Impaired Mitochondrial Fatty Acid Synthesis Leads to Neurodegeneration in Mice.

There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties ∼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.

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.

Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.

Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both ß-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

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.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

Cholesterol is catabolized to bile acids by peroxisomal ß-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this ß-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

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.

Exploration of dietary interventions to treat mitochondrial fatty acid disorders in a mouse model.

Mitochondrial fatty acids synthesis (mtFAS) is a conserved metabolic pathway essential for mitochondrial respiration. The best characterized mtFAS product is the medium-chain fatty acid octanoate (C8) used as a substrate in the synthesis of lipoic acid (LA), a cofactor required by several mitochondrial enzyme complexes. In humans, mutations in the mtFAS component enoyl reductase MECR cause childhood-onset neurodegenerative disorder MEPAN. A complete deletion of Mecr in mice is embryonically lethal, while selective deletion of Mecr in cerebellar Purkinje cells causes neurodegeneration in these cells. A fundamental question in the research of mtFAS deficiency is if the defect is amenable to treatment by supplementation with known mtFAS products. Here we used the Purkinje-cell specific mtFAS deficiency neurodegeneration model mice to study if feeding the mice with a medium-chain triacylglycerol-rich formula supplemented with LA could slow down or prevent the neurodegeneration in Purkinje cell-specific Mecr KO mice. Feeding started at the age of 4 weeks and continued until the age of 9 months. The neurological status on the mice was assessed at the age of 3, 6, and 9 months with behavioral tests and the state of the Purkinje cell deterioration in the cerebellum was studied histologically. We showed that feeding the mice with medium chain triacylglycerols and LA affected fatty acid profiles in the cerebellum and plasma but did not prevent the development of neurodegeneration in these mice. Our results indicate that dietary supplementation with medium chain fatty acids and LA alone is not an efficient way to treat mtFAS disorders.

An engineered variant of MECR reductase reveals indispensability of long-chain acyl-ACPs for mitochondrial respiration.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function.

Mitochondrial 2,4-dienoyl-CoA reductase deficiency in mice results in severe hypoglycemia with stress intolerance and unimpaired ketogenesis.

The mitochondrial beta-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids, we created a DECR-deficient mouse line. In Decr(-/-) mice, the mitochondrial beta-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr(-/-) mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C(18:2)), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr(-/-) mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal beta-oxidation and microsomal omega-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1alpha and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.

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.

Mitochondrial Fatty Acids and Neurodegenerative Disorders.

Fatty acids in mitochondria, in sensu stricto, arise either as ß-oxidation substrates imported via the carnitine shuttle or through de novo synthesis by the mitochondrial fatty acid synthesis (mtFAS) pathway. Defects in mtFAS or processes involved in the generation of the mtFAS product derivative lipoic acid (LA), including iron-sulfur cluster synthesis required for functional LA synthase, have emerged only recently as etiology for neurodegenerative disease. Intriguingly, mtFAS deficiencies very specifically affect CNS function, while LA synthesis and attachment defects have a pleiotropic presentation beyond neurodegeneration. Typical mtFAS defect presentations include optical atrophy, as well as basal ganglia defects associated with dystonia. The phenotype display of patients with mtFAS defects can resemble the presentation of disorders associated with coenzyme A (CoA) synthesis. A recent publication links these processes together based on the requirement of CoA for acyl carrier protein maturation. MtFAS defects, CoA synthesis- as well as Fe-S cluster-deficiencies share lack of LA as a common symptom.

Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2 kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117-Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.

Correction to: Binding of Brucella protein, Bp26, to select extracellular matrix molecules.

Following publication of the original article [1], an error was reported in the tagging of Eugene H. Johnson and Remya R. Nair in the author group.

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 phenotype 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.

17B-hydroxysteroid dehydrogenases as acyl thioester metabolizing enzymes.

17ß-Hydroxysteroid dehydrogenases (HSD17B) catalyze the oxidation/reduction of 17ß-hydroxy/keto group in position C17 in C18- and C19 steroids. Most HSD17Bs are also catalytically active with substrates other than steroids. A subset of these enzymes is able to process thioesters of carboxylic acids. This group of enzymes includes HSD17B4, HSD17B8, HSD17B10 and HSD17B12, which execute reactions in intermediary metabolism, participating in peroxisomal ß-oxidation of fatty acids, mitochondrial oxidation of 3R-hydroxyacyl-groups, breakdown of isoleucine and fatty acid chain elongation in endoplasmic reticulum. Divergent substrate acceptance capabilities exemplify acquirement of catalytic site adaptiveness during evolution. As an additional common feature these HSD17Bs are multifunctional enzymes that arose either via gene fusions (HSD17B4) or are incorporated as subunits into multifunctional protein complexes (HSD17B8 and HSD17B10). Crystal structures of HSD17B4, HSD17B8 and HSD17B10 give insight into their structure-function relationships. Thus far, deficiencies of HSD17B4 and HSD17B10 have been assigned to inborn errors in humans, underlining their significance as enzymes of metabolism.

Mitochondrial acyl carrier protein (ACP) at the interface of metabolic state sensing and mitochondrial function.

Acyl carrier protein (ACP) is a principal partner in the cytosolic and mitochondrial fatty acid synthesis (FAS) pathways. The active form holo-ACP serves as FAS platform, using its 4'-phosphopantetheine group to present covalently attached FAS intermediates to the enzymes responsible for the acyl chain elongation process. Mitochondrial unacylated holo-ACP is a component of mammalian mitoribosomes, and acylated ACP species participate as interaction partners in several ACP-LYRM (leucine-tyrosine-arginine motif)-protein heterodimers that act either as assembly factors or subunits of the electron transport chain and Fe-S cluster assembly complexes. Moreover, octanoyl-ACP provides the C8 backbone for endogenous lipoic acid synthesis. Accumulating evidence suggests that mtFAS-generated acyl-ACPs act as signaling molecules in an intramitochondrial metabolic state sensing circuit, coordinating mitochondrial acetyl-CoA levels with mitochondrial respiration, Fe-S cluster biogenesis and protein lipoylation.

Mitochondrial 2,4-dienoyl-CoA reductase (Decr) deficiency and impairment of thermogenesis in mouse brown adipose tissue.

A large number of studies have demonstrated significance of polyunsaturated fatty acids (PUFAs) for human health. However, many aspects on signals translating PUFA-sensing into body homeostasis have remained enigmatic. To shed light on PUFA physiology, we have generated a mouse line defective in mitochondrial dienoyl-CoA reductase (Decr), which is a key enzyme required for ß-oxidation of PUFAs. Previously, we have shown that these mice, whose oxidation of saturated fatty acid is intact but break-down of unsaturated fatty acids is blunted, develop severe hypoglycemia during metabolic stresses and fatal hypothermia upon acute cold challenge. In the current work, indirect calorimetry and thermography suggested that cold intolerance of Decr-/- mice is due to failure in maintaining appropriate heat production at least partly due to failure of brown adipose tissue (BAT) thermogenesis. Magnetic resonance imaging, electron microscopy, mass spectrometry and biochemical analysis showed attenuation in activation of lipolysis despite of functional NE-signaling and inappropriate expression of genes contributing to thermogenesis in iBAT when the Decr-/- mice were exposed to cold. We hypothesize that the failure in turning on BAT thermogenesis occurs due to accumulation of unsaturated long-chain fatty acids or their metabolites in Decr-/- mice BAT suppressing down-stream propagation of NE-signaling.

Binding of Brucella protein, Bp26, to select extracellular matrix molecules.

BACKGROUND: Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). RESULTS: ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein. CONCLUSIONS: Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

CoA-dependent activation of mitochondrial acyl carrier protein links four neurodegenerative diseases.

PKAN, CoPAN, MePAN, and PDH-E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH-E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA-dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4'-phosphopantetheine moiety required for the posttranslational 4'-phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4'-phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA-mtACP-PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re-activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases.

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.