells with elevated aerobic metabolism and in epithelial cells that actively transport nutrients and ions. Deletion of DAPIT is known to induce loss of mitochondrial ATP synthase but the effects of its over-expression are obscure. Results In order to study the consequences of high expression of DAPIT, we constructed a transgenic cell line that constitutively expressed DAPIT in human embryonal kidney cells, HEK293T. Enhanced DAPIT expression decreased mtDNA content and mitochondrial mass, and saturated respiratory chain by decreasing H+-ATP synthase activity. DAPIT over-expression also increased mitochondrial membrane potential and superoxide level, and translocated the transcription factors hypoxia inducible factor 1α (Hif1α) and β-catenin to the nucleus. Accordingly, cells over-expressing DAPIT used more glucose and generated a larger amount of lactate compared to control cells. Interestingly, these changes were associated with an epithelial to mesenchymal (EMT)-like transition by changing E-cadherin to N-cadherin and up-regulating several key junction/adhesion proteins. At physiological level, DAPIT over-expression slowed down cell growth by G1 arrest and migration, and enhanced cell detachment. Several cancers also showed an increase in genomic copy number of Usmg5 (gene encoding DAPIT), thereby providing strong correlative evidence for DAPIT possibly having oncogenic function in cancers. Conclusions DAPIT over-expression thus appears to modulate mitochondrial functions and alter cellular regulations, promote anaerobic metabolism and induce EMT-like transition. We propose that DAPIT over-expression couples the changes in mitochondrial metabolism to physiological and pathophysiological regulations, and suggest it could play a critical role in H+-ATP synthase dysfunctions. Public Library of Science open access