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Metabolic Switching, March 2012
Edited by James Murray, PhD
Thousands of researchers around the world are studying the connection between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlight how Abcam MitoSciences research tools have contribute to this effort. The full list of 20 original research papers published this month using MitoSciences range of products can be found here.
Past issues are available for review in the archives.
Table of Contents
I. Metabolic Switching
II. Mitochondrial Disease
III. OXPHOS, Supercomplexes and the Respirasome
I. Metabolic Switching
Metabolic changes are known to occur in some cancers. For example, normal glucose oxidation in the cytoplasm with subsequent mitochondrial ATP generation by the TCA cycle and oxidative phosphorylation, is replaced with ATP and lactate generation by a less efficient glycolytic process performed only in the cytoplasm, without mitochondrial involvement. Developing views of this, the Warburg effect, propose that mitochondria are not damaged, but instead cancer cells benefit by generating ATP rapidly by glycolysis and also repurpose their mitochondria to provide increased TCA cycle intermediates for the biosynthesis of lipids, proteins and nucleic acids required for accelerated growth and cell division. As a consequence of these changes, the mitochondrion and the cell become less sensitive to apoptosis by mechanisms that remain unclear. Therapies that return a cancer cell's metabolism to an oxidative state have the potential to limit its capacity to grow and divide while also restoring sensitivity to apoptosis.
Pyruvate dehydrogenase kinases (PDK) are potentially druggable target enzymes in the metabolic pathway, since phosphorylation of the mitochondrial pyruvate dehydrogenase complex by these kinases prevents the metabolism of pyruvate and entry of acetyl CoA entry into the TCA cycle. Dichloroacetate (DCA) is a small molecule inhibitor of PDH kinases and therefore has the potential to return metabolism to an oxidative state, restore apoptotic sensitivity and may be the basis for future anticancer agents if toxicity concerns can be addressed. In their recent paper, Xue et al. showed that a common chemo-therapeutic drug, cisplatin, coupled to two DCA moieties and termed ‘Mitaplatin' was able to kill previously cisplatin-insensitive cells. These researchers showed that, after intracellular reduction, the DCA moieties specifically inhibited the kinases by decreasing phosphorylation of PDH at all three regulatory sites. This was shown using a PDH phosphoserine in-cell ELISA approach.
The effect of this was to reduce glucose utilization, reduce mitochondrial membrane polarization, and increase the sensitivity of the cells to cisplatin-induced apoptosis. The dual action approach described may allow the development of more successful anti-cancer drugs that are effective at lower concentrations and more easily tolerated.
Mitaplatin increases sensitivity of tumor cells to Cisplatin by inducing mitochondrial dysfunction. Xue X, You S, Zhang Q, Wu Y, Zou GZ, Wang PC, Zhao YL, Xu Y, Jia L, Zhang X, Liang XJ.
A glycolytic switch was also recently observed in a model of mitochondrial genetic disease by Desquiret-Dumas et al. Mutations may be present in only a fraction of the copies of mitochondrial DNA in a cell, a phenomenon known as heteroplasmy. The authors of this work created cybrid cells from neuroblastoma SH-SY5Y cells harboring approximately 70% and 100% loads of the mtDNA mutation 3243A>G, a common cause of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). These authors observed that the 100% mutant cells had significant reduction in mitochondrial OXPHOS subunits and assembled Complexes I / IV and determined by Western blotting and blue native electrophoresis, but with an accompanying increase in glycolytic metabolism to ensure normal cellular ATP levels. Switching to a low glucose media decreased the mutant load to 90% and significantly increased the assembly and activity of Complexes I and IV. Culture media was also supplemented with the NO-donor L-arginine to compensate for decreased NO synthase activity in cybrids. This lead to an increase in Complex I activity in 100% mutant load cybrids, however the mechanism remains to be studied. Using these two approaches to affect metabolism and restore mitochondrial function may provide a promising approach to therapy in MELAS and other mitochondrial conditions.
Metabolically induced heteroplasmy shifting and l-arginine treatment reduce the energetic defect in a neuronal-like model of MELAS. Desquiret-Dumas V, Gueguen N, Barth M, Chevrollier A, Hancock S, Wallace DC, Amati-Bonneau P, Henrion D, Bonneau D, Reynier P, Procaccio V.
Also this month:
Glucose-modulated mitochondria adaptation in tumor cells: a focus on ATP synthase and inhibitor factor 1. Domenis R, Bisetto E, Rossi D, Comelli M, Mavelli I.
Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS. Chen V, Staub RE, Fong S, Tagliaferri M, Cohen I, Shtivelman E.
Mitochondrial biogenesis and increased uncoupling protein 1 in brown adipose tissue of mice fed a ketone ester diet. Srivastava S, Kashiwaya Y, King MT, Baxa U, Tam J, Niu G, Chen X, Clarke K, Veech RL.
II. Mitochondrial Disease
Next generation sequencing (NGS) promises significant advances in the identification of disease causing mutations. Future applications of sequencing may include routine genome wide screening to identify causal pathogenic alleles in patients, as distinct from the variants that exist between all individuals. In a study this month by Calvo et al., NGS was designed for the 'MitoExome' a collection of genes including the mitochondrial DNA plus the 1034 genes thought to encode the mitochondrial proteome. Samples were analyzed from 42 unrelated patients exhibiting clinical and biochemical deficiency consistent with mitochondrial oxidative phosphorylation disease that had been prescreened for common mutations. These researchers found only 24% (10/42) of cases were due to mutations in known disease causing loci, and 31% (13/42) harbored new, rare mutations not previously linked to OXPHOS disease and were determined as likely causal. Support for one of these genes, CI - NDUFB3, as pathogenic was provided by deficiency in specific CI Western blotting and Complex I/Complex IV activity dipstick analyses. Enzyme activity was rescued by protein expression complementation.
The authors conclude that approximately 50% of all cases will remain a challenge to interpretation from sequencing data until more disease causing alleles are identified, more data is available on variations within the general population, and reduced cost permits the complete genome sequencing of patient and parents. Furthermore, interpretation of sequence variants will continue to be most valuable when combined with clinical and biochemical data.
Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing. Calvo SE, Compton AG, Hershman SG, Lim SC, Lieber DS, Tucker EJ, Laskowski A, Garone C, Liu S, Jaffe DB, Christodoulou J, Fletcher JM, Bruno DL, Goldblatt J, Dimauro S, Thorburn DR, Mootha VK.
Also this month:
A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome. Assouline Z, Jambou M, Rio M, Bole-Feysot C, de Lonlay P, Barnerias C, Desguerre I, Bonnemains C, Guillermet C, Steffan J, Munnich A, Bonnefont JP, Rötig A, Lebre AS.
Mitochondrial complex III stabilizes complex I in the absence of NDUFS4 to provide partial activity. Calvaruso MA, Willems P, van den Brand M, Valsecchi F, Kruse S, Palmiter R, Smeitink J, Nijtmans L.
Combined OXPHOS complex I and IV defect, due to mutated complex I assembly factor C20ORF7. Saada A, Edvardson S, Shaag A, Chung WK, Segel R, Miller C, Jalas C, Elpeleg O.
III. OXPHOS, Supercomplexes and the Respirasome
As all Mitochondriacs know, the oxidative phosphorylation system (OXPHOS) is comprised of the respiratory chain (Complexes I-IV) and the ATP synthase (Complex V) within the inner mitochondrial membrane. Electron transfer by the respiratory chain is coupled to proton gradient generation across the inner membrane. The proton gradient is dissipated through the ATP synthase and generates ATP by a rotational catalytic mechanism. With so many component complexes transferring high energy intermediates it is then not surprising that the system is organized into supercomplexes which form the respirasome. This assembly may confer increased stability, enhancement of transfer efficiency and the decrease of electron and proton leakage.
A description of the biosynthetic pathway of the respirasome was proposed in a paper this month by Moreno-Lastres et al. The authors used an antibiotic to inhibit mitochondrial protein translation and deplete OXPHOS assembly. Upon removal of the antibiotic, supercomplex formation could occur, which was followed by blue-native electrophoresis and blotting using a host of anti-Complex I, II, III and IV antibodies. They found that CIII and CIV assemble onto the scaffold of an 830 kDa intermediate of Complex I. The assembly factor NDUFAF2 likely completes the assembly of Complex I by inserting the subunits NDUFV1/NDUFS4 and therefore could be considered a supercomplex assembly factor. These findings explain why CI activity is often reduced in CIV patient cell lines but reduced CI levels do not usually lead to CIII or CIV assembly and functional defects.
Mitochondrial complex I plays an essential role in human respirasome assembly. Moreno-Lastres D, Fontanesi F, García-Consuegra I, Martín MA, Arenas J, Barrientos A, Ugalde C.
Also this month:
Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV. Diaz F, Enríquez JA, Moraes CT.
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