Kaija J. Autio

Mitochondrial Fatty Acid Synthesis Type II: More than Just Fatty Acids

Eukaryotes harbor a highly conserved mitochondrial pathway for fatty acid synthesis (FAS), which is completely independent of the eukaryotic cytosolic FAS apparatus. The activities of the mitochondrial FAS system are catalyzed by soluble enzymes, and the pathway thus resembles its prokaryotic counterparts. Except for octanoic acid, which is the direct precursor for lipoic acid synthesis, other end products and functions of the mitochondrial FAS pathway are still largely enigmatic. In addition to low cellular levels of lipoic acid, disruption of genes encoding mitochondrial FAS enzymes in yeast results in a respiratory-deficient phenotype and small rudimentary mitochondria. Recently, two distinct links between mitochondrial FAS and RNA processing have been discovered in vertebrates and yeast, respectively. In vertebrates, the mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase and the RPP14 subunit of RNase P are encoded by the same bicistronic transcript in an evolutionarily conserved arrangement that is unusual for eukaryotes. In yeast, defects in mitochondrial FAS result in inefficient RNase P cleavage in the organelle. The intersection of mitochondrial FAS and RNA metabolism in both systems provides a novel mechanism for the coordination of intermediary metabolism in eukaryotic cells.

Mitochondrial fatty acid synthesis and respiration

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl‐CoA dependent manner. We describe here the identification of the 3‐hydroxyacyl‐ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony‐colour‐sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non‐fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology,  were  found  to  have  a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase‐isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3‐hydroxyacyl‐thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.

An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing

In bacteria, functionally related gene products are often encoded by a common transcript. Such polycistronic transcripts are rare in eukaryotes. Here we isolated several clones from human cDNA libraries, which rescued the respiratory‐deficient phe‐notype of a yeast mitochondrial 3‐hydroxyacyl thioester dehydratase 2 (htd2) mutant strain. All complementing cDNAs were derived from the RPP14 transcript previously described to encode the RPP14 subunit of the human ribonuclease P (RNase P) complex. We identified a second, 3′ open reading frame (ORF) on the RPP14 transcript encoding a protein showing similarity to known dehydratases and hydratase 2 enzymes. The protein was localized in mitochondria, and the recombinant enzyme exhibited (3R)‐specific hydratase 2 activity. Based on our results, we named the protein human 3‐hydroxyacyl‐thioester dehydratase 2 (HsHTD2), which is involved in mitochondrial fatty acid synthesis. The bicistronic arrangement of RPP14 and HsHTD2, as well as the general exon structure of the gene, is conserved in vertebrates from fish to humans, indicating a genetic link conserved for 400 million years between RNA processing and mitochondrial fatty acid synthesis.—Autio, K. J., Kastaniotis, A. J., Pospiech, H., Miinalainen, I. J., Schonauer, M. S., Dieckmann, C. L., Hiltunen, J. K. An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing. FASEB J. 22, 569–578 (2008)

Mitochondrial fatty acid synthesis and maintenance of respiratory competent mitochondria in yeast.

Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.

The 3-hydroxyacyl-ACP dehydratase of mitochondrial fatty acid synthesis in Trypanosoma brucei

The trypanosomatid parasite Trypanosoma brucei synthesizes fatty acids in the mitochondrion using the type II fatty acid synthesis (FAS) machinery. When mitochondrial FAS was characterized in T. brucei, all of the enzymatic components were identified based on their homology to yeast mitochondrial FAS enzymes, except for 3‐hydroxyacyl‐ACP dehydratase. Here we describe the characterization of T. brucei mitochondrial 3‐hydroxyacyl‐ACP dehydratase (TbHTD2), which was identified by its similarity to the human mitochondrial dehydratase. TbHTD2 can rescue the respiratory deficient phenotype of the yeast knock‐out strain and restore the lipoic acid content, is localized in the mitochondrion and exhibits hydratase 2 activity.

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

Metabolic adaptation allows Amacr-deficient mice to remain symptom-free despite low levels of mature bile acids

Bile acids play multiple roles in the physiology of vertebrates; they facilitate lipid absorption, serve as signaling molecules to control carbohydrate and lipid metabolism, and provide a disposal route for cholesterol. Unexpectedly, the α-methylacyl-CoA racemase (Amacr) deficient mice, which are unable to complete the peroxisomal cleavage of C27-precursors to the mature C24-bile acids, are physiologically asymptomatic when maintained on a standard laboratory diet. The aim of this study was to uncover the underlying adaptive mechanism with special reference to cholesterol and bile acid metabolism that allows these mice to have a normal life span. Intestinal cholesterol absorption in Amacr-/- mice is decreased resulting in a 2-fold increase in daily cholesterol excretion. Also fecal excretion of bile acids (mainly C27-sterols) is enhanced 3-fold. However, the body cholesterol pool remains unchanged, although Amacr-deficiency accelerates hepatic sterol synthesis 5-fold. Changes in lipoprotein profiles are mainly due to decreased phospholipid transfer protein activity. Thus Amacr-deficient mice provide a unique example of metabolic regulation, which allows them to have a normal lifespan in spite of the disruption of a major metabolic pathway. This metabolic adjustment can be mainly explained by setting cholesterol and bile acid metabolism to a new balanced level in the Amacr-deficient mouse.

Phytol is lethal for Amacr-deficient mice.

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks 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. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. 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 to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.

Genetic modifications of Mecr reveal a role for mitochondrial 2-enoyl-CoA/ACP reductase in placental development in mice

Mitochondrial fatty acid synthesis (mtFAS) is an underappreciated but highly conserved metabolic process, indispensable for mitochondrial respiration. It was recently reported that dysfunction of mtFAS causes childhood onset of dystonia and optic atrophy in humans (MEPAN). To study the role of mtFAS in mammals, we generated three different mouse lines with modifications of the Mecr gene, encoding mitochondrial enoyl-CoA/ACP reductase (Mecr). A knock-out-first type mutation, relying on insertion of a strong transcriptional terminator between the first two exons of Mecr, displayed embryonic lethality over a broad window of time and due to a variety of causes. Complete removal of exon 2 or replacing endogenous Mecr by its functional prokaryotic analogue fabI (Mecrtm(fabI)) led to more consistent lethality phenotypes and revealed a hypoplastic placenta. Analyses of several mitochondrial parameters indicate that mitochondrial capacity for aerobic metabolism is reduced upon disrupting mtFAS function. Further analysis of the synthetic Mecrtm(fabI) models disclosed defects in development of placental trophoblasts consistent with disturbed peroxisome proliferator-activated receptor signalling. We conclude that disrupted mtFAS leads to deficiency in mitochondrial respiration, which lies at the root of the observed pantropic effects on embryonic and placental development in these mouse models.