ATP synthase | EPFL Graph Search

ATP synthase is a protein that catalyzes the formation of the energy storage molecule adenosine triphosphate (ATP) using adenosine diphosphate (ADP) and inorganic phosphate (Pi). ATP synthase is a molecular machine. The overall reaction catalyzed by ATP synthase is:

ADP + Pi + 2H+out ATP + H2O + 2H+in

ATP synthase lies across a cellular membrane and forms an aperture that protons can cross from areas of high concentration to areas of low concentration, imparting energy for the synthesis of ATP. This electrochemical gradient is generated by the electron transport chain and allows cells to store energy in ATP for later use. In prokaryotic cells ATP synthase lies across the plasma membrane, while in eukaryotic cells it lies across the inner mitochondrial membrane. Organisms capable of photosynthesis also have ATP synthase across the thylakoid membrane, which in plants is located in the chloroplast and in cyanobacteria is located in the cytoplasm. Eukaryotic ATP synthases are F-ATP

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

Tuberculosis is an ancient human disease affecting millions of people every year. The common causative agent, Mycobacterium tuberculosis, is a peculiar pathogen that can persist in the host tissues for decades. Many of the key attributes of M. tuberculosis can be traced to its metabolism. Therefore, novel insights into the metabolism of this bacterium could lead to the identification of new drug targets as well as better methods to understand the mechanism of action of anti-tuberculosis drugs. Analysis of the metabolic network of this bacterium using a genome-scale model offers an unconventional approach to understanding utilization of metabolic pathways of this pathogen during growth and non-replicating persistence. Metabolic adaptation of a pathogen is central to its life style in vivo. To understand the capacity and limitations of M. tuberculosis, we developed an updated and thermodynamically curated genome scale model – iOS783. This model has been verified with respect to the observed phenotypes for targeted gene deletion studies reported previously. Evaluation of iOS783 with thermodynamics based flux balance analysis (TFBA) improved overall reliability of the model results and led to identification of possible bottleneck reactions. In addition, several new hypotheses were generated with iOS783, involving the role of isocitrate lyase enzymes, propionate toxicity, and energy metabolism of this pathogen. Analysis of iOS783 for conditional gene essentiality emphasizes the importance of the carbon source available to the pathogen. Several genes show a conditional impact on biomass production rate of the model, depending on the availability of carbohydrates or fatty acids as the carbon substrate. Conditional essentiality is even more pronounced when ATP replenishment capacity is used as a measure of a pathogen’s ability to survive under stress in a non-growing state. As expected, ATP synthase plays a major role in estimations of ATP replenishment capacity based on iOS783. Curiously, computational analysis of the metabolic network with ATP synthase activity blocked indicates that the damaged network can use substrate-level phosphorylation reactions to sustain ATP production to fulfill non-growth-associated ATP requirements when glucose is available. In order to establish the dependence of M. tuberculosis on ATP synthase, we attempted to construct a deletion of the chromosomal atp operon. Our results show that the native copy of these genes can be deleted only when a second set of genes are introduced elsewhere in the genome, indicating essentiality of the ATP synthase for growth. Although our results indicate that ATP synthase is essential for growth, we observed carbon source specific efficacy of an ATP synthase inhibitor, bedaquiline, on M. tuberculosis. These experimental results are consistent with ATP turnover estimations based on modeling results. Furthermore, analysis of iOS783 suggests a novel role in energy metabolism for recently described glycine dehydrogenase (GDH) on energy metabolism. Deletion of ald gene responsible for GDH activity reduces the ATP replenishment capacity of iOS783, especially during metabolism of fatty acid derivative carbon sources. Consistent with these modeling results, wetlab experiments designed to test this hypothesis show that bedaquiline has a stronger bactericidal effect on an M. tuberculosis ∆ald mutant, compared to the wild-type parental strain, when cells are cultured in medium containing acetate as the carbon source. Metabolic modelling is already well established as an invaluable tool for engineering of strains with desired characteristics. For pathogenic species, however, the application of this approach has so far been quite limited. The work presented in this thesis exemplifies an interdisciplinary approach combining computational modeling and experimental studies of M. tuberculosis metabolism. First, iOS783 was utilized to interpret existing experimental results and to generate novel hypotheses, thereby providing the impetus for new wetlab experiments. Conversely, results from iOS783-inspired wetlab experiments were used to further refine and expand the iOS783 model. This cyclic exchange between computational modeling and wetlab experimentation illustrates how a close interdisciplinary partnership can create a positive feedback loop that enriches both disciplines.

EPFL2013

Quantifying and Localizing the Mitochondrial Proteome Across Five Tissues in A Mouse Population

Ruedi Aebersold, Johan Auwerx, Moaraj Hasan, Pooja Jha, Jun Yong Kim, Jiayi Lan, Dongryeol Ryu, Evan Graehl Williams

We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles. Key functional differences among the 3648 genes were also observed across tissues, with electron transport chain (ETC) genes particularly investigated. ETC complex proteins covary and form strong gene networks according to tissue, but their equivalent transcripts do not. Certain physiological consequences, such as the depletion of ATP synthase in BAT, are thus obscured in transcript data. Lastly, we compared the quantitative proteomic measurements between the total cell and mitochondrial fractions for the five tissues. The resulting enrichment score highlighted several hundred proteins which were strongly enriched in mitochondria, which included several dozen proteins were not reported in literature to be mitochondrially localized. Four of these candidates were selected for biochemical validation, where we found MTAP, SOAT2, and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane. These findings demonstrate the synergies of a multi-omics approach to study complex metabolic processes, and this provides a resource for further discovery and analysis of proteoforms, modified proteins, and protein localization.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC2018

Delayed bactericidal response of Mycobacterium tuberculosis to bedaquiline involves remodelling of bacterial metabolism

Neeraj Dhar, Ping Lu, John McKinney, Muhittin Emre Özdemir

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.