Asymmetric Distribution of Plasmalogens and Their Roles-A Mini Review.

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. The synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. Plasmalogens are transported to the post-Golgi compartment, including endosomes and plasma membranes, in a manner dependent on ATP, but not vesicular transport. Plasmalogens are preferentially localized in the inner leaflet of the plasma membrane in a manner dependent on P4-type ATPase ATP8B2, that associates with the CDC50 subunit. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and controls the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. The physiological consequences of such asymmetric localization and homeostasis of plasmalogens are discussed in this review.

Analysis of Peroxisome Biogenesis by Phos-Tag SDS-PAGE.

Phos-tag, a selective phosphate-binding molecule, and Phos-tag-based methodologies have been developed to investigate the phosphoproteome. In various analytical techniques using Phos-tag derivatives, phosphate-affinity electrophoresis using Phos-tag acrylamide, called Phos-tag SDS-PAGE, enables separation of phosphorylated proteins with a slower migration from non-phosphorylated proteins in polyacrylamide gels. The procedures for Phos-tag SDS-PAGE are largely common to those for conventional SDS-PAGE, thus being readily available for all laboratories. Phos-tag SDS-PAGE is widely applied to quantitative analysis of the overall phosphorylation state depending on the number and/or sites of the phosphate group. Phos-tag SDS-PAGE has also been introduced to the field of peroxisome study, including oxidative stress-induced and mitosis-specific phosphorylation of Pex14, a central component of the translocation machinery complex for peroxisomal matrix proteins. Here, we describe a practical protocol for Phos-tag SDS-PAGE and its application to peroxisome biogenesis research.

Alterations in ether lipid metabolism and the consequences for the mouse lipidome.

Alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1) are enzymes involved in ether lipid metabolism. While AGMO degrades plasmanyl lipids by oxidative cleavage of the ether bond, PEDS1 exclusively synthesizes a specific subclass of ether lipids, the plasmalogens, by introducing a vinyl ether double bond into plasmanylethanolamine phospholipids. Ether lipids are characterized by an ether linkage at the sn-1 position of the glycerol backbone and they are found in membranes of different cell types. Decreased plasmalogen levels have been associated with neurological diseases like Alzheimer's disease. Agmo-deficient mice do not present an obvious phenotype under unchallenged conditions. In contrast, Peds1 knockout mice display a growth phenotype. To investigate the molecular consequences of Agmo and Peds1 deficiency on the mouse lipidome, five tissues from each mouse model were isolated and subjected to high resolution mass spectrometry allowing the characterization of up to 2013 lipid species from 42 lipid subclasses. Agmo knockout mice moderately accumulated plasmanyl and plasmenyl lipid species. Peds1-deficient mice manifested striking changes characterized by a strong reduction of plasmenyl lipids and a concomitant massive accumulation of plasmanyl lipids resulting in increased total ether lipid levels in the analyzed tissues except for the class of phosphatidylethanolamines where total levels remained remarkably constant also in Peds1 knockout mice. The rate-limiting enzyme in ether lipid metabolism, FAR1, was not upregulated in Peds1-deficient mice, indicating that the selective loss of plasmalogens is not sufficient to activate the feedback mechanism observed in total ether lipid deficiency.

Regulation of plasmalogen biosynthesis in mammalian cells and tissues.

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. Synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. The absence of plasmalogens in several organs of patients with deficiency in peroxisome biogenesis suggests that de novo synthesis of plasmalogens contributes significantly to plasmalogen homeostasis in humans. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. Dysregulation of plasmalogen synthesis impairs cholesterol synthesis in cells and brain, resulting in the reduced expression of genes such as mRNA encoding myelin basic protein, a phenotype found in the cerebellum of plasmalogen-deficient mice. In this review, we summarize the current knowledge of molecular mechanisms underlying the regulation of plasmalogen biosynthesis and the link between plasmalogen homeostasis and cholesterol biosynthesis, and address the pathogenesis of impaired plasmalogen homeostasis in rodent and humans.

Genetic defects in peroxisome morphogenesis (Pex11ß, dynamin-like protein 1, and nucleoside diphosphate kinase 3) affect docosahexaenoic acid-phospholipid metabolism.

Peroxisomes are essential organelles involved in lipid metabolisms including plasmalogen biosynthesis and ß-oxidation of very long-chain fatty acids. Peroxisomes proliferate by the growth and division of pre-existing peroxisomes. The peroxisomal membrane is elongated by Pex11ß and then divided by the dynamin-like GTPase, DLP1 (also known as DRP1 encoded by DNM1L gene), which also functions as a fission factor for mitochondria. Nucleoside diphosphate kinase 3 (NME3) localized in both peroxisomes and mitochondria generates GTP for DLP1 activity. Deficiencies of either of these factors induce abnormal morphology of peroxisomes and/or mitochondria, and are associated with central nervous system dysfunction. To investigate whether the impaired division of peroxisomes affects lipid metabolisms, we assessed the phospholipid composition of cells lacking each of the different division factors. In fibroblasts from the patients deficient in DLP1, NME3, or Pex11ß, docosahexaenoic acid (DHA, C22:6)-containing phospholipids were found to be decreased. Conversely, the levels of several fatty acids such as arachidonic acid (AA, C20:4) and oleic acid (C18:1) were elevated. Mouse embryonic fibroblasts from Drp1- and Pex11ß-knockout mice also showed a decrease in the levels of phospholipids containing DHA and AA. Collectively, these results suggest that the dynamics of organelle morphology exert marked effects on the fatty acid composition of phospholipids.

Purification and characterization of protease M, a yeast mitochondrial nucleotide-stimulated metal protease: its identification as CYM1 gene product, a mitochondrial presequence peptidase.

A chelator-sensitive protease in the mitochondrial matrix of the yeast, Saccharomyces cerevisiae (Biochem. Biophys. Res. Commun. 144, 277, 1987), was purified and characterized. The purified enzyme, termed protease M, specifically hydrolyzes peptide substrates on the N-side of the paired basic residues. When mastoparan was used as substrate, it cleaved Ala8-Leu9 and Lys11-Lys12 bonds as well as the N-side of Lys11-Lys12 residues. Nucleotide triphosphates stimulated the activity 3-fold at 2.5 mM. The genomic DNA sequence showed that protease M was a gene product of CYM1 known as mitochondrial presequence protease homologue in S. cerevisiae, encoding a 989-amino acid-long precursor protein. The N-terminal sequence of the purified enzyme indicated that protease M has 16-residue signal sequence and the 'mature' protein consists of 973 amino acids with a molecular mass of 110 kDa. Protease M contained consensus sequence motifs of ATP-binding site very near the carboxyl terminus. The alignment of the two ATP-binding motifs is an inverted version of the common alignment. Gene disruption of the enzyme generates mixed subunits in tetrameric MnSOD formed with 23-kDa mature and 24-kDa partial presequence-containing subunits. This report describes newly identified enzyme properties of the CYM1 gene product, protease M and abnormal MnSOD complex formation of the disruption mutant.

Molecular insights into peroxisome homeostasis and peroxisome biogenesis disorders.

Peroxisomes are single-membrane organelles essential for cell metabolism including the ß-oxidation of fatty acids, synthesis of etherlipid plasmalogens, and redox homeostasis. Investigations into peroxisome biogenesis and the human peroxisome biogenesis disorders (PBDs) have identified 14 PEX genes encoding peroxins involved in peroxisome biogenesis and the mutation of PEX genes is responsible for the PBDs. Many recent findings have further advanced our understanding of the biology, physiology, and consequences of a functional deficit of peroxisomes. In this Review, we discuss cell defense mechanisms that counteract oxidative stress by 1) a proapoptotic Bcl-2 factor BAK-mediated release to the cytosol of H2O2-degrading catalase from peroxisomes and 2) peroxisomal import suppression of catalase by Ser232-phosphorylation of Pex14, a docking protein for the Pex5-PTS1 complex. With respect to peroxisome division, the important issue of how the energy-rich GTP is produced and supplied for the division process was recently addressed by the discovery of a nucleoside diphosphate kinase-like protein, termed DYNAMO1 in a lower eukaryote, which has a mammalian homologue NME3. In regard to the mechanisms underlying the pathogenesis of PBDs, a new PBD model mouse defective in Pex14 manifests a dysregulated brain-derived neurotrophic factor (BDNF)-TrkB pathway, an important signaling pathway for cerebellar morphogenesis. Communications between peroxisomes and other organelles are also addressed.

ATP8B2-Mediated Asymmetric Distribution of Plasmalogens Regulates Plasmalogen Homeostasis and Plays a Role in Intracellular Signaling.

Plasmalogens are a subclass of glycerophospholipid containing vinyl-ether bond at the sn-1 position of glycerol backbone. Ethanolamine-containing plasmalogens (plasmalogens) are major constituents of cellular membranes in mammalian cells and de novo synthesis of plasmalogens largely contributes to the homeostasis of plasmalogens. Plasmalogen biosynthesis is regulated by a feedback mechanism that senses the plasmalogen level in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (Far1), a rate-limiting enzyme for plasmalogen biosynthesis. However, the molecular mechanism underlying the localization of plasmalogens in cytoplasmic leaflet of plasma membrane remains unknown. To address this issue, we attempted to identify a potential transporter of plasmalogens from the outer to the inner leaflet of plasma membrane by focusing on phospholipid flippases, type-IV P-type adenosine triphosphatases (P4-ATPase), localized in the plasma membranes. We herein show that knockdown of ATP8B2 belonging to the class-1 P4-ATPase enhances localization of plasmalogens but not phosphatidylethanolamine in the extracellular leaflet and impairs plasmalogen-dependent degradation of Far1. Furthermore, phosphorylation of protein kinase B (AKT) is downregulated by lowering the expression of ATP8B2, which leads to suppression of cell growth. Taken together, these results suggest that enrichment of plasmalogens in the cytoplasmic leaflet of plasma membranes is mediated by ATP8B2 and this asymmetric distribution of plasmalogens is required for sensing plasmalogens as well as phosphorylation of AKT.

De novo formation and maintenance of mammalian peroxisomes in cultured PEX16-knockout cells generated by CRISPR/Cas9.

Mammalian PEX16 has been considered essential for generating and maintaining peroxisomal membranes. This view is based primarily on the finding that fibroblasts from several PEX16-deficient patients are devoid of peroxisomal structures but can form peroxisomes upon expression of PEX16. However, unlike these patient-derived cells, pex16 mutants in other model organisms contain partially functional peroxisomes. Here, we report that PEX16-knockout (KO) cells derived from three mammalian cultured cell lines comprise cells containing a fewer number of enlarged peroxisomes and cells lacking peroxisomes. We also suggest that PEX16 accelerates the process by which peroxisome-less cells form peroxisomal membranes and subsequently establish mature peroxisomes, independently of its ability to mediate peroxisomal targeting of PEX3. Nevertheless, PEX16 is not absolutely required for this process. Moreover, a well-known patient-derived PEX16 mutant inhibits the de novo formation of peroxisomal membranes. Our findings suggest that although PEX16 is undoubtedly important for optimal peroxisomal membrane biogenesis, mammalian cells may be able to form peroxisomes de novo and maintain the organelles without the aid of PEX16.

Identification of a Homozygous PEX26 Mutation in a Heimler Syndrome Patient.

This study aimed to identify the molecular genetic etiology of an 8-year-old boy with amelogenesis imperfecta in permanent dentition. Bilateral cochlear implants were placed due to sensorineural hearing loss, and there was no other family member with a similar phenotype. Peripheral blood samples were collected with the understanding and written consent of the participating family members. A constitutional chromosome study was performed for the proband. Genomic DNA was isolated, and whole exome sequencing was performed. A series of bioinformatic analyses were performed with the obtained paired-end sequencing reads, and the variants were filtered and annotated with dbSNP147. There was no abnormality in the constitutional chromosome study. Whole exome sequencing analysis with trio samples identified a homozygous mutation (c.506T>C, p. (Leu169Pro)) in the PEX26 gene. We verified "temperature sensitivity (ts)" of patient-derived Pex26-L169P by expression in pex26 CHO mutant ZP167 cells to determine the effect of the L169P mutation on Pex26 function. The L169P mutation causes a mild ts-cellular phenotype representing the decreased peroxisomal import of catalase. This study supports the finding that the recessive mutations in PEX26 are associated with Heimler syndrome and demonstrates the importance of an early and correct diagnosis.

A New Paradigm in Catalase Research.

Recent findings provide evidence for dynamic and highly regulated dual subcellular localization of catalase, a hydrogen peroxide (H2O2)-metabolizing enzyme, in peroxisomes and the cytosol. These data suggest a number of important implications for the field of oxidative stress biology.

Peroxisome Deficiency Impairs BDNF Signaling and Memory.

Peroxisome is an intracellular organelle that functions in essential metabolic pathways including ß-oxidation of very-long-chain fatty acids and biosynthesis of plasmalogens. Peroxisome biogenesis disorders (PBDs) manifest severe dysfunction in multiple organs including central nervous system (CNS), whilst the pathogenic mechanisms are largely unknown. We recently reported that peroxisome-deficient neural cells secrete an increased level of brain-derived neurotrophic factor (BDNF), resulting in the cerebellar malformation. Peroxisomal functions in adulthood brain have been little investigated. To induce the peroxisome deficiency in adulthood brain, we here established tamoxifen-inducible conditional Pex2-knockout mouse. Peroxisome deficiency in the conditional Pex2-knockout adult mouse brain induces the upregulated expression of BDNF and its inactive receptor TrkB-T1 in hippocampus, which notably results in memory disturbance. Our results suggest that peroxisome deficiency gives rise to the dysfunction of hippocampal circuit via the impaired BDNF signaling.

Distinct Functions of Acyl/Alkyl Dihydroxyacetonephosphate Reductase in Peroxisomes and Endoplasmic Reticulum.

Plasmalogens are a subclass of ether glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Plasmalogen biosynthesis is initiated in peroxisomes. At the third step of plasmalogen synthesis, alkyl-dihydroxyacetonephosphate (DHAP) is enzymatically reduced to 1-alkyl-sn-glycero-3-phospate by acyl/alkyl DHAP reductase (ADHAPR), whose activity is found in both peroxisomes and microsomes. We herein show that knockdown of ADHAPR in HeLa cells reduced the synthesis of ethanolamine plasmalogen (PlsEtn), similar to the Chinese hamster ovary cell mutant FAA.K1B deficient in ADHAPR activity. Endogenous ADHAPR and ectopically expressed FLAG-tagged ADHAPR were localized to peroxisomes and endoplasmic reticulum (ER) as a type I integral membrane protein in HeLa cells. ADHAPR targets to peroxisomes via a Pex19p-dependent class I pathway. In addition, it is also inserted into the ER via the SRP-dependent mechanism. The ADHAPR mutant lacking the N-terminal domain preferentially targets to the ER, restoring the reduced level of PlsEtn synthesis in FAA.K1B cell. In contrast, the expression of full-length ADHAPR in the mutant cells elevates the synthesis of phosphatidylethanolamine, but not PlsEtn. Taken together, these results suggest that the third step of plasmalogen synthesis is mediated by ER-localized ADHAPR.

Mammalian Homologue NME3 of DYNAMO1 Regulates Peroxisome Division.

Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology.

Molecular Basis of Mitochondrial and Peroxisomal Division Machineries.

Mitochondria and peroxisomes are ubiquitous subcellular organelles that are highly dynamic and possess a high degree of plasticity. These organelles proliferate through division of pre-existing organelles. Studies on yeast, mammalian cells, and unicellular algae have led to a surprising finding that mitochondria and peroxisomes share the components of their division machineries. At the heart of the mitochondrial and peroxisomal division machineries is a GTPase dynamin-like protein, Dnm1/Drp1, which forms a contractile ring around the neck of the dividing organelles. During division, Dnm1/Drp1 functions as a motor protein and constricts the membrane. This mechanochemical work is achieved by utilizing energy from GTP hydrolysis. Over the last two decades, studies have focused on the structure and assembly of Dnm1/Drp1 molecules around the neck. However, the regulation of GTP during the division of mitochondrion and peroxisome is not well understood. Here, we review the current understanding of Dnm1/Drp1-mediated divisions of mitochondria and peroxisomes, exploring the mechanisms of GTP regulation during the Dnm1/Drp1 function, and provide new perspectives on their potential contribution to mitochondrial and peroxisomal biogenesis.

The peroxisome counteracts oxidative stresses by suppressing catalase import via Pex14 phosphorylation.

Most of peroxisomal matrix proteins including a hydrogen peroxide (H2O2)-decomposing enzyme, catalase, are imported in a peroxisome-targeting signal type-1 (PTS1)-dependent manner. However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H2O2 in mammalian cells. The H2O2-induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively impairs in vitro the interaction of catalase with the Pex14-Pex5 complex. A phosphomimetic mutant Pex14-S232D elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H2O2. We thus suggest that the H2O2-induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.

Mitotic phosphorylation of Pex14p regulates peroxisomal import machinery.

Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis.

Recent insights into peroxisome biogenesis and associated diseases.

Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs. A number of recent findings have advanced our understanding of the biology, physiology and consequences of functional defects in peroxisomes. In this Review, we discuss a cooperative cell defense mechanisms against oxidative stress that involves the localization of BAK (also known as BAK1) to peroxisomes, which alters peroxisomal membrane permeability, resulting in the export of catalase, a peroxisomal enzyme. Another important recent finding is the discovery of a nucleoside diphosphate kinase-like protein that has been shown to be essential for how the energy GTP is generated and provided for the fission of peroxisomes. With regard to PBDs, we newly identified a mild mutation, Pex26-F51L that causes only hearing loss. We will also discuss findings from a new PBD model mouse defective in Pex14, which manifested dysregulation of the BDNF-TrkB pathway, an essential signaling pathway in cerebellar morphogenesis. Here, we thus aim to provide a current view of peroxisome biogenesis and the molecular pathogenesis of PBDs.

A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid ß-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid ß-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway.

Reactive oxygen species (ROS) play critical roles in metabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators of oxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress.