Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase.

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded FO domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS175N) prevents FO-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors (aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20-30 Å) from the original mutation. The possibility to compensate through long-range effects the aS175N mutation is an interesting observation that holds promise for the development of therapeutic molecules.

Overexpression of mitochondrial oxodicarboxylate carrier (ODC1) preserves oxidative phosphorylation in a yeast model of Barth syndrome.

Cardiolipin (CL) is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS), presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ) of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1Δ yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS.

Yeast models of mutations in the mitochondrial ATP6 gene found in human cancer cells.

Since the discovery of somatic mtDNA mutations in tumor cells, multiple studies have focused on establishing a causal relationship between those changes and alterations in energy metabolism, a hallmark of cancer cells. Yet the consequences of these mutations on mitochondrial function remain largely unknown. In this study, Saccharomyces cerevisiae has been used as a model to investigate the functional consequences of four cancer-associated missense mutations (8914C>A, 8932C>T, 8953A>G, 9131T>C) found in the mitochondrial MT-ATP6 gene. This gene encodes the a-subunit of F1FO-ATP synthase, which catalyzes the last steps of ATP production in mitochondria. Although the four studied mutations affected well-conserved residues of the a-subunit, only one of them (8932C>T) had a significant impact on mitochondrial function, due to a less efficient incorporation of the a-subunit into ATP synthase. Our findings indicate that these ATP6 genetic variants found in human tumors are neutral mitochondrial genome substitutions with a limited, if any, impact on the energetic function of mitochondria.

N52 monodeamidated Bcl­xL shows impaired oncogenic properties in vivo and in vitro.

Bcl-xL is a member of the Bcl-2 family, playing a critical role in the survival of tumor cells. Here, we show that Bcl-xL oncogenic function can be uncoupled from its anti-apoptotic activity when it is regulated by the post-translational deamidation of its Asn52.Bcl-xL activity can be regulated by post-translational modifications: deamidation of Asn52 and 66 into Asp residues was reported to occur exclusively in response to DNA damage, and to cripple its anti-apoptotic activity. Our work reports for the first time the spontaneous occurrence of monodeamidated Asp52Bcl-xL in control conditions, in vivo and in vitro. In the normal and cancer cell lines tested, no less than 30% and up to 56% of Bcl-xL was singly deamidated on Asn52. Functional analyses revealed that singly deamidated Bcl-xL retains anti-apoptotic functions, and exhibits enhanced autophagic activity while harboring impaired clonogenic and tumorigenic properties compared to native Bcl-xL. Additionally, Asp52Bcl-xL remains phosphorylatable, and thus is still an eligible target of anti-neoplasic agents. Altogether our results complement the existing data on Bcl-xL deamidation: they challenge the common acceptance that Asn52 and Asn66 are equally eligible for deamidation, and provide a valuable improvement of our knowledge on the regulation of Bcl-xLoncogenic functions by deamidation.

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C.

Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase. Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production. An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C.

The energetic state of mitochondria modulates complex III biogenesis through the ATP-dependent activity of Bcs1.

Our understanding of the mechanisms involved in mitochondrial biogenesis has continuously expanded during the last decades, yet little is known about how they are modulated to optimize the functioning of mitochondria. Here, we show that mutations in the ATP binding domain of Bcs1, a chaperone involved in the assembly of complex III, can be rescued by mutations that decrease the ATP hydrolytic activity of the ATP synthase. Our results reveal a Bcs1-mediated control loop in which the biogenesis of complex III is modulated by the energy-transducing activity of mitochondria. Although ATP is well known as a regulator of a number of cellular activities, we show here that ATP can be also used to modulate the biogenesis of an enzyme by controlling a specific chaperone involved in its assembly. Our study further highlights the intramitochondrial adenine nucleotide pool as a potential target for the treatment of Bcs1-based disorders.

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells.

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-ß estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.

Complexomics study of two Helicobacter pylori strains of two pathological origins: potential targets for vaccine development and new insight in bacteria metabolism.

Helicobacter pylori infection plays a causal role in the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma (LG-MALT) and duodenal ulcer (DU). Although many virulence factors have been associated with DU, many questions remain unanswered regarding the evolution of the infection toward this exceptional event, LG-MALT. The present study describes and compares the complexome of two H. pylori strains, strain J99 associated with DU and strain B38 associated with LG-MALT, using the two-dimensional blue native/SDS-PAGE method. It was possible to identify 90 different complexes (49 and 41 in the B38 and J99 strains, respectively); 12 of these complexes were common to both strains (seven and five in the membrane and cytoplasm, respectively), reflecting the variability of H. pylori strains. The 44 membrane complexes included numerous outer membrane proteins, such as the major adhesins BabA and SabA retrieved from a complex in the B38 strain, and also proteins from the hor family rarely studied. BabA and BabB adhesins were found to interact independently with HopM/N in the B38 and J99 strains, respectively. The 46 cytosolic complexes essentially comprised proteins involved in H. pylori physiology. Some orphan proteins were retrieved from heterooligomeric complexes, and a function could be proposed for a number of them via the identification of their partners, such as JHP0119, which may be involved in the flagellar function. Overall, this study gave new insights into the membrane and cytoplasm structure, and those which could help in the design of molecules for vaccine and/or antimicrobial agent development are highlighted.

Effects of the endocrine disruptors atrazine and PCB 153 on the protein expression of MCF-7 human cells.

Polychlorinated biphenyls (PCBs) and a number of pesticides can act as endocrine disrupting compounds (EDCs). These molecules exhibit hormonal activity in vivo, and can therefore interact and perturb normal physiological functions. Many of these compounds are persistent in the environment, and their bioaccumulation may constitute a significant threat for human health. Physiological abnormalities following exposure to these xenobiotic compounds go along with alterations at the protein level of individual cells. In this study, MCF-7 cells were exposed to environmentally relevant concentrations of atrazine, PCB153 (100 ppb, respectively), 17-beta estradiol (positive control, 10 nM) and a negative control (solvent) for t = 24 h (n = 3 replicates/exposure group). After trizol extraction and protein solubilization, protein expression levels were studied by 2D-DIGE. Proteins differentially expressed were excised, trypsin-digested, and identified by MALDI-ToF-ToF, followed by NCBInr database search. 2D-DIGE experiments demonstrated that 49 spots corresponding to 29 proteins were significantly differentially expressed in MCF-7 cells (>1.5-fold, P < 0.05, Student's paired t test). These proteins belonged to various cellular compartments (nucleus, cytosol, membrane), and varied in function; 88% of proteins were down-regulated during atrazine exposure, whereas 75% of proteins were up-regulated by PCB153. Affected proteins included those regulating oxidative stress such as superoxide dismutase and structural proteins such as actin or tropomyosin, which may explain morphological changes of cells already observed under the microscope. This study highlights the susceptibility of human cells to compounds with endocrine disrupting properties.

Cancer immunomics: from serological proteome analysis to multiple affinity protein profiling.

Cancer remains one of the leading causes of death worldwide. Thus, to identify any useful biomarkers is still a need. We performed "cancer immunomics" to identify autoantibody signatures produced in response to the presence of either breast or colorectal cancer. SERological proteome analysis (SERPA) was performed by two-dimensional (2-D) electrophoresis separation, immunoblotting, image analysis, and mass spectrometry. Alternatively, to identify the antigens recognized by the autoantibodies of cancer patients, we developed an approach combining 2-D immunoaffinity chromatography, enzymatic digestion of the isolated antigens, nano flow separation of the resulting peptides, and identification: MAPPing (multiple affinity protein profiling). By these approaches we identified both proteins recognized by autoantibodies independently of a cancer status, and a limited number of proteins reacting preferentially with cancer sera.

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling.

Patients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures.

Proteomic Analysis of the MCF7 Breast Cancer Cell Line.

BACKGROUND: The MCF7 breast cancer cell line is a cellular model for breast cancer studies and marker discovery. Therefore, a better knowledge of its proteome is a prerequisite for a more efficient use of this model. MATERIALS AND METHODS: Proteins expressed during the exponential growth phase of MCF7 cells were analyzed and mapped using two-dimensional gel electrophoresis and mass spectrometry. RESULTS: From the spots excised from preparative gels of whole-cell extracts, a subset of 368 different polypeptides, corresponding to 249 different proteins, was identified. These polypeptides were positioned on a silver-stained gel to construct a reference map. CONCLUSION: The data allowed the construction of the most extensive reference map for MCF7 published to date, with 189 novel proteins, which had not been previously listed on maps, and are now accessible on World 2D-PAGE database, providing a basis for further studies on MCF7.

Absence of accessory subunit in the DNA polymerase gamma purified from yeast mitochondria.

In yeast and animals, replication of the mitochondrial genome is carried out by the DNA polymerase gamma. In mammals this polymerase is composed of a catalytic and an accessory subunit. Yeast DNA polymerase gamma was purified over 6600-fold from mitochondria. The catalytic polypeptide of this enzyme was identified as a 135-kDa protein by a photochemical crosslinking procedure and its native molecular weight was estimated between 120 and 140 kDa by gel filtration and glycerol gradient sedimentation. These results indicate that yeast DNA polymerase gamma contains only one subunit and thus has a different quaternary structure from its counterpart in animals.