All tags Metabolism MitoNews, Volume 10, Issue 3

MitoNews, Volume 10, Issue 3

Mitochondria, Epigenetics and Cell cycle regulation

Edited by James Murray, PhD.

Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's MitoSciences range of research tools has contributed to this effort. Read the full list of 42 original research papers published in recent months.

Gene expression patterns are not only brought about by differences in DNA sequence, but also by changes in histone modification and DNA methylation in a process known as epigenetic regulation.  These changes may be induced spontaneously, as a consequence of specific mutations, or induced by environmental factors and the metabolic state of the cell. 

In a recent paper published in Cell, Sutendra, et al. demonstrated that a partial or intact mitochondrial pyruvate dehydrogenase complex (PDC) translocates to the nucleus in order to generate a separate pool of acetyl CoA for histone acetylation.  The increase in histone acetylation promotes entry of the cell into S phase by relaxing the wound nucleic acid allowing DNA replication.   Therefore migration to the nucleus of an inhibitor resistant variant of the PDC may be an important aspect of epigenetic regulation and DNA replication, as well as a key element in the uncontrolled cell division observed in carcinogenesis.

First, using z-stacked images in confocal microscopy, the presence of the PDC-E1a subunit was clearly identified in the nucleus in a number of available human cell lines.  A distinct population of PDC outside the nucleus was also observed and determined to be the canonical mitochondrial population by co-localization with a mitochondrial stain.    

Nuclear staining of PDC-E1a showed a similar staining pattern to histone H3 by confocal microscopy and electron microscopy, validating the authenticity of the nuclear localization.  Additionally, all four subunits of the PDC, specifically E1a, E1b, E2E2/E3bp, were observed by western blotting (WB) of highly pure, nuclear preparations lacking mitochondrial and non-mitochondrial contaminants (citrate synthase, isocitrate dehydrogenase 2, lamin, succinyl CoA synthetase and succinate dehydrogenase). 

PDC-E1a was knocked down by siRNA resulting in the loss of both WB signal and PDC activity, measured using a PDH-complex activity dipstick assay. PDC in siRNA scrambled control was unaffected. Nuclei treated with 13C2-pyruvate generated a significant quantity of 13C2-acetyl CoA in the scrambled siRNA control versus the targeted E1a knockdown. Next, histone acetylation was synchronized by glucose deprivation in PDC-E1a control and knockdown siRNA cells. After addition of pyruvate, an increased pattern of acetylated histones was observed in the scrambled control, which did not occur in the E1a knockdown cell line. These experiments indicate that a functional PDC is necessary for nuclear PDH activity, acetyl CoA generation and histone modification. 

PDC activity is down-regulated by phosphorylation at three serine residues in the E1a subunit. The primary or master regulator is phosphorylation of serine 293. Four kinases and two phosphatases exist in tissue specific expression patterns to regulate the phosphorylation of the E1a subunit. The authors found the presence of a small amount of PDC phosphatase I in the nucleus of A549 cells but not the corresponding kinases. Finally, the phosphorylated E1a subunit was not found in the nucleus, indicating that the nuclear PDC might be constitutively active. 

In cell-cycle synchronized cultures after serum stimulation, levels of nuclear PDC and acetylated histone H3 lysine 9 increased and decreased in concert as the cell progressed through the cell cycle. Nuclear PDC increased as mitochondrial PDC decreased, while total cellular levels remained the same. This suggests that PDC is migrating from mitochondria into the nucleus. Inhibition of new protein translation in the ER did not affect this pattern of PDC localization, supporting the notion that the PDC is not newly synthesized but translocating within the cell. 

Using knockdown experiments, the authors confirmed that mitochondrial proteases are essential for correct pre-sequence processing of the PDC found in the nucleus.  Therefore the PDC subunits are processed in the mitochondrion before being exported to the nucleus.  Next knockdown of only one PDC subunit significantly reduced levels of all other nuclear PDC components, both findings indicate that the subunits do not arrive at the nucleus independently, but are more likely assembled and exported to the nucleus as a complex. 

Epidermal growth factor (an S-phase regulator) and rotenone (a mitochondrial Complex I inhibitor and stressor) both increased translocation of PDC to the nucleus. Mitochondrial stress also increased the amount of heat shock proteins such as HSP70.  Both PDC-E1 and PDC-E2 were shown to co-immunoprecipitate with HSP70. A binding site was proposed for HSP70 in the PDC that may share the same location as the PDC kinase binding site, suggesting that HSP70 binding may compete with kinase binding to maintain PDC activity. 

In summary, the authors provide a compelling case for PDC translocating from the mitochondria to the nucleus promoted by the chaperone HSP70 in order to autonomously generate acetyl CoA and regulate the cell cycle. Furthermore, nuclear PDC activity is regulated differently and does not exhibit the same level of activity as the mitochondrial enzyme within the same cells. The authors propose that, in cancer, inactive mitochondria and inhibited PDC prevent apoptosis and cell death, while a simultaneously active nuclear PDC promotes cell cycle progression and division. These two phenomena may be important features of the dysregulated cell division seen in cancer. These interactions, regulations and translocations may provide opportunities for therapeutic intervention in cancer and will certainly augment our understanding of the interconnected nature of cellular organelles, metabolism and nuclear regulation. 

A Nuclear Pyruvate Dehydrogenase Complex Is Important for the Generation of Acetyl-CoA and Histone Acetylation. Cell. 2014. Sutendra G, Kinnaird A, Dromparis P, Paulin R, Stenson TH, Haromy A, Hashimoto K, Zhang N3 Flaim E and Michelakis ED.

 See also

c-Myc Programs Fatty Acid Metabolism and Dictates Acetyl-CoA Abundance and Fate. J. Bio Chem. 2014. Edmunds LR, Sharma L, Kang A, Lu J1, Vockley J, Basu S, Uppala R, Goetzman ES, Beck ME, Scott D, Prochownik EV.

Epigenetic therapy for Friedreich ataxia. Ann Neurol. 2014. Soragni E, Miao W, Iudicello M, Jacoby D, De Mercanti S, Clerico M, Longo F, Piga A, Ku S, Campau E, Du J, Penalver P, Rai M, Madara JC, Nazor K, O'Connor M, Maximov A, Loring JF, Pandolfo M, Durelli L, Gottesfeld JM, Rusche JR.

Frataxin silencing inactivates mitochondrial Complex I in NSC34 motoneuronal cells and alters glutathione homeostasis. Int J Mol Sci. 2014. Carletti B, Piermarini E, Tozzi G, Travaglini L, Torraco A, Pastore A, Sparaco M, Petrillo S, Carrozzo R, Bertini E, Piemonte F.

Sirt3 regulates metabolic flexibility of skeletal muscle through reversible enzymatic deacetylation. Diabetes. 2014. Jing E, O'Neill BT, Rardin MJ, Kleinridders A, Ilkeyeva OR, Ussar S, Bain JR, Lee KY, Verdin EM, Newgard CB, Gibson BW, Kahn CR.

Structure-guided Development of Specific Pyruvate Dehydrogenase Kinase Inhibitors Targeting the ATP-binding Pocket. J. Bio Chem. 2014. Tso SC, Qi X, Gui WJ, Wu CY, Chuang JL, Wernstedt-Asterholm I, Morlock LK, Owens KR, Scherer PE, Williams NS, Tambar UK, Wynn RM, Chuang DT.

Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice. PLOSOne, 2014. Turunen MP, Husso T, Musthafa H, Laidinen S, Dragneva G, Laham-Karam N, Honkanen S, Paakinaho A, Laakkonen JP, Gao E, Vihinen-Ranta M, Liimatainen T, Ylä-Herttuala S.