Prof. Kalervo Hiltunen, M.D., Ph.D.
Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu
A long-standing problem in the biology of mammalian peroxisomes is the role of their membrane as a permeability barrier to metabolic intermediates. Studies have identified an ATP transporter and the three human peroxisomal half-ABC transporters are considered to participate in peroxisomal lipid transport. Our contribution to solving the puzzle on material exchange between the cytosol and peroxisomes is identification of the peroxisomal membrane protein Pxmp2 as a channel allowing transmembrane traffic for molecules less 500 Da.
Among the recently recognized features of mitochondrial function is their ability to synthesize fatty acids in an acyl carrier protein (ACP)-dependent manner. Failure of mitochondrial fatty acid synthesis (mtFAS) in yeast leads to loss of mitochondrial respiratory function and incapability of growing on non-fermentable carbon sources, and to defective mitochondrial RNA processing. A striking finding is that mtFAS is also operational in mammals, and there is also mounting evidence pointing to indispensable functions of mtFAS for the well-being of mammals. This notion is in line with observations that disruption of mtFAS results in embryonic lethality in mice.
The enzyme α-methylacyl-CoA racemase (Amacr) catalyses interconversion of the R and S forms of the 2-methyl groups of various 2-methylacyl-CoA molecules, a reaction required for degradation of methyl-branched fatty acids and other isoprenoid-derived compounds such as cholesterol and its metabolites. Patients with Amacr deficiency have been described. Amacr has also recently been identified as a tissue biomarker of prostate and colon cancer tissues.
More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes – ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. Our work on yeast Pex11 revealed an unexpected novel function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membranes. The amino acid sequence shows sequence similarity to transient receptor potential (TRP) cation-selective channels. In vivo, detection of the rate of β-oxidation revealed involvement of the Pex11 channel in transmembrane transfer of metabolites and in regulation of peroxisomal metabolic processes. As a whole, the data show that Pex11 is a multipurpose protein performing distinct functions in peroxisome biogenesis and metabolism.
Based on sequence similarity between Pxmp2 and other members of the family we suggest that all these proteins are transmembrane channels. In line with this hypothesis, we turned to human mitochondrial Mpv17p and tested its function as a potential channel-forming protein. Mutations in human Mpv17 cause hepatocerebral mitochondrial DNA (mtDNA) depletion syndrome (MDDS) that progresses at an early age and is characterized by developmental delay, sensory and motor neuropathy, and metabolic abnormalities. Mice with deleted Mpv17 show signs of premature aging: grey coat early in adulthood, high blood pressure, glomerulosclerosis, sensorineural deafness, depletion of mtDNA and a decrease in mitochondrial cytochromes. We expressed human mitochondrial Mpv17p in yeast (Pichia pastoris) cells, and the data obtained with the purified recombinant protein indicated that Mpv17 forms a non-selective channel with a pore size of 1.8 nm and we located the channel’s selective filter. Voltage-dependent gating of the channel was found to be regulated by redox conditions and pH. The results suggest that the function of the channel is modulation of membrane potential to preserve homeostasis in mitochondria and to conduct quality control of these organelles.
In contrast to humans, Amarc-deficient mice are clinically symptomless on a standard laboratory diet, but fail to thrive when fed phytol-enriched chow. We have studied the effect and the mechanism behind the phytol-feeding-associated disease state in Amarc-deficient mice. All Amarc−/− mice died within 36 weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death, accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. A phytol diet for two weeks in this study resulted in 60-fold elevation of phytanic and pristanic acid in the liver tissue of Amacr−/− mice. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared with a control diet. This indicates that detoxification of phytol metabolites in the liver is accompanied by activation of multiple pathways at the molecular level and Amacr−/− mice are not able to respond adequately. The results of this study show that the primary physiological function of Amacr is detoxification of α-methyl branched chain fatty acids. A secondary role of Amacr is in the bile acid synthesis pathway, which can be compensated for by alternative pathways when Amacr activity is missing. The findings in this study also suggest that strict dietary intervention, avoiding phytol and its metabolites, might restrain the emergence of pathological symptoms in patients suffering from Amacr deficiency.
Several mouse models to investigate mtFAS deficiency have been established and a priority of our work is characterization of our mouse mtFAS dysfunction model. Complete knockout of the Mecr enoyl reductase gene in mice causes early embryonic lethality. We have replaced this gene with a construct encoding a mitochondrially localized bacterial enoyl reductase. Embryos carrying this transgene survive two days longer than complete KO lines, and are currently under study to determine the effects of this construct on the development of the embryo, mitochondrial markers and respiration. Data collection on mouse lines with tissue-specific deletions (forebrain and cerebellum) is ongoing. A second goal is characterization and possibly reconstruction of the mitochondrial lipoylation pathway in yeast to help our understanding of this process.
A large number of studies have demonstrated the significance of polyunsaturated fatty acids (PUFAs) for human health. In spite of intensive international research, the molecular mechanisms translating PUFA sensing into changes in gene expression have remained largely enigmatic. As one approach to shed light on the responses of animals to PUFAs we have recently generated a mouse line defective in mitochondrial dienoyl-CoA reductase (Decr), which is a key enzyme required for mitochondrial breakdown of PUFAs. These mice developed severe hypoglycemia, as do many other animal models of fatty acid oxidation disorders during metabolic stress. In contrast to other models, where hypoglycemia is associated with hypoketonemia, absence of Decr activity did not alter the ketogenic response to fasting. We will expand our investigation of the molecular mechanisms leading to the physiological consequences of Decr deletion in mice.
Antonenkov VD, Isomursu A, Mennerich D, Vapola MH, Weiher H, Kietzmann T, Hiltunen JK. The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential. J Biol Chem 290(22):13840-61, 2015.
Jokinen R, Lahtinen T, Marttinen P, Myöhänen M, Ruotsalainen P, Yeung N, Shvetsova A., Kastaniotis AJ, Hiltunen JK, Öhman T, Nyman TA, Weiler H, Battersby B J. Quantitative changes in Gimap3 and Gimap5 expression modify mitochondrial DNA segregation in mice. Genetics 200: 221-235, 2015.
Liu P, Wang X, Hiltunen K, Chen Z. Controllable drug release system in living cells triggered by enzyme-substrate recognition. ACS Appl Mater Interfaces 7:26811-26818, 2015.
Liu PC, Wang H, Hiltunen JK, Chen ZJ, Shen JC. Cross-linked proteins with gold nanoclusters: A dual-purpose pH-responsive material for controllable cell imaging and antibiotic delivery. Part Part Syst Char 32:749-755, 2015.
Selkälä EM, Nair RR, Schmitz W, Kvist A-P, Baes M, Hiltunen JK, Autio KJ. Phytol is lethal for Amacr-deficient mice. Biochim Biophys Acta 1851: 1394-13405, 2015.
Mindthoff S, Grunau S, Steinfort L, Girzalsky W, Hiltunen JK, Erdmann R, Antonenkov VD. Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels. BBA - Molecular Cell Research 1863: 271-283, 2016.
Kalervo Hiltunen, M.D., Ph.D., Professor (University of Oulu)
Senior and Post-doctoral Investigators:
Vasily Antonenkov, M.D., Ph.D., Visiting Professor (University of Oulu)
Alexander Kastaniotis, Ph.D., Docent, group leader (Academy of Finland)
Kaija Autio, Ph.D. (Academy of Finland)
Antonina Shvetsova, Ph.D. (Foundation, a part of the year)
Jahangir Alam, B.Sc. (Biocenter Oulu)
Guangyu Jiang, M.Sc. (Foundations)
Juha Kerätär, M.Sc. (Biocenter Oulu)
Geoffray Monteuuis, M.Sc. (Academy of Finland)
Anne Mäkelä, M.Sc. (Academy of Finland)
Laura Pietikäinen, M.Sc. (Biocenter Oulu)
Remya R. Nair, M.V.Sc. (Academy of Finland)
Eija Selkälä, M.D., M.Sc. (Foundations)
Foreign Scientists, 7
Group Members Who Spent More Than Two Weeks in Foreign Laboratories During 2015
Kaija Autio, Ph.D. University of Arizona at Tucson, Tucson, Arizona, USA (1.9.2015 –)
Visiting Researchers in 2015 (over two weeks)
Carol Dieckman, University of Arizona, Tucson, AZ, USA (6.7. – 1.8.2015)
Han Ding, Jilin University, Changchun, China (7.1.2015 - 31.5.2015)
Joana Schröder, Hamburg School of Life Sciences, Hamburg (5.1.2015 – 30.4.2015)
Thimo Meyer, Hamburg School of Life Sciences, Hamburg, Germany (1.9.2015 – 31.12.2015)
Karina Kürpick, Friedrich‐Schiller‐University Jena, Jena, Germany (3.10. ‐ 20.12.2015)
Last updated: 16.9.2016