Language：English   Publishing type：Research paper (scientific journal)  

Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

DOI： 10.1074/jbc.M115.656983

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.

DOI： 10.1074/jbc.M113.498345

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the
past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In
the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated
from patients that carry defects in peroxisomal fatty acid b-oxidation, peroxisomes are much less abundant than normal cells. Treatment
of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced
peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with
dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyperoligomerization
of Pex11pb and the formation of Pex11pb-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of
peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction
followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that
DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

DOI： 10.1242/​jcs.087452

Other Link： http://jcs.biologists.org/content/125/3/589.long

Language：English   Publishing type：Research paper (scientific journal)  

We earlier isolated peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, ZPEG241, by the 9-(1′-pyrene)nonanol/ultraviolet selection method, from TKaEG2, the wild-type CHO-K1 cells transformed with two cDNAs encoding rat Pex2p and peroxisome targeting signal type 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). Peroxisomal localization of PTS2-EGFP was specifically impaired in ZPEG241 due to the failure of Pex5pL expression. Analysis of partial genomic sequence of PEX5 revealed one-point nucleotide-mutation from G to A in the 3′-acceptor splice site located at 1 nt upstream of exon 7 encoding Pex5pL specific 37-amino acid insertion, thereby generating 21-nt deleted mRNA of PEX5L in ZPEG241. When ZPEG241-derived Pex5pL was ectopically expressed in ZPEG241, PTS2 import was not restored because of no interaction with Pex7p. Together, we confirm the pivotal role of Pex5pL in PTS2 import, showing that the N-terminal 7-amino acid residues in the 37-amino acid insertion of Pex5pL are essential for the binding to Pex7p.

DOI： 10.1093/jb/mvq130

Language：English   Publishing type：Research paper (scientific journal)  

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.

DOI： 10.1074/jbc.M109.083311

Language：English   Publishing type：Research paper (scientific journal)  

We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PlsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-dihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PlsEtn, we investigated its subcellular localization. PlsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PlsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PlsEtn was not essential for lipid-raft architecture in CHO cells.

DOI： 10.1016/j.bbamcr.2008.05.018

Language：English   Publishing type：Research paper (scientific journal)  

Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant membranes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose, we sought to investigate whether extraction with different detergents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRMs prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol over glycerophospholipids as well as saturated over unsaturated phosphatidylcholine. In addition, we observed cell type-dependent variations of the molecular characteristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.

DOI： 10.1073/pnas.0631579100

Language：English   Publishing type：Research paper (scientific journal)  

Previously we isolated human PEX16 encoding 336-amino acid-long peroxin Pex16p and showed that its dysfunction was responsible for Zellweger syndrome of complementation group D (group 9). Here we have determined the membrane topology of Pex16p by differential permeabilization method: both N- and C-terminal parts are exposed to the cytosol. In the search for Pex16p topogenic sequence, basic amino acids clustered sequence, RKELRKKLPVSLSQQK, at positions 66-81 and the first transmembrane segment locating far downstream, nearly by 40 amino acids, of this basic region were defined to be essential for integration into peroxisome membranes. Localization to peroxisomes of membrane proteins such as Pex14p, Pex13p, and PMP70 was interfered with in CHO-K1 cells by a higher level expression of the pex16 patient-derived dysfunctional but topogenically active Pex16pR176ter comprising resides 1-176 or of the C-terminal cytoplasmic part starting from residues at 244 to the C terminus. Furthermore, Pex16p C-terminal cytoplasmic part severely abrogated peroxisome restoration in pex mutants such as matrix protein import-defective pex12 and membrane assembly impaired pex3 by respective PEX12 and PEX3 expression, whereas the N-terminal cytosolic region did not affect restoration. These results imply that Pex16p functions in peroxisome membrane assembly, more likely upstream of Pex3p.

DOI： 10.1074/jbc.M206139200

Language：English   Publishing type：Research paper (scientific journal)  

Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii. We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein. PMP34 was characterized as an integral membrane protein of peroxisomes. Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol. Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity. Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes. Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal.

DOI： 10.1074/jbc.M003304200

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which >10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope- tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C→T at position 526 in a codon (CGA) for ¹⁷⁶ Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.

DOI： 10.1086/302161

Language：English   Publishing type：Research paper (scientific journal)  

Cytochrome b₅ (b5), a typical tail-anchored protein of the endoplasmic reticulum (ER) membrane, is composed of three functionally different domains: amino-terminal heine-containing catalytic, central hydrophobic membrane- anchoring, and carboxyl-terminal ER-targeting domains (Mitoma, J., and Ito, A. (1992) EMBO J. 11, 41974203). To analyze the potential retention signal of b5, mutant proteins were prepared to replace each domain with natural or artificial sequences, and subcellular localizations were examined using immunofluorescence microscopy and cell fractionation. The transmembrane domain functioned to retain the cytochrome in the ER, and the mutation of all or part of the transmembrane domain with an artificial hydrophobic sequence had practically no effect on intracellular distribution of the cytochrome. However, when the transmembrane domain was extended systematically, a substantial portion of the protein with the domain of over 22 amino acid residues leaked from the organelle. Thus, the transmembrane length functions as the retention signal. When cytochromes with mutations at the carboxyl- terminal end were overexpressed in cells, a substantial portion of the protein was transported to the plasma membrane, indicating that the carboxyl- terminal luminal domain also has a role in retention of b5 in the ER. Carbohydrate moiety of the glycosylatably-mutated b5 was sensitive to endoglycosidase H but resistant to endoglycosidase D. Therefore, both transmembrane and carboxylterminal portions seems to function as the static retention signal.

DOI： 10.1074/jbc.273.33.20860

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers in Cell and Developmental Biology  

Plasmalogens (Pls) are specialized phospholipids integral to brain health, whose decline due to aging and stress contributes to cognitive impairment and neuroinflammation. This study explores the potential of a novel Pls derivative, KIT-13 (1-O-octadecyl-2-arachidonoyl-sn-glycerol-3-phosphoethanolamine), in mitigating neuroinflammation and enhancing cognition. When administered to mice, KIT-13 exhibited potent memory enhancement attributed to upregulated brain-derived neurotrophic factor (BDNF), a key player in cognitive processes. In vitro experiments with neuronal cells revealed KIT-13's ability to induce robust cellular signaling, surpassing natural plasmalogens. KIT-13 also promoted neurogenesis and inhibited apoptosis of neuronal-like cells, highlighting its potential in fostering neuronal growth and plasticity. Additionally, KIT-13 treatments reduced pro-inflammatory cytokine expression and attenuated glial activation in the brain. KIT-13's superior efficacy over natural Pls positions it as a promising therapeutic candidate for neurodegenerative conditions such as Alzheimer's disease, characterized by cognitive decline and neuroinflammation. This study presents KIT-13 as an innovative approach for addressing cognitive impairment and neuroinflammatory pathologies.

DOI： 10.3389/fcell.2024.1443536

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Resource Announcements  

The genome sequence of Lentilactobacillus buchneri subsp. silagei MGR2-32, isolated from guinea grass silage, is 2,540,137 bp, has a GC content of 44%, and contains 2,393 predicted protein-coding genes. Pairwise average nucleotide identity and digital DNA-DNA hybridization values between MGR2-32 and the type strain were 99.75% and 99.90%, respectively.

DOI： 10.1128/mra.00695-23

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Language：English   Publishing type：Research paper (scientific journal)  

The whole-genome sequence of strain K-4, isolated from grass silage in Thailand, which constitutes a chromosome and two plasmids, is 2,914,933 bp long, has a GC content of 37.5%, and contains 2,734 predicted protein-coding genes. Average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) values indicated that the strain K-4 was closely related to Enterococcus faecalis.

DOI： 10.1128/mra.00814-22

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbalip.2023.159285

Language：English   Publishing type：Research paper (scientific journal)  

Ethanolamine-containing alkenyl ether glycerophospholipids, plasmalogens, are major cell membrane components of mammalian cells that activate membrane protein receptors such as ion transporters and G-protein coupled receptors. However, the mechanism by which plasmalogens modulate receptor function is unknown. Here, we found that exogenously added plasmalogens activate transient receptor potential cation channel subfamily C member 4 (TRPC4) to increase Ca2+ influx, followed by calcium/calmodulin-dependent protein kinase 2-mediated phosphorylation of AMP-activated protein kinase (AMPK). Upon topical application of plasmalogens to the skin of mice, AMPK activation was observed in TRPC4-expressing hair bulbs and hair follicles. Here, TRPC4 was co-localized with the leucine-rich repeat containing G protein-coupled receptor 5, a marker of hair-follicle stem cells, leading to hair growth. Collectively, this study indicates that plasmalogens could function as gate openers for TRPC4, followed by activating AMPK, which likely accelerates hair growth in mice.

DOI： 10.3389/fmolb.2022.1008626

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.3389/fmolb.2022.915457

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome is an intracellular organelle that functions in essential metabolic pathways including β-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.

DOI： 10.3389/fcell.2020.567017

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1083/jcb.202001003

Other Link： https://rupress.org/jcb/article/219/10/e202001003/152047/Mitotic-phosphorylation-of-Pex14p-regulates?searchresult=1

Language：English   Publishing type：Research paper (scientific journal)  

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-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 β-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.

DOI： 10.1074/jbc.RA119.011989

Other Link： https://doi.org/10.1074/jbc.RA119.011989

Language：English   Publishing type：Research paper (scientific journal)  

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. Despite its importance in metabolism and disease, a comprehensive analysis of the cellular response to oxidative stress is lacking. Here, Dubreuil et al. use genome-wide screens to identify cellular regulators of oxidative stress. They investigate paradoxical mechanisms by which disruption of the pentose phosphate and peroxisomal import pathways protect cells.

DOI： 10.1016/j.celrep.2020.01.013

Language：English   Publishing type：Research paper (scientific journal)  

Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

DOI： 10.1242/jcs.223016

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome biogenesis disorders (PBDs) manifest as neurological deficits in the central nervous system, including neuronal migration defects and abnormal cerebellum development. However, the mechanisms underlying pathogenesis remain enigmatic. Here, to investigate how peroxisome deficiency causes neurological defects of PBDs, we established a new PBD model mouse defective in peroxisome assembly factor Pex14p, termed Pex14^ΔC/ΔC mouse. Pex14^ΔC/Δ^C mouse manifests a severe symptom such as disorganization of cortical laminar structure and dies shortly after birth, although peroxisomal biogenesis and metabolism are partially defective. The Pex14^ΔC/Δ^C mouse also shows malformation of the cerebellum including the impaired dendritic development of Purkinje cells. Moreover, extracellular signal-regulated kinase and AKT signaling are attenuated in this mutant mouse by an elevated level of brain-derived neurotrophic factor (BDNF) together with the enhanced expression of TrkB-T1, a dominant-negative isoform of the BDNF receptor. Our results suggest that dysregulation of the BDNF-TrkB pathway, an essential signaling for cerebellar morphogenesis, gives rise to the pathogenesis of the cerebellum in PBDs.

DOI： 10.26508/lsa.201800062

Language：English   Publishing type：Research paper (scientific journal)  

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance.

DOI： 10.1038/s41467-018-07009-z

Language：English   Publishing type：Research paper (scientific journal)  

Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer’s disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto the Gnpat promoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation of Gnpat in the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism of Gnpat reduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases.

DOI： 10.1523/JNEUROSCI.3941-15.2017

Language：English   Publishing type：Research paper (scientific journal)  

Organelle division is executed through contraction of a ring-shaped supramolecular dividing machinery. A core component of the machinery is the dynamin-based ring conserved during the division of mitochondrion, plastid and peroxisome. Here, using isolated peroxisome-dividing (POD) machinery from a unicellular red algae, Cyanidioschyzon merolae, we identified a dynamin-based ring organizing center (DOC) that acts as an initiation point for formation of the dynamin-based ring. C. merolae contains a single peroxisome, the division of which can be highly synchronized by light-dark stimulation; thus, intact POD machinery can be isolated in bulk. Dynamin-based ring homeostasis is maintained by the turnover of the GTP-bound form of the dynamin-related protein Dnm1 between the cytosol and division machinery via the DOC. A single DOC is formed on the POD machinery with a diameter of 500-700 nm, and the dynamin-based ring is unidirectionally elongated from the DOC in a manner that is dependent on GTP concentration. During the later step of membrane fission, the second DOC is formed and constructs the double dynamin-based ring to make the machinery thicker. These findings provide new insights to define fundamental mechanisms underlying the dynamin-based membrane fission in eukaryotic cells.

DOI： 10.1242/jcs.199182

Language：English   Publishing type：Research paper (scientific journal)  

Pex11p family proteins are key players in peroxisomal fission, but their molecular mechanisms remains mostly unknown. In the present study, overexpression of Pex11pb caused substantial vesiculation of peroxisomes in mammalian cells. This vesicle formation was dependent on dynamin-like protein 1 (DLP1) and mitochondrial fission factor (Mff), as knockdown of these proteins diminished peroxisomal fission after Pex11pb overexpression. The fission-deficient peroxisomes exhibited an elongated morphology, and peroxisomal marker proteins, such as Pex14p or matrix proteins harboring peroxisomal targeting signal 1, were discernible in a segmented staining pattern, like beads on a string. Endogenous Pex11pb was also distributed a striped pattern, but which was not coincide with Pex14p and PTS1 matrix proteins. Altered morphology of the lipid membrane was observed when recombinant Pex11p proteins were introduced into proteo-liposomes. Constriction of proteo-liposomes was observed under confocal microscopy and electron microscopy, and the reconstituted Pex11pb protein localized to the membrane constriction site. Introducing point mutations into the N-terminal amphiphathic helix of Pex11pb strongly reduced peroxisomal fission, and decreased the oligomer formation. These results suggest that Pex11p contributes to the morphogenesis of the peroxisomal membrane, which is required for subsequent fission by DLP1.

DOI： 10.1242/bio.201410801

Language：English   Publishing type：Research paper (scientific journal)  

Rhizomelic chondrodysplasia punctata (RCDP) is an autosomal recessive disorder due to the deficiency in ether lipid synthesis. RCDP type 1, the most prominent type, is caused by the dysfunction of the receptor of peroxisome targeting signal type 2, Pex7 (peroxisomal biogenesis factor 7), and the rest of the patients, RCDP types 2 and 3, have defects in peroxisomal enzymes catalyzing the initial two steps of alkyl-phospholipid synthesis, glyceronephosphate O-acyltransferase and alkylglycerone phosphate synthase (Agps). We herein investigated defects of two patients with RCDP type 3. Patient 1 had a novel missense mutation, T1533G, resulting in the I511M substitution in Agps. The plasmalogen level was mildly reduced, whereas the protein level and peroxisomal localization of Agps-I511M in fibroblasts were normal as in the control fibroblasts. Structure prediction analysis suggested that the mutated residue was located in the helix α15 on the surface of V-shaped active site tunnel in Agps, likely accounting for the mild defects of plasmalogen synthesis. These results strongly suggest that an individual with mildly affected level of plasmalogen synthesis develops RCDP. In fibroblasts from patient 2, the expression of AGPS mRNA and Agps protein was severely affected, thereby giving rise to the strong reduction of plasmalogen synthesis.

DOI： 10.1038/jhg.2014.39

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.

DOI： 10.1016/j.bbalip.2014.01.001

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisomal division comprises three steps: elongation, constriction, and fission. Translocation of dynamin-like protein 1 (DLP1), a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission; however, the molecular machinery for peroxisomal targeting of DLP1 remains unclear. This study investigated whether mitochondrial fission factor (Mff), which targets DLP1 to mitochondria, may also recruit DLP1 to peroxisomes. Results show that endogenous Mff is localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes, in addition to mitochondria. Knockdown of MFF abrogates the fission stage of peroxisomal division and is associated with failure to recruit DLP1 to peroxisomes, while ectopic expression of MFF increases the peroxisomal targeting of DLP1. Co-expression of MFF and PEX11β, the latter being a key player in peroxisomal elongation, increases peroxisome abundance. Overexpression of MFF also increases the interaction between DLP1 and Pex11pβ, which knockdown of MFF, but not Fis1, abolishes. Moreover, results show that Pex11pβ interacts with Mff in a DLP1-dependent manner. In conclusion, Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pβ and DLP1.

DOI： 10.1242/bio.20135298

Language：English   Publishing type：Research paper (scientific journal)  

The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin- and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic β-amyloid peptide (Aβ), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of Aβ. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing.

DOI： 10.1523/JNEUROSCI.5345-07.2008

Language：English   Publishing type：Research paper (scientific journal)  

Although the exact etiology of Alzheimer's disease (AD) is a topic of debate, the consensus is that the accumulation of β-amyloid (Aβ) peptides in the senile plaques is one of the hallmarks of the progression of the disease. The Aβ peptide is formed by the amyloidogenic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. The endocytic system has been implicated, in the cleavages leading to the formation of Aβ. However, the identity of the intracellular compartment where the amyloidogenic secretases cleave and the mechanism by which the intracellularly generated Aβ is released into the extracellular milieu are not clear. Here, we show that β-cleavage occurs in early endosomes followed by routing of Aβ to multivesicular bodies (MVBs) in HeLa and N2a cells. Subsequently, a minute fraction of Aβ peptides can be secreted from the cells in association with exosomes, intraluminal vesicles of MVBs that are released into the extracellular space as a result of fusion of MVBs with the plasma membrane. Exosomal proteins were found to accumulate in the plaques of AD patient brains, suggesting a role in the pathogenesis of AD.

DOI： 10.1073/pnas.0603838103

Language：English   Publishing type：Research paper (scientific journal)  

RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell.

DOI： 10.1073/pnas.0401285101

Language：English   Publishing type：Research paper (scientific journal)  

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.

DOI： 10.1016/S0014-5793(03)00135-2

Language：English   Publishing type：Research paper (scientific journal)  

Subfractionation studies showed that cytochrome b₅ (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

DOI： 10.1093/jb/mvg009

Language：English   Publishing type：Research paper (scientific journal)  

Peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy are autosomal recessive diseases caused by defects in peroxisome assembly, for which 13 genotypes have been identified. Expression of the human peroxin Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in fibroblasts from PBDG-02, a patient with complementation group G (CG-G) ZS. Patient PBDG-02 carried a homozygous, inactivating mutation - a 97-bp deletion of nucleotide residues at positions 942-1038 - resulting in a 32-amino-acid truncation and in a frameshift inducing both a 3-amino-acid substitution and a termination codon. Genomic PCR analysis revealed mutation of T→G at eight bases upstream of the splicing site at the boundary of intron 10 and exon 11 of PEX3 gene, giving rise to a deletion of all of exon 11. When assessed by expression in a pex3 mutant of Chinese hamster ovary cells and the patient's fibroblasts, PBDG-02-derived PEX3 cDNA was found to be defective in peroxisome-restoring activity. These results provide evidence that PEX3 is a novel, pathogenic gene responsible for CG-G PBDs.

DOI： 10.1086/303086

Language：English   Publishing type：Research paper (scientific journal)  

In mammals, two isoforms of the peroxisome targeting signal (PTS) type 1 receptor Pex5p, i.e. Pex5pS and Pex5pL with an internal 37-amino acid insertion, have previously been identified. Expression of either type of Pex5p complements the impaired PTS1 import in Chinese hamster ovary pex5 mutants, but only Pex5pL can rescue the PTS2 import defect noted in a subgroup of pex5 mutants such as ZP105. In this work, we found that Pex5pL directly interacts with the PTS2 receptor Pex7p, carrying its cargo PTS2 protein in the cytosol. Pex5pL, but not Pex5pS, mediated the binding of PTS2 protein to Pex14p by translocating Pex7p, demonstrating that Pex5pL plays a pivotal role in peroxisomal PTS2 import. Pex5p was localized mostly in the cytosol in wild-type CHO-K1 and Pex14p-deficient mutant cells, whereas it accumulated in the peroxisomal remnants in cell mutants defective in Pex13p or the RING family peroxins such as Pex2p and Pex12p. Furthermore, overexpression of Pex14p, but not Pex10p, Pex12p, or Pex13p, caused accumulation of Pex5p in peroxisomal membranes, with concomitant interference with PTS1 and PTS2 import. Therefore, Pex5p carrying the cargoes most likely docks with the initial site (Pex14p) in a putative import machinery, subsequently translocating to other components such as Pex13p, Pex2p, Pex10p, and Pex12p.

DOI： 10.1074/jbc.M000720200

Language：English   Publishing type：Research paper (scientific journal)  

Outer mitochondrial membrane cytochrome b₅ (OMb), which is an isoform of cytochrome b₅ (cyt b₅) in the endoplasmic reticulum, is a typical tail- anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b₅ that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amine acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b₅, in which the acidic amine acid in its carboxyl-terminal end was replaced by basic amine acid, could be transported to the mitochondria. It would thus seem that charged amine acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.

DOI： 10.1074/jbc.273.47.31097

Language：English   Publishing type：Research paper (scientific journal)  

Cytochrome b₅ is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions. In situ topology of cytochrome b₅ in the ER membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b₅) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b₅ and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b₅ antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immune-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b₅ was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b₅ only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.

DOI： 10.1093/oxfordjournals.jbchem.a021486

Responsible for pages：Medical Science Digest Vol 48 (1) , 2022   Language：Japanese  

Responsible for pages：Lizard G. (eds) Peroxisome Biology: Experimental Models, Peroxisomal Disorders and Neurological Diseases. Advances in Experimental Medicine and Biology, vol 1299, 3-17   Language：English  

Peroxisome is an organelle conserved in almost all eukaryotic cells with a variety of functions in cellular metabolism, including fatty acid β-oxidation, synthesis of ether glycerolipid plasmalogens, and redox homeostasis. Such metabolic functions and the exclusive importance of peroxisomes have been highlighted in fatal human genetic disease called peroxisomal biogenesis disorders (PBDs). Recent advances in this field have identified over 30 PEX genes encoding peroxins as essential factors for peroxisome biogenesis in various species from yeast to humans. Functional delineation of the peroxins has revealed that peroxisome biogenesis comprises the processes, involving peroxisomal membrane assembly, matrix protein import, division, and proliferation. Catalase, the most abundant peroxisomal enzyme, catalyzes decomposition of hydrogen peroxide. Peroxisome plays pivotal roles in the cellular redox homeostasis and the response to oxidative stresses, depending on intracellular localization of catalase.

DOI： 10.1007/978-3-030-60204-8_1

Responsible for pages：Lizard G. (eds) Peroxisome Biology: Experimental Models, Peroxisomal Disorders and Neurological Diseases. Advances in Experimental Medicine and Biology, vol 1299, 45-54   Language：English  

Peroxisomes are presented in all eukaryotic cells and play essential roles in many of lipid metabolic pathways, including β-oxidation of fatty acids and synthesis of ether-linked glycerophospholipids, such as plasmalogens. Impaired peroxisome biogenesis, including defects of membrane assembly, import of peroxisomal matrix proteins, and division of peroxisome, causes peroxisome biogenesis disorders (PBDs). Fourteen complementation groups of PBDs are found, and their complementing genes termed PEXs are isolated. Several new mutations in peroxins from patients with mild PBD phenotype or patients with phenotypes unrelated to the commonly observed impairments of PBD patients are found by next-generation sequencing. Exploring a dysfunctional step(s) caused by the mutation is important for unveiling the pathogenesis of novel mutation by means of cellular and biochemical analyses.

DOI： 10.1007/978-3-030-60204-8_4

Responsible for pages：Lizard G. (eds) Peroxisome Biology: Experimental Models, Peroxisomal Disorders and Neurological Diseases. Advances in Experimental Medicine and Biology, vol 1299, 119-143   Language：English  

Fourteen PEX genes are currently identified as genes responsible for peroxisome biogenesis disorders (PBDs). Patients with PBDs manifest as neurodegenerative symptoms such as neuronal migration defect and malformation of the cerebellum. To address molecular mechanisms underlying the pathogenesis of PBDs, mouse models for the PBDs have been generated by targeted disruption of Pex genes. Pathological phenotypes and metabolic abnormalities in Pex-knockout mice well resemble those of the patients with PBDs. The mice with tissue- or cell type-specific inactivation of Pex genes have also been established by using a Cre-loxP system. The genetically modified mice reveal that pathological phenotypes of PBDs are mediated by interorgan and intercellular communications. Despite the illustrations of detailed pathological phenotypes in the mutant mice, mechanistic insights into pathogenesis of PBDs are still underway. In this chapter, we overview the phenotypes of Pex-inactivated mice and the current understanding of the pathogenesis underlying PBDs.

DOI： 10.1007/978-3-030-60204-8_10

Responsible for pages：実験医学 7月号特集『ユビキチン化を介したオルガネロファジー』 Vol. 35 No. 11, 1824-1831  

Responsible for pages：Schrader, M. (ed.) Peroxisomes: Methods and Protocols, Methods in Molecular Biology 213-219   Language：English  

Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including β-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.

DOI： 10.1007/978-1-4939-6937-1_20

Responsible for pages：Schrader, M. (ed.) Peroxisomes: Methods and Protocols, Methods in Molecular Biology 207-212   Language：English  

The Pex19p- and Pex3p-dependent direct import of peroxisomal membrane proteins (PMPs), termed the class I pathway, can be reconstituted in vitro by incubating cell-free synthesized PMPs with highly purified peroxisomes at 26 °C for 1 h. This method ensures that the proteins targeted to peroxisomes are imported directly without involvement of other organelles.

DOI： 10.1007/978-1-4939-6937-1_19

Responsible for pages：別冊・医学のあゆみ 特集「ストレスシグナルと疾患―細胞恒常性維持機構の破綻と病態」 75-79   Language：Japanese  

Responsible for pages：機能性食品と薬理栄養 特集「プラズマローゲンと神経機能」 Vol. 9 No. 5, 322-327   Language：Japanese  

Responsible for pages：医学のあゆみ　特集「細胞内小器官とストレス」 Vol. 254 No. 5, 397-401   Language：Japanese  

Responsible for pages：Encyclopedia of Life Sciences   Language：English  

Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β‐oxidation of very long‐chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes.

DOI： 10.1002/9780470015902.a0002618.pub2

Responsible for pages：Brocard, C. and Hartig, A. (eds) Molecular machines involved in peroxisome biogenesis and maintenance, Springer-Verlag, Wien, Austria. 391-401   Language：English  

Peroxisomal division comprises three stages: elongation, constriction, and fission. Potential candidates thus far studied for the factors involved in these stages include Pex11pβ, dynamin-like protein 1 (DLP1), mitochondrial fission factor (Mff), and Fission 1 (Fis1). A poly-unsaturated fatty acid of peroxisomal β-oxidation metabolites, docosahexaenoic acid (C22:6n-3), augments hyper-oligomerization of Pex11pβ that gives rise to peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division. Translocation of DLP1, a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission. However, the molecular machinery for peroxisomal targeting of DLP1 remains elusive. Mff is also localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes. Knockdown of Mff abrogates the fission stage of peroxisomal division and fails to recruit DLP1 to peroxisomes, while ectopic expression of Mff increases the peroxisomal targeting of DLP1. Co-expression of Mff and Pex11pβ increases peroxisome abundance. Overexpression of Mff also increases the interaction between DLP1 and Pex11pβ, which knockdown of Mff, but not Fis1, abolishes. Moreover, Pex11pβ interacts with Mff in a DLP1-dependent manner. Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pβ and DLP1. The investigations performed to date suggest that a functional complex comprising Pex11pβ, Mff, and DLP1 promotes Mff-mediated fission during peroxisomal division. With regard to peroxisome morphogenesis, we address recent issues and findings and propose a model for peroxisome division.

DOI： 10.1007/978-3-7091-1788-0_17

Responsible for pages：医学のあゆみVol. 248 No. 13, 1143-1149   Language：Japanese  

Responsible for pages：生体の科学　特集「細胞の分子構造と機能―核以外の細胞小器官」 Vol. 63 No. 5, 448-451   Language：Japanese  

DOI： 10.11477/mf.2425101352

Responsible for pages：実験医学 Vol. 28 No. 13, 2094-2101   Language：Japanese  

Responsible for pages：Cell Biology : A Laboratory Handbook 2, 5-9   Language：English  

DOI： 10.1016/B978-012164730-8/50073-3

Responsible for pages：実験医学増刊 Vol. 21 No. 14, 1904-1911   Language：Japanese  

Event date： 2012.12

Venue：福岡県福岡市   Country：Japan  

Event date： 2012.6

Venue：福岡県福岡市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2009.10

Venue：兵庫県神戸市   Country：Japan  

Event date： 2009.5

Presentation type：Oral presentation (invited, special)  

Venue：福岡市   Country：Japan  

Event date： 2008.12

Venue：兵庫県神戸市   Country：Japan  

Event date： 2008.5

Venue：福岡市   Country：Japan  

Event date： 2000.10 - 2000.11

Venue：福岡市   Country：Japan  

Event date： 2024.11

Language：Japanese   Presentation type：Poster presentation  

Venue：横浜市   Country：Japan  

researchmap

Event date： 2024.10

Language：English  

Venue：Gold Coast, QLD, Australia.   Country：Australia  

researchmap

Event date： 2023.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東洋大学（東京都北区）   Country：Japan  

Event date： 2023.10 - 2023.11

Language：Japanese  

Venue：福岡国際会議場・マリンメッセ福岡（福岡市）   Country：Japan  

Event date： 2023.9

Language：English  

Venue：Medical University Vienna Center for Brain Research (Vienna)   Country：Austria  

Event date： 2022.11

Language：Japanese  

Venue：愛知県名古屋市   Country：Japan  

Event date： 2022.11

Language：Japanese  

Venue：愛知県名古屋市   Country：Japan  

Event date： 2022.10

Language：Japanese  

Venue：WEB   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：WEB   Country：Japan  

Event date： 2020.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：WEB   Country：Japan  

Event date： 2020.9

Language：Japanese  

Venue：WEB   Country：Japan  

Event date： 2019.12

Venue：福岡国際会議場、マリンメッセ福岡   Country：Japan  

Event date： 2019.12

Venue：福岡国際会議場、マリンメッセ福岡   Country：Japan  

Event date： 2019.12

Venue：福岡国際会議場、マリンメッセ福岡   Country：Japan  

Event date： 2019.10

Presentation type：Oral presentation (general)  

Venue：Abbaye de Talloires, Talloires   Country：France  

Event date： 2019.10

Presentation type：Oral presentation (general)  

Venue：Abbaye de Talloires, Talloires   Country：France  

Event date： 2019.10

Presentation type：Oral presentation (general)  

Venue：Abbaye de Talloires, Talloires   Country：France  

Event date： 2019.9

Venue：パシフィコ横浜   Country：Japan  

Event date： 2019.9

Venue：パシフィコ横浜   Country：Japan  

Event date： 2019.9

Venue：パシフィコ横浜   Country：Japan  

Event date： 2019.3

Venue：Eden Roc Hotel, Sant Feliu de Guixols   Country：Spain  

Event date： 2018.12

Venue：San Diego Convention Center, San Diego  

Event date： 2018.11

Presentation type：Symposium, workshop panel (public)  

Venue：パシフィコ横浜（横浜市）   Country：Japan  

Event date： 2018.8

Venue：Hotel Enryakuji Kaikan, Shiga   Country：Japan  

Event date： 2018.6 - 2018.7

Venue：九州大学医学部百年講堂   Country：Japan  

Event date： 2017.12

Venue：神戸ポートアイランド（神戸市）   Country：Japan  

Event date： 2017.6

Venue：京都大学百周年時計台記念館 (京都市）   Country：Japan  

Event date： 2017.6

Venue：仙台国際センター【仙台市】   Country：Japan  

Event date： 2016.11 - 2016.12

Venue：横浜パシフィコ（横浜市）   Country：Japan  

Event date： 2016.11 - 2016.12

Venue：横浜パシフィコ（横浜市）   Country：Japan  

Event date： 2016.11

Venue：Centennial Hall Kyushu University School of Medicine, Fukuoka   Country：Japan  

Event date： 2016.11

Venue：Centennial Hall Kyushu University School of Medicine, Fukuoka   Country：Japan  

Event date： 2016.11

Venue：Centennial Hall Kyushu University School of Medicine, Fukuoka   Country：Japan  

Event date： 2016.9

Venue：仙台国際センター／東北大学川内北キャンパス   Country：Japan  

Event date： 2016.9

Venue：仙台国際センター／東北大学川内北キャンパス   Country：Japan  

Event date： 2016.9

Venue：沖縄コンベンションセンター（沖縄県宜野湾市）   Country：Japan  

Event date： 2016.9

Venue：Medical University of Vienna,Vienna   Country：Austria  

Event date： 2016.7

Venue：東京大学　本郷キャンパス   Country：Japan  

Event date： 2016.2

Venue：Weizmann Institute of Science, Rehovot   Country：Israel  

Event date： 2015.12

Venue：神戸ポートアイランド（神戸市）   Country：Japan  

Event date： 2015.12

Venue：神戸ポートアイランド（神戸市）   Country：Japan  

Event date： 2015.7

Venue：Omaha, Nebraska  

Event date： 2015.6 - 2015.7

Venue：タワーホール船堀（東京都）   Country：Japan  

Event date： 2015.6

Venue：一橋大学一橋講堂（東京都千代田区）   Country：Japan  

Event date： 2015.2

Presentation type：Oral presentation (general)  

Venue：Keio Plaza Hotel, Tokyo   Country：Japan  

Event date： 2014.11

Venue：横浜市（横浜パシフィコ）   Country：Japan  

Event date： 2014.10

Venue：京都市（国立京都国際会館・グランドプリンスホテル京都）   Country：Japan  

Event date： 2014.10

Venue：京都市（国立京都国際会館・グランドプリンスホテル京都）   Country：Japan  

Event date： 2014.10

Venue：京都市（国立京都国際会館・グランドプリンスホテル京都）   Country：Japan  

Event date： 2014.10

Venue：京都市（国立京都国際会館・グランドプリンスホテル京都）   Country：Japan  

Event date： 2013.9

Venue：Comundo Conference Center, Neuss   Country：Germany  

Event date： 2013.9

Venue：神奈川県横浜市   Country：Japan  

Event date： 2013.9

Venue：神奈川県横浜市   Country：Japan  

Event date： 2013.9

Venue：神奈川県横浜市   Country：Japan  

Event date： 2012.12

Venue：福岡県福岡市   Country：Japan  

Event date： 2012.12

Venue：福岡県福岡市   Country：Japan  

Event date： 2012.12

Venue：福岡県福岡市   Country：Japan  

Event date： 2012.5

Venue：福岡県福岡市   Country：Japan  

Event date： 2011.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2011.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2011.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2011.12

Venue：Denver, USA  

Event date： 2011.11

Venue：福岡   Country：Japan  

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1), a peroxisomal C-tail anchored protein, supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.

Event date： 2011.11

Presentation type：Oral presentation (general)  

Venue：Fukuoka   Country：Japan  

Event date： 2011.9

Venue：京都府京都市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2010.12

Venue：神戸市   Country：Japan  

Event date： 2010.9

Venue：Nara   Country：Japan  

Event date： 2010.9

Venue：Nara   Country：Japan  

Event date： 2009.12

Venue：San Diego  

Event date： 2009.11

Venue：Seattle  

Event date： 2009.11

Venue：Seattle  

Event date： 2009.10

Venue：兵庫県神戸市   Country：Japan  

Event date： 2009.6

Venue：愛知県名古屋市   Country：Japan  

Event date： 2009.5

Venue：福岡市   Country：Japan  

Event date： 2008.12

Venue：San Francisco  

Event date： 2008.12

Venue：兵庫県神戸市   Country：Japan  

Event date： 2008.12

Venue：兵庫県神戸市   Country：Japan  

Event date： 2008.12

Venue：兵庫県神戸市   Country：Japan  

Event date： 2008.6 - 2008.7

Venue：神奈川県横浜市   Country：Japan  

Event date： 2007.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2007.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2007.12

Venue：神奈川県横浜市   Country：Japan  

Event date： 2007.5

Venue：福岡市   Country：Japan  

Event date： 2006.6

Venue：Kyoto   Country：Japan  

Event date： 2006.5

Venue：福岡市   Country：Japan  

Event date： 2005.12

Venue：福岡市   Country：Japan  

Event date： 2005.12

Venue：福岡市   Country：Japan  

Event date： 2005.10 - 2005.11

Venue：Awaji   Country：Japan  

Event date： 2005.10

Venue：兵庫県神戸市   Country：Japan  

Event date： 2005.10

Presentation type：Symposium, workshop panel (public)  

Venue：兵庫県神戸市   Country：Japan  

Event date： 2002.12

Venue：San Francisco  

Event date： 2002.10

Venue：京都市   Country：Japan  

Event date： 2002.10

Presentation type：Symposium, workshop panel (public)  

Venue：京都市   Country：Japan  

Event date： 2001.10

Presentation type：Symposium, workshop panel (public)  

Venue：京都市   Country：Japan  

Event date： 2000.12

Venue：神戸国際展示場・神戸国際会議場・神戸ポートピアホテル   Country：Japan  

Event date： 2000.10 - 2000.11

Venue：福岡市   Country：Japan  

Event date： 1999.12

Presentation type：Symposium, workshop panel (public)  

Venue：福岡ドーム・シーホークホテル&リゾート・福岡SRPセンターホール・国立病院九州医療センター   Country：Japan  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Peroxisomes are single-membrane organelles essential for cell metabolism including the β-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.

DOI： 10.1016/j.bbamcr.2022.119330

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.5685/plmorphol.32.59

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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.

DOI： 10.1242/jcs.236943

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

To investigate peroxisome assembly and human peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, thirteen different complementation groups (CGs) of Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis have been isolated and established as a model research system. Successful gene-cloning studies by a forward genetic approach utilized a rapid functional complementation assay of CHO cell mutants led to isolation of human peroxin (PEX) genes. Search for pathogenic genes responsible for PBDs of all 14 CGs is now completed together with the homology search by screening the human expressed sequence tag database using yeast PEX genes. Peroxins are divided into three groups: (1) peroxins including Pex3p, Pex16p, and Pex19p, are responsible for peroxisome membrane biogenesis via classes I and II pathways; (2) peroxins that function in matrix protein import; (3) those such as three forms of Pex11p, Pex11pα, Pex11pβ, and Pex11pγ, are involved in peroxisome proliferation where DLP1, Mff, and Fis1 coordinately function. In membrane assembly, Pex19p forms complexes in the cytosol with newly synthesized PMPs including Pex16p and transports them to the receptor Pex3p, whereby peroxisomal membrane is formed (Class I pathway). Pex19p likewise forms a complex with newly made Pex3p and translocates it to the Pex3p receptor, Pex16p (Class II pathway). In matrix protein import, newly synthesized proteins harboring peroxisome targeting signal type 1 or 2 are recognized by Pex5p or Pex7p in the cytoplasm and are imported to peroxisomes via translocation machinery. In regard to peroxisome-cytoplasmic shuttling of Pex5p, Pex5p initially targets to an 800-kDa docking complex consisting of Pex14p and Pex13p and then translocates to a 500-kDa RING translocation complex. At the terminal step, Pex1p and Pex6p of the AAA family mediate the export of Pex5p, where Cys-ubiquitination of Pex5p is essential for the Pex5p exit.

DOI： 10.3389/fphys.2014.00307