CoA‐dependent activation of mitochondrial acyl carrier protein links four neurodegenerative diseases
2019; Springer Nature; Volume: 11; Issue: 12 Linguagem: Inglês
10.15252/emmm.201910488
ISSN1757-4684
AutoresRoald A. Lambrechts, Hein Schepers, Yi Yu, Marianne van der Zwaag, Kaija J. Autio, Marcel A. Vieira‐Lara, Barbara M. Bakker, Marina A.J. Tijssen, Susan J. Hayflick, Nicola A. Grzeschik, Ody C.M. Sibon,
Tópico(s)Hereditary Neurological Disorders
ResumoArticle7 November 2019Open Access Source DataTransparent process CoA-dependent activation of mitochondrial acyl carrier protein links four neurodegenerative diseases Roald A Lambrechts Roald A Lambrechts Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Hein Schepers Hein Schepers Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Yi Yu Yi Yu Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Marianne van der Zwaag Marianne van der Zwaag Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Kaija J Autio Kaija J Autio Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland Search for more papers by this author Marcel A Vieira-Lara Marcel A Vieira-Lara Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Barbara M Bakker Barbara M Bakker Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Marina A Tijssen Marina A Tijssen Neurology Department, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Susan J Hayflick Susan J Hayflick orcid.org/0000-0003-2595-3943 Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Nicola A Grzeschik Nicola A Grzeschik Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Ody CM Sibon Corresponding Author Ody CM Sibon [email protected] orcid.org/0000-0002-6836-6063 Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Roald A Lambrechts Roald A Lambrechts Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Hein Schepers Hein Schepers Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Yi Yu Yi Yu Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Marianne van der Zwaag Marianne van der Zwaag Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Kaija J Autio Kaija J Autio Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland Search for more papers by this author Marcel A Vieira-Lara Marcel A Vieira-Lara Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Barbara M Bakker Barbara M Bakker Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Marina A Tijssen Marina A Tijssen Neurology Department, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Susan J Hayflick Susan J Hayflick orcid.org/0000-0003-2595-3943 Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA Search for more papers by this author Nicola A Grzeschik Nicola A Grzeschik Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Ody CM Sibon Corresponding Author Ody CM Sibon [email protected] orcid.org/0000-0002-6836-6063 Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Author Information Roald A Lambrechts1, Hein Schepers1,‡, Yi Yu1,‡, Marianne van der Zwaag1, Kaija J Autio2, Marcel A Vieira-Lara3, Barbara M Bakker3, Marina A Tijssen4, Susan J Hayflick5, Nicola A Grzeschik1 and Ody CM Sibon *,1 1Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 2Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland 3Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 4Neurology Department, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 5Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +31 503616111; E-mail: [email protected] EMBO Mol Med (2019)11:e10488https://doi.org/10.15252/emmm.201910488 See also: SY Jeong et al (December 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis PKAN, CoPAN, MePAN and PDH-E2 deficiency are neurodegenerative diseases that damage a specific area of the brain and in which mutated genes encode enzymes that play a role in intermediary metabolism. Restoring PDH activity rescues abnormalities caused by CoA biosynthesis defects in disease models. PKAN, CoPAN, MePAN and PDH-E2 deficiency are linked by a metabolic pathway, which when disturbed causes a common phenotype. This pathway explains how impaired CoA biosynthesis (in PKAN/CoPAN) decreases mitochondrial acyl carrier protein activity, leading to decreased protein lipoylation (in MePAN) and decreased activity of PDH (in PDH-E2 deficiency). Restoration of PDH activity, the final common step of the proposed linked metabolic pathway, rescues abnormalities caused by defects in CoA biosynthesis, the first step of the pathway. This pathway explains not only the overlapping clinical characteristics of the four diseases but also the brain iron accumulation in PKAN/CoPAN and the absence of iron in MePAN/PDH-E2 deficiency. These results suggest disease-specific treatment strategies. Introduction Coenzyme A (CoA) is an essential cofactor participating in approximately 9% of all cellular metabolic reactions, such as the tricarboxylic acid (TCA) cycle, and fatty acid synthesis and degradation (Leonardi et al, 2005; Strauss, 2010). CoA is synthesized de novo in cells, utilizing vitamin B5 as a starting molecule and requiring five enzymatic reactions. These are carried out by pantothenate kinase (PANK), phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK), respectively (Leonardi et al, 2005; Strauss, 2010). In some organisms, including Drosophila melanogaster, mice, and humans, PPAT and DPCK enzyme activities are performed by a single bifunctional protein, referred to as CoA synthase or COASY (Fig 1). The intermediate products that are being sequentially formed from vitamin B5 during the CoA de novo biosynthesis pathway are as follows: 4′-phosphopantothenate, 4′-phosphopantothenoylcysteine, 4′-phosphopantetheine, dephospho-CoA, and CoA (Fig 1). Enzymes of the CoA de novo biosynthesis pathway are evolutionarily conserved, further underscoring the importance of this pathway for all living organisms. Figure 1. Metabolic pathways in which coenzyme A is formed, re-used, or consumed and their interconnections A. De novo biosynthesis pathway of coenzyme A (CoA) is a pathway during which CoA is produced. Vitamin B5 is taken up by cells and converted into CoA by the action of five enzymatic reactions (Leonardi et al, 2005; Strauss, 2010). These are carried out by pantothenate kinase (PANK), phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK), respectively. In Drosophila melanogaster, mice, and humans, PPAT and DPCK enzyme activities are carried out by a single bifunctional protein, CoA synthase or COASY. Abbreviations of the enzymes are provided. The starting product vitamin B5/pantothenate, the intermediate, 4′-phosphopantetheine, and the final product CoA are depicted. PKAN and CoPAN are inherited recessive diseases caused by homozygous mutations in PANK2 and COASY, respectively. B. Formation of holo-mtACP (active form of mitochondrial acyl carrier protein) is a CoA consuming metabolic reaction (Beld et al, 2014). 4′-phosphopantetheinylation of inactive apo-mtACP is required to produce the active holo-mtACP form. During this process, CoA serves as the source for the 4′-phosphopantetheinylation, and hereby, a CoA molecule is degraded and adenosine 3′–5′-biphosphate is released. Holo-mtACP plays a key role in mitochondrial fatty acid synthesis, which can be visualized in (C). C. Fatty acid synthesis is a metabolic pathway in which CoA is re-used (Kastaniotis et al, 2017). Mitochondrial fatty acid synthesis (mtFASII) is required for the synthesis of lipoic acid, a pathway that is dependent on the activity of holo-mtACP. In this pathway, malonic acid is converted to malonyl-CoA, which requires holo-mtACP (or NDUFABI in humans) for the formation of malonyl-holo-ACP as well as for subsequent downstream steps. Mitochondrial Enoyl-[acyl-carrier-protein] reductase (MECR, defective enzyme in MEPAN) is required for the formation of acyl-holo-mtACP as indicated. Via this pathway lipoic acid is formed from octanoyl-holo-mtACP by lipoic acid synthetase (LIAS). This product is then required for the formation of lipoylated pyruvate dehydrogenase (PDH), lipoylated α-ketoglutarate dehydrogenase (αKGDH), lipoylated branched-chain alpha-keto acid dehydrogenase (BCKDH), and lipoylated glycine cleavage system (GCV). Lipoylation of the above-mentioned proteins is necessary for catalysis of their respective reactions to occur. D. Fatty acid (FA) degradation or β-oxidation is an example of a degradation pathway in which CoA is re-used. The starting mitochondrial precursor acyl-carnitine, the intermediates, and the end product of this pathway are indicated, as well as reactions in which CoA is required (but not consumed) and released. E. In the glycolysis and TCA cycle, CoA is re-used. Pyruvate dehydrogenase (PDH) catalyzes the oxidative decarboxylation of pyruvate to produce acetyl that is coupled to CoA to produce acetyl-CoA. Impaired function of this enzyme leads to PDH-E2 deficiency. The product of the PDH reaction, acetyl-CoA, is the fuel for the TCA cycle which is also a CoA re-using pathway as indicated. OAA: oxaloacetate. Download figure Download PowerPoint Two autosomal recessive neurodegenerative diseases are caused by mutations in genes encoding enzymes of the CoA pathway. Pathogenic variants in PANK2 and COASY lead to two early-onset neurodegenerative diseases: pantothenate kinase-associated neurodegeneration (PKAN) and CoA synthase protein-associated neurodegeneration (CoPAN). The human genome contains four genes encoding pantothenate kinase homologs, PANK1-4, and only mutations in PANK2 are associated with PKAN. PKAN and CoPAN patients accumulate iron in the globus pallidus, a basal ganglia structure in the brain (Hayflick et al, 2003; Dusi et al, 2014). Iron accumulation is visible on T2-weighted imaging as a hypointense signal on MRI. In PKAN and CoPAN areas, T2-hyperintense signals are also seen at the globus pallidus, indicating edema and tissue damage (Kruer et al, 2012). These CoA-linked diseases are characterized by progressive motor dysfunction and severe dystonia. Damage to the globus pallidus occurs in other inborn errors of metabolism as well. MePAN, a third childhood-onset neurodegenerative disorder, manifests with damage to the globus pallidus, which is also visible on brain MRI as hyperintense signal on T2-weighted imaging although without the iron-associated signal abnormalities (Heimer et al, 2016). MePAN patients carry mutations in the gene encoding mitochondrial enoyl-[acyl-carrier-protein] reductase (MECR), one of four enzymes involved in the elongation of fatty acids in mitochondria to form octanoic acid, a precursor of lipoic acid (Fig 1; Heimer et al, 2016). In eukaryotic cells, this process of mitochondrial fatty acid synthesis (mtFAS-type II) is required for lipoic acid production and subsequently for lipoylation of proteins (Brody et al, 1997; Wada et al, 1997; Feng et al, 2009). Finally, mutations causing impairment of a component of the pyruvate dehydrogenase complex, PDH-E2, lead to PDH-E2 deficiency and cause a form of Leigh disease, in which neuroradiographic abnormalities are again observed specifically in the globus pallidus (Head et al, 2005; McWilliam et al, 2010; Leoni et al, 2012). PDH catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA, thereby linking glycolysis to the TCA cycle (Fig 1). The symptoms, signs, and MRI characteristics of PKAN and PDH-E2 deficiency can be similar. In patients with a clinical suspicion of PKAN, PDH-E2 deficiency should also be considered in the differential diagnosis and vice versa (Head et al, 2005; McWilliam et al, 2010; Leoni et al, 2012). The clinical and neuroradiographic overlapping features of PKAN, CoPAN, MePAN, and PDH-E2 deficiency suggest a common element in their pathogeneses. The pathways of CoA biosynthesis, mitochondrial fatty acid synthesis, and glycolysis/TCA cycle show interdependency and interconnectivity (Leonardi et al, 2005; Strauss, 2010; Beld et al, 2014; Kastaniotis et al, 2017; Fig 1), but a specific molecular etiology common to these four disorders has been lacking. Herein, we propose a common underlying pathway directly connecting these four diseases. In most CoA-dependent metabolic reactions, CoA acts as an acyl carrier; the acyl moiety is transferred between CoA and another molecule (e.g., carnitine), leaving CoA intact and available for further transfer reactions. CoA-dependent acyl transfer occurs during the TCA cycle, fatty acid synthesis, and fatty acid degradation (Fig 1). Because CoA is re-used as acyl carrier component, these reactions do not lead to reduced levels of total CoA. In contrast, one specific form of posttranslational modification "consumes" CoA. 4′-phosphopantetheinylation adds a 4′-phosphopantetheine moiety to specific proteins resulting in their activation. For this modification, the 4′-phosphopantetheine moiety is derived from CoA (Beld et al, 2014). Therefore, this reaction forms a 4′-phosphopantetheinylated protein, and adenosine 3′,5′-bisphosphate is released and thereby causes a net loss of CoA (Elovson & Vagelos, 1968). In humans, a select group of proteins requires this 4′-phosphopantetheine moiety in order to function, e.g. 10-formyltetrahydrofolate dehydrogenase, an enzyme of folate metabolism (Strickland et al, 2010), cytosolic fatty acid synthase, and mitochondrial acyl carrier protein (mtACP; Joshi et al, 2003; Beld et al, 2014). We focussed on mtACP because human PANK2, COASY, MECR, and PDH-E2 are mitochondrial proteins (Kotzbauer et al, 2005; Dusi et al, 2014) and mitochondria are defective in various PKAN animal models (Rana et al, 2010; Brunetti et al, 2014; Orellana et al, 2016; Jeong et al, 2019). The active 4′-phosphopantetheinylated form of mtACP is referred to as holo-mtACP. mtACP, known in humans as NDUFAB1, is one of the subunits of the respiratory chain complex I and plays a central role in mitochondrial fatty acid synthesis (Brody et al, 1997; Feng et al, 2009). In this latter process, the thiol group of the 4′-phosphopantetheine prosthetic group forms the attachment site for a growing carbon chain (Beld et al, 2014). Octanoate formed in this way is converted to lipoic acid and used to modify mitochondrial proteins, among which are the E2 subunits of three enzyme complexes (pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (αKGDH), branched-chain α-keto acid dehydrogenase (BCKDH), and the glycine cleavage system (GCV); Cronan, 2016; Rowland et al, 2018). In humans, octanoic acid is transferred to its target proteins by the lipoyl transferases LIPT2 and LIPT1. By the action of the conserved enzyme lipoic acid synthase (LIAS in humans), protein-bound octanoate is transformed into lipoic acid (LA) by the insertion of two sulfhydryl groups (Booker, 2004; Hiltunen et al, 2010; Solmonson & DeBerardinis, 2018), enabling the now-lipoylated proteins to function (Fig 1; Hiltunen et al, 2010). Based on these reports, we hypothesized that, because 4′-phosphopantetheinylation of mtACP consumes CoA, this reaction may be most sensitive to impaired CoA biosynthesis. Consequently, downstream processes are predicted to be affected as well, including decreased lipoylation and activity of PDH (Fig 2). This hypothesis could explain how defects in PANK2 and COASY might lead to a phenotype similar to that arising from defects in MECR and PDH (Fig 2). A central tenet of this hypothesis is that impaired de novo biosynthesis of CoA leads to decreased levels of holo-mtACP. Here, we investigated this hypothesis. Figure 2. De novo CoA biosynthesis pathway and key downstream steps to link PKAN, CoPAN, MePAN, and PDH-E2 deficiencyLeft part: Proposed linear pathway linking CoA-mtACP-PDH. From top to bottom: The de novo CoA biosynthesis pathway starts with the cellular uptake of pantothenate (Vitamin B5). Pantothenate kinase (PANK), phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and coenzyme A synthase (COASY) are enzymes required for the de novo biosynthesis of CoA. Mitochondrial acyl carrier protein (mtACP) undergoes a posttranslational modification and active holo-mtACP is formed. This posttranslational modification consists of 4′-phosphopantetheine, which is derived from CoA. Holo-mtACP in turn is required for lipoylation of PDH-E2, a modification necessary for activation of the PDH complex. It is hypothesized that a decrease in CoA biosynthesis leads to decreased amounts of holo-ACP, decreased lipoylation of PDH-E2, and decreased activity of PDH.Right part: Steps that are affected in PKAN, CoPAN, MePAN, and PDH-E2 deficiency. Primary affected steps caused by the genetic defects are depicted as the most upstream steps as well as the hypothesized downstream steps for CoPAN, PKAN, and MePAN. Download figure Download PowerPoint The de novo CoA biosynthesis pathway and the metabolic reactions presented in Figs 1 and 2) are highly conserved between human and Drosophila melanogaster (Leonardi et al, 2005; Bosveld et al, 2008). Our combined approach employing the versatile genetic tools of Drosophila melanogaster and mammalian cells enabled us to demonstrate that impaired CoA biosynthesis leads to decreased levels of active, 4′-phosphopantetheinylated mtACP. This observation was associated with decreased lipoylation of PDH-E2 and decreased PDH activity. Our results revealed the presence of a CoA-mtACP-PDH pathway in which the 4′-phosphopantetheinylation of mtACP is a key step. Next, we showed that stimulation of PDH rescued phenotypes caused by impaired CoA biosynthesis, highlighting PDH as a possible common target for ameliorating diseases induced by defects in the CoA-mtACP-PDH pathway. Our findings combined with those reported by Jeong et al suggest therapeutic approaches for PKAN, CoPAN, MePAN, and PDH-E2 deficiency. Results holo-mtACP levels are reduced by impeding CoA biosynthesis PKAN and CoPAN patients carry mutations in genes coding for pantothenate kinase 2 and COASY, enzymes required for CoA biosynthesis (Fig 1). To test our hypothesis (presented in Fig 2), we first investigated consequences of impaired CoA biosynthesis on 4′-phosphopantetheinylation of mtACP. For this, we chose Drosophila melanogaster because of its conserved metabolic steps and genes and its versatile genetic tools. mtACP requires activation in order to function; the active holo form is generated by enzymatic transfer of a negatively charged 4′-phosphopantetheine moiety to a conserved serine residue of the inactive apo form (Elovson & Vagelos, 1968; Jung et al, 2016; Fig 3A). For our study we manipulated mtacp, the Drosophila melanogaster gene encoding mtACP, containing Ser-99, which is predicted to bind 4-phosphopantetheine (Ragone et al, 1999). In order to be able to identify and distinguish the two forms of mtACP (holo versus apo), we generated constructs encoding mutant proteins that would be refractory to 4-phosphopantetheinylation and observed their mobility differences using gel electrophoresis. We mutated the crucial serine residue: one to mimic the uncharged apo form (S99A) and two negatively charged forms (S99D and S99E) to mimic the charged holo form of mtACP (Fig 3A). Overexpression of wild-type mtACP constructs in Drosophila S2 Schneider cells enabled the visualization of protein bands that correspond to endogenous apo- and holo-mtACP forms. By comparing these bands to the apo-mimetic S99A and holo-mimetics S99D and S99E, we were able to prove the identity of the bands visualized under control and mtACP wild-type overexpressing conditions. Under physiologic conditions, endogenous holo-mtACP was detected (Fig 3B and C). In contrast, no endogenous apo-mtACP protein was visible, consistent with previous observations in other organisms that the inactive apo-mtACP form is not stable (Jackowski & Rock, 1983; Post-Beittenmiller et al, 1989). Figure 3. Decreased levels of CoA are associated with decreased levels of holo-mtACP and lipoylated PDH-E2 A. Schematic presentation of endogenous active and inactive forms of mtACP and synthesized mutant forms of mtACP. For the endogenous form, the S represents the serine residue that is 4′-phosphopantetheinylated, while D and L represent the flanking amino acids. Inactive form of mtACP (apo-mtACP) is indicated with 1. CoA is the source for 4′-phosphopantetheine and is required for 4′-phosphopantetheinylation of mtACP occurring on the serine residue. This posttranslational modification results in an active form of mtACP, holo-mtACP, which is negatively charged and indicated with 2. Three mtACP constructs were generated: One in which serine 99 was modified to an alanine, indicated with a 3 and indicated as S99A (non-4′-phosphopantetheinylatable form); one in which serine 99 was modified into aspartate, indicated with a 4 and indicated as S99D (phosphomimetic); one in which serine 99 was modified into glutamate, indicated with a 5 and indicated as S99E (phosphomimetic). S99D and S99E are negatively charged, mimicking the negatively charged holo-mtACP. The red circle indicates the presence of a negative charge. B. Western blot analysis of S2 cells overexpressing wild-type constructs of mtACP or the various mutant forms. First lane: lysates of control cells, resulting in the detection of holo-mtACP visible after long exposure. Second lane: overexpression (OE) of mtACP WT results in the detection of an apo-mtACP form and a holo-mtACP form, indicated with 1 and 2, respectively. Third lane: overexpression of mtACP S99A mutant form resulted in the detection of an apo-mtACP (non-4′-phosphopantetheinylatable) band only, indicated with 3. Fourth lane: overexpression of mtACP S99D resulted in the detection of a phosphomimetic form of mtACP only, migrating at the same mobility as holo-mtACP, indicated with 4. Fifth lane: overexpression of mtACP S99E results in the detection of a phosphomimetic form of mtACP only, migrating at the same mobility as holo-mtACP, indicated with 5. For visualization, a low exposure and high exposure blot are shown. Note that overexposed blots were required to visualize endogenous forms of mtACP. C. Western blot analysis of mtACP forms under control conditions, under conditions of HoPan treatment, and under conditions of HoPan + CoA treatment. Lanes showing overexpression of mtACP WT, mtACP S99A, and mtACP S99D were used to allow identification of the holo- and the apo-forms of mtACP. α-Tubulin was used as a loading control. Various exposure times of the blots are presented to allow identification of mtACP under all conditions. D. Western blot analysis showing lipoylated proteins under control conditions, after HoPan treatment and after HoPan + CoA treatment. S2 cells were treated with HoPan or HoPan + CoA for 4 days; non-treated cells were used as control. Antibodies specifically recognizing lipoylated proteins or PDH-E2 were used. Arrow heads indicate lipoylated PDH-E2 (left panel) or total PDH-E2 (right panel). E. Quantification of lipoylated PDH-E2 from (D). Mean ± SD is given. n = 3 for all samples. F. PDH activity was measured in control cells, HoPan-treated cells or HoPan-treated cells rescued with CoA or DCA. Mean ± SD of five biological replicates, each composed of three technical replicates and corrected for protein concentration. Data information: For (E and F), two-tailed Student's t-test was performed to calculate statistical significance for the indicated subsets. Source data are available online for this figure. Source Data for Figure 3 [emmm201910488-sup-0002-SDataFig3.pdf] Download figure Download PowerPoint We proceeded to investigate whether levels of active holo-mtACP would decrease upon CoA deprivation, a key assumption of our hypothesis. Treating S2 cells with the PANK inhibitor hopantenate (HoPan) leads to reduced CoA biosynthesis and to reduced levels of total CoA (Rana et al, 2010; Siudeja et al, 2011; Srinivasan et al, 2015). Under these conditions, we observed reduced levels of endogenous holo-mtACP (Fig 3C), and addition of CoA to the medium of HoPan-treated cells reverted this phenotype, consistent with previous studies in which administration of extracellular CoA is able to rescue phenotypes associated with reduced intracellular CoA biosynthesis (Rana et al, 2010; Siudeja et al, 2011; Srinivasan et al, 2015). These results demonstrate that impaired CoA biosynthesis is associated with decreased levels of 4′-phosphopantetheinylation of mtACP. Protein lipoylation is reduced by impeding CoA biosynthesis To further investigate the consequences of impaired CoA biosynthesis and reduced levels of 4′-phosphopantetheinylated mtACP, we examined mtACP-dependent processes, specifically those linked to MePAN and PDH-E2 deficiency. holo-mtACP and MECR are required for protein lipoylation (Fig 1), a process that is therefore affected in MePAN patients (Heimer et al, 2016). Four evolutionary conserved lipoylated enzyme complexes have been identified: PDH, α-KGDH, BCKDH, and GCV. To assess whether lipoylation was affected under conditions of impaired CoA biosynthesis, total protein lipoylation was analyzed using Western blot analysis. Incubation with an antibody that detects protein-bound lipoic acid revealed decreased levels of various protein bands under conditions of reduced CoA levels, an effect that was rescued when CoA was supplemented to the medium (Fig 3D, Appendix Fig S1). The availability of an antibody recognizing the Drosophila PDH-E2 subunit allowed the analysis of lipoylated PDH-E2 specifically, demonstrating
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