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Naomi Kondo - One of the best experts on this subject based on the ideXlab platform.
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the common phospholipid binding activity of the n terminal domains of PEX1 and vcp p97
FEBS Journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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The common phospholipid-binding activity of the N-terminal domains of PEX1 and VCP/p97.
The FEBS journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype-genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37 degrees C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634-690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p-Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Temperature-Sensitive Mutation of PEX6 in Peroxisome Biogenesis Disorders in Complementation Group C (CG-C): Comparative Study of PEX6 and PEX1
Pediatric Research, 2000Co-Authors: Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Zhongyi Zhang, Toshiro Tsukamoto, Yukio Fujiki, Tadao Orii, Takashi Osumi, Ronald J A Wanders, Naomi KondoAbstract:Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as PEX1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30°C but not at 37°C. This patient was homozygous for a missense mutation, T→C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1 -deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1 , the counterpart to L57P in PEX6 , showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6 , the counterpart to the temperature-sensitive mutation G843D in PEX1 , revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and PEX1p.
Yukio Fujiki - One of the best experts on this subject based on the ideXlab platform.
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AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys‐Monoubiquitinated Pex5 and Pex6 AAA ATPase
Traffic (Copenhagen Denmark), 2011Co-Authors: Non Miyata, Kanji Okumoto, Satoru Mukai, Masafumi Noguchi, Yukio FujikiAbstract:During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with PEX1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in PEX1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.
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The pathogenic peroxin Pex26p recruits the PEX1p-Pex6p AAA-ATPase complexes to peroxisomes
Nature cell biology, 2003Co-Authors: Naomi Matsumoto, Shigehiko Tamura, Yukio FujikiAbstract:Peroxisomes are ubiquitous organelles with a single membrane that contain over 50 different enzymes that catalyse various metabolic pathways, including beta-oxidation and lipid synthesis. Peroxisome biogenesis disorders (PBDs), such as Zellweger syndrome and neonatal adrenoleukodystrophy, are fatal genetic diseases that are autosomal recessive. Among the PBDs of the 12 complementation groups (CGs), 11 associated PEX genes have been isolated. Accordingly, only the PBD pathogenic gene for CG8 (also called CG-A) remains unidentified. Here we have isolated human PEX26 encoding a type II peroxisomal membrane protein of relative molecular mass 34,000 (M(r) 34K) by using ZP167 cells, a Chinese hamster ovary (CHO) mutant cell line. Expression of PEX26 restores peroxisomal protein import in the fibroblasts of an individual with PBD of CG8. This individual possesses a homozygous, inactivating pathogenic point mutation, Arg98Trp, in Pex26. Pex6 and PEX1 of the AAA ATPase family co-immunoprecipitate with Pex26. Epitope-tagged Pex6 and PEX1 are discernible as puncta in normal CHO-K1 cells, but not in PEX26-defective cells. PEX26 expression in ZP167 cells re-establishes colocalization of Pex6 and PEX1 with Pex26, in a Pex6-dependent manner. Thus, Pex26 recruits Pex6-PEX1 complexes to peroxisomes.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype-genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37 degrees C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634-690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p-Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Temperature-Sensitive Mutation of PEX6 in Peroxisome Biogenesis Disorders in Complementation Group C (CG-C): Comparative Study of PEX6 and PEX1
Pediatric Research, 2000Co-Authors: Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Zhongyi Zhang, Toshiro Tsukamoto, Yukio Fujiki, Tadao Orii, Takashi Osumi, Ronald J A Wanders, Naomi KondoAbstract:Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as PEX1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30°C but not at 37°C. This patient was homozygous for a missense mutation, T→C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1 -deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1 , the counterpart to L57P in PEX6 , showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6 , the counterpart to the temperature-sensitive mutation G843D in PEX1 , revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and PEX1p.
Hidekazu Hiroaki - One of the best experts on this subject based on the ideXlab platform.
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the common phospholipid binding activity of the n terminal domains of PEX1 and vcp p97
FEBS Journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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The common phospholipid-binding activity of the N-terminal domains of PEX1 and VCP/p97.
The FEBS journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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structure of the n terminal domain of PEX1 aaa atpase characterization of a putative adaptor binding domain
Journal of Biological Chemistry, 2004Co-Authors: Kumiko Shiozawa, Nobuo Maita, Kentaro Tomii, Azusa Seto, Natsuko Goda, Yutaka Akiyama, Toshiyuki Shimizu, Masahiro Shirakawa, Hidekazu HiroakiAbstract:Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (<10% identity) of PEX1 NTD to the N-terminal domains of other membrane-related type II AAA-ATPases, such as VCP (p97) and NSF. We have determined the crystal structure of mouse PEX1 NTD at 2.05-A resolution, which clearly demonstrated that the domain belongs to the double-psi-barrel fold family found in the other AAA-ATPases. The N-domains of both VCP and NSF are structural neighbors of PEX1 NTD with a 2.7- and 2.1-A root mean square deviation of backbone atoms, respectively. Our findings suggest that the supradomain architecture, which is composed of a single N-terminal domain followed by tandem AAA domains, is a common feature of organellar membrane-associating AAA-ATPases. We propose that PEX1 functions as a protein unfoldase in peroxisomal biogenesis, using its N-terminal putative adaptor-binding domain.
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Crystallographic characterization of the N-terminal domain of PEX1.
Acta Crystallographica Section D Biological Crystallography, 2004Co-Authors: Kumiko Shiozawa, Nobuo Maita, Kentaro Tomii, Azusa Seto, Natsuko Goda, Yutaka Akiyama, Toshiyuki Shimizu, Masahiro Shirakawa, Hidekazu HiroakiAbstract:Peroxisomal enzymes are responsible for several primary metabolism pathways, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases and both are necessary for the import of more than 50 peroxisomal resident proteins from the cytosol into peroxisomes. In this study, PEX1 N-terminal domain crystals have been prepared. The crystals belong to space group P3(1) or P3(2), with unit-cell parameters a = b = 63.5 A, c = 33.5 A, and contain one protein molecule per crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.05 A.
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Structure of the N-terminal domain of PEX1 AAA-ATPase. Characterization of a putative adaptor-binding domain.
The Journal of biological chemistry, 2004Co-Authors: Kumiko Shiozawa, Nobuo Maita, Kentaro Tomii, Azusa Seto, Natsuko Goda, Yutaka Akiyama, Toshiyuki Shimizu, Masahiro Shirakawa, Hidekazu HiroakiAbstract:Peroxisomes are responsible for several pathways in primary metabolism, including beta-oxidation and lipid biosynthesis. PEX1 and PEX6 are hexameric AAA-type ATPases, both of which are indispensable in targeting over 50 peroxisomal resident proteins from the cytosol to the peroxisomes. Although the tandem AAA-ATPase domains in the central region of PEX1 and PEX6 are highly similar, the N-terminal sequences are unique. To better understand the distinct molecular function of these two proteins, we analyzed the unique N-terminal domain (NTD) of PEX1. Extensive computational analysis revealed weak similarity (
Nobuyuki Shimozawa - One of the best experts on this subject based on the ideXlab platform.
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the common phospholipid binding activity of the n terminal domains of PEX1 and vcp p97
FEBS Journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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The common phospholipid-binding activity of the N-terminal domains of PEX1 and VCP/p97.
The FEBS journal, 2006Co-Authors: Kumiko Shiozawa, Natsuko Goda, Toshiyuki Shimizu, Masahiro Shirakawa, Nobuyuki Shimozawa, Naomi Kondo, Kenji Mizuguchi, Hidekazu HiroakiAbstract:PEX1 is a type II AAA-ATPase that is indispensable for biogenesis and maintenance of the peroxisome, an organelle responsible for the primary metabolism of lipids, such as β-oxidation and lipid biosynthesis. Recently, we demonstrated a striking structural similarity between its N-terminal domain and those of other membrane-related AAA-ATPases, such as valosin-containing protein (p97). The N-terminal domain of valosine-containing protein serves as an interface to its adaptor proteins p47 and Ufd1, whereas the physiologic interaction partner of the N-terminal domain of PEX1 remains unknown. Here we found that N-terminal domains isolated from valosine-containing protein, as well as from PEX1, bind phosphoinositides. The N-terminal domain of PEX1 appears to preferentially bind phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate, whereas the N-terminal domain of valosine-containing protein displays broad and nonspecific lipid binding. Although N-ethylmaleimide-sensitive fusion protein, CDC48 and Ufd1 have structures similar to that of valosine-containing protein, they displayed lipid specificity similar to that of the N-terminal domain of PEX1 in the assays. By mutational analysis, we demonstrate that a conserved arginine surrounded by hydrophobic residues is essential for lipid binding, despite very low sequence similarity between PEX1 and valosine-containing protein.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype-genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37 degrees C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634-690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p-Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by PEX1p–Pex6p interaction
Biochemical Journal, 2001Co-Authors: Shigehiko Tamura, Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Naomi Kondo, Naomi Matsumoto, Yukio FujikiAbstract:The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin PEX1p of the AAA ATPase family. It has been also reported that PEX1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. PEX1p from IRD such as PEX1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of PEX1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. PEX1p-G843D interacted with Pex6p at approx. 50% of the level of normal PEX1p, whereas PEX1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of PEX1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in PEX1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
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Temperature-Sensitive Mutation of PEX6 in Peroxisome Biogenesis Disorders in Complementation Group C (CG-C): Comparative Study of PEX6 and PEX1
Pediatric Research, 2000Co-Authors: Atsushi Imamura, Nobuyuki Shimozawa, Yasuyuki Suzuki, Zhongyi Zhang, Toshiro Tsukamoto, Yukio Fujiki, Tadao Orii, Takashi Osumi, Ronald J A Wanders, Naomi KondoAbstract:Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as PEX1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30°C but not at 37°C. This patient was homozygous for a missense mutation, T→C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1 -deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1 , the counterpart to L57P in PEX6 , showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6 , the counterpart to the temperature-sensitive mutation G843D in PEX1 , revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and PEX1p.
Thomas Walz - One of the best experts on this subject based on the ideXlab platform.
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Structures of the double-ring AAA ATPase PEX1-Pex6 involved in peroxisome biogenesis.
The FEBS journal, 2015Co-Authors: Dongyan Tan, Neil B. Blok, Tom A. Rapoport, Thomas WalzAbstract:The PEX1 and Pex6 proteins are members of the AAA family of ATPases and are involved in peroxisome biogenesis. Recently, cryo-electron microscopy structures of the PEX1-Pex6 complex in different nucleotide states have been determined. This Structural Snapshot describes the structural features of the complex and their implications for its function, as well as questions that still await answers.
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unique double ring structure of the peroxisomal PEX1 pex6 atpase complex revealed by cryo electron microscopy
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Neil B. Blok, Tom A. Rapoport, Ray Yuruei Wang, Pawel A Penczek, David Baker, Frank Dimaio, Thomas WalzAbstract:Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the PEX1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. PEX1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of PEX1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between PEX1/Pex6 and p97, supporting the hypothesis that the PEX1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.
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unique double ring structure of the peroxisomal PEX1 pex6 atpase complex revealed by cryo electron microscopy
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Neil B. Blok, Dongyan Tan, Tom A. Rapoport, Ray Yuruei Wang, Pawel A Penczek, David Baker, Frank Dimaio, Thomas WalzAbstract:Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the PEX1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. PEX1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of PEX1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between PEX1/Pex6 and p97, supporting the hypothesis that the PEX1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.
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Unique double-ring structure of the peroxisomal PEX1/Pex6 ATPase complex revealed by cryo-electron microscopy
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Neil B. Blok, Dongyan Tan, Tom A. Rapoport, Ray Yuruei Wang, Pawel A Penczek, David Baker, Frank Dimaio, Thomas WalzAbstract:Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the PEX1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. PEX1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of PEX1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between PEX1/Pex6 and p97, supporting the hypothesis that the PEX1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.