A hunt for OM45 synthetic petite interactions in Saccharomyces cerevisiae reveals a role for Miro GTPase Gem1p in cristae structure maintenance.

Om45 is a major protein of the yeast's outer mitochondrial membrane under respiratory conditions. However, the cellular role of the protein has remained obscure. Previously, deletion mutant phenotypes have not been found, and clear amino acid sequence similarities that would allow inferring its functional role are not available. In this work, we describe synthetic petite mutants of GEM1 and UGO1 that depend on the presence of OM45 for respiratory growth, as well as the identification of several multicopy suppressors of the synthetic petite phenotypes. In the analysis of our mutants, we demonstrate that Om45p and Gem1p have a collaborative role in the maintenance of mitochondrial morphology, cristae structure, and mitochondrial DNA maintenance. A group of multicopy suppressors rescuing the synthetic lethal phenotypes of the mutants on non-fermentable carbon sources additionally supports this result. Our results imply that the synthetic petite phenotypes we observed are due to the disturbance of the inner mitochondrial membrane and point to this mitochondrial sub-compartment as the main target of action of Om45p, Ugo1p, and the yeast Miro GTPase Gem1p.

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.

Genetic dissection of the mitochondrial lipoylation pathway in yeast.

BACKGROUND: Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate. RESULTS: We show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA. CONCLUSIONS: In the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.

Crystallographic binding studies of rat peroxisomal multifunctional enzyme type 1 with 3-ketodecanoyl-CoA: capturing active and inactive states of its hydratase and dehydrogenase catalytic sites.

The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the ß-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Šresolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.

Estradiol Valerate in COC Has More Favorable Inflammatory Profile Than Synthetic Ethinyl Estradiol: A Randomized Trial.

CONTEXT: Combined oral contraceptives (COCs) alter inflammatory status and lipid metabolism. Whether different estrogens have different effects is poorly understood. OBJECTIVE: We compared the effects of COCs containing ethinyl estradiol (EE) or estradiol valerate (EV) and dienogest (DNG) with those containing DNG only on inflammation and lipid metabolism. DESIGN: Randomized, controlled, open-label clinical trial. SETTING: Two-center study in Helsinki and Oulu University Hospitals. PARTICIPANTS: Fifty-nine healthy, young, nonsmoking women with regular menstrual cycles. Age, body mass index, and waist-to-hip ratio were comparable in all study groups at the beginning. Fifty-six women completed the study (EV + DNG, n = 20; EE + DNG, n = 19; DNG only, n = 17). INTERVENTIONS: Nine-week continuous use of COCs containing either EV + DNG or EE + DNG, or DNG only as control. MAIN OUTCOME MEASURES: Parameters of chronic inflammation (high-sensitivity C-reactive protein [hs-CRP], and pentraxin 3 [PTX-3]) and lipid profile (high-density lipoprotein [HDL], low-density lipoprotein [LDL], triglycerides, and total cholesterol). RESULTS: Serum hs-CRP increased after 9-week use of EE + DNG (mean change ± standard deviation 1.10 ± 2.11 mg/L) compared with EV + DNG (-0.06 ± 0.97 mg/L, P = 0.001) or DNG only (0.13 ± 0.68 mg/L, P = 0.021). Also, PTX-3 increased in the EE + DNG group compared with EV + DNG and DNG-only groups (P = 0.017 and P = 0.003, respectively). In the EE + DNG group, HDL and triglycerides increased compared with other groups (HDL: EE + DNG 0.20 ± 0.24 mmol/L vs EV + DNG 0.02 ± 0.20 mmol/L [P = 0.002] vs DNG 0.02 ± 0.18 mmol/L [P = 0.002]; triglycerides: EE + DNG 0.45 ± 0.21 mmol/L vs EV + DNG 0.18 ± 0.36 mmol/L [P = 0.003] vs DNG 0.06 ± 0.18 mmol/L [P < 0.001]). CONCLUSIONS: EV + DNG and DNG only had a neutral effect on inflammation and lipids, while EE + DNG increased both hs-CRP and PTX-3 levels as well as triglycerides and HDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT02352090.

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.

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.

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.

Non-electron transfer chain mitochondrial defects differently regulate HIF-1α degradation and transcription.

Mitochondria are the main consumers of molecular O2 in a cell as well as an abundant source of reactive oxygen species (ROS). Both, molecular oxygen and ROS are powerful regulators of the hypoxia-inducible factor-1α-subunit (HIF-α). While a number of mechanisms in the oxygen-dependent HIF-α regulation are quite well known, the view with respect to mitochondria is less clear. Several approaches using pharmacological or genetic tools targeting the mitochondrial electron transport chain (ETC) indicated that ROS, mainly formed at the Rieske cluster of complex III of the ETC, are drivers of HIF-1α activation. However, studies investigating non-ETC located mitochondrial defects and their effects on HIF-1α regulation are scarce, if at all existing. Thus, in the present study we examined three cell lines with non-ETC mitochondrial defects and focused on HIF-1α degradation and transcription, target gene expression, as well as ROS levels. We found that cells lacking the key enzyme 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (MECR), and cells lacking manganese superoxide dismutase (MnSOD) showed a reduced induction of HIF-1α under long-term (20h) hypoxia. By contrast, cells lacking the mitochondrial DNA depletion syndrome channel protein Mpv17 displayed enhanced levels of HIF-1α already under normoxic conditions. Further, we show that ROS do not exert a uniform pattern when mediating their effects on HIF-1α, although all mitochondrial defects in the used cell types increased ROS formation. Moreover, all defects caused a different HIF-1α regulation via promoting HIF-1α degradation as well as via changes in HIF-1α transcription. Thereby, MECR- and MnSOD-deficient cells showed a reduction in HIF-1α mRNA levels whereas the Mpv17 lacking cells displayed enhanced HIF-1α mRNA levels under normoxia and hypoxia. Altogether, our study shows for the first time that mitochondrial defects which are not related to the ETC and Krebs cycle contribute differently to HIF-1α regulation by affecting HIF-1α degradation and HIF-1α transcription where ROS play not a major role.

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.

MECR Mutations Cause Childhood-Onset Dystonia and Optic Atrophy, a Mitochondrial Fatty Acid Synthesis Disorder.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

Trim37-deficient mice recapitulate several features of the multi-organ disorder Mulibrey nanism.

Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37(-/-)) model for MUL. Trim37(-/-) mice were viable and had normal weight development until approximately 12 months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37(-/-) mice as compared with wild-type. Both male and female Trim37(-/-) mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37(-/-) mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37(-/-) mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5 years Trim37(-/-) mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37(-/-) mice. The most consistently seen phenotypes in Trim37(-/-) mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37(-/-) mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis.

Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells.

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.

Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels.

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. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ=4.1 nS in 1.0M KCl is moderately cation-selective (PK(+)/PCl(-)=1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r~0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300-400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids ß-oxidation in intact cells but not in the corresponding lysates. The ß-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the ß-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of ß-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal ß-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis.

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.

The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential.

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.