RESUMEN

Urokinase-type plasminogen activator (u-PA) and plasminogen play a primary role in liver repair through the accumulation of macrophages and alteration of their phenotype. However, it is still unclear whether u-PA and plasminogen mediate the activation of macrophage phagocytosis during liver repair. Herein, we investigated the morphological changes in macrophages that accumulated at the edge of damaged tissue induced by a photochemical reaction or hepatic ischaemia-reperfusion in mice with u-PA ( u-PA-/- ) or plasminogen ( Plg-/- ) gene deficiency by using transmission electron and fluorescence microscopy. In wild-type mice, the macrophages aligned at the edge of the damaged tissue and extended a large number of long pseudopodia. These macrophages clearly engulfed cellular debris and showed well-developed organelles, including lysosome-like vacuoles, nuclei, and Golgi complexes. In wild-type mice, the distribution of the Golgi complex in these macrophages was biased towards the direction of the damaged tissue, indicating the extension of their pseudopodia in this direction. Conversely, in u-PA-/- and Plg-/- mice, the macrophages located at the edge of the damaged tissue had few pseudopodia and less developed organelles. The Golgi complex was randomly distributed in these macrophages in u-PA-/- mice. Furthermore, interferon γ and IL-4 were expressed at a low level at the border region of the damaged tissue in u-PA-/- mice. Our data provide novel evidence that u-PA and plasminogen are essential for the phagocytosis of cellular debris by macrophages during liver repair. Furthermore, u-PA plays a critical role in the induction of macrophage polarity by affecting the microenvironment at the edge of damaged tissue.

Asunto(s)

Regulación de la Expresión Génica , Hígado/metabolismo , Macrófagos/metabolismo , Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Femenino , Aparato de Golgi/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión/métodos , Modelos Genéticos , Fagocitosis , Plasminógeno/genética , Seudópodos/metabolismo , Daño por Reperfusión , Activador de Plasminógeno de Tipo Uroquinasa/genética

RESUMEN

SUMMARY BACKGROUND: The involvement of plasminogen in liver repair has been reported, but its exact role in promoting this process is unknown. OBJECTIVE: To elucidate the underlying mechanism, we examined the dynamics of liver repair by using a reproducible liver injury model in plasminogen gene-deficient mice and their wild-type littermates. METHODS: Liver injury was induced by photochemical reaction and the subsequent responses were histologically analyzed. RESULTS: In wild-type animals, the area of the damage successively decreased, and the repair process was associated with macrophage accumulation at its border. Neutrophils were also attracted to the damaged region on day 1 and were evident only at its border by day 4, which spatially and temporally coincided with the expression of macrophage chemoattractant protein-1 (MCP-1). Neutrophil depletion suppressed recruitment of macrophages at the border between the damaged and the normal tissues. These changes were followed by activated hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis at the boundaries of the injured zone. In contrast, in plasminogen gene-deficient mice, the decrease in the area of damage, macrophage accumulation, late-phase neutrophil recruitment, hepatic stellate cell accumulation, collagen fiber deposition and angiogenesis were all impaired. CONCLUSION: Our data suggest that accumulated neutrophils at the border of the damaged area may contribute to macrophage accumulation at granulation tissue via the production of MCP-1 after liver injury. The plasminogen system is critical for liver repair by facilitating macrophage accumulation and triggering a cascade of subsequent repair events.

Asunto(s)

Enfermedad Hepática Inducida por Sustancias y Drogas , Tejido de Granulación/crecimiento & desarrollo , Regeneración Hepática , Plasminógeno/fisiología , Animales , Movimiento Celular , Quimiocina CCL2/biosíntesis , Células Estrelladas Hepáticas , Macrófagos/fisiología , Ratones , Ratones Noqueados , Neutrófilos/fisiología , Plasminógeno/genética

RESUMEN

The three peroxin genes, PEX12, PEX2, and PEX10, encode peroxisomal integral membrane proteins with RING finger at the C-terminal part and are responsible for human peroxisome biogenesis disorders. Mutation analysis in PEX12 of Chinese hamster ovary cell mutants revealed a homozygous nonsense mutation at residue Trp263Ter in ZP104 cells and a pair of heterozygous nonsense mutations, Trp170Ter and Trp114Ter, in ZP109. This result and domain mapping of Pex12p showed that RING finger is essential for peroxisome-restoring activity of Pex12p but not necessary for targeting to peroxisomes. The N-terminal region of Pex12p, including amino acid residues at positions 17-76, was required for localization to peroxisomes, while the sequence 17-76 was not sufficient for peroxisomal targeting. Peroxins interacting with RING finger of Pex2p, Pex10p, and Pex12p were investigated by yeast two-hybrid as well as in vitro binding assays. The RING finger of Pex12p bound to Pex10p and the PTS1-receptor Pex5p. Pex10p also interacted with Pex2p and Pex5p in vitro. Moreover, Pex12p was co-immunoprecipitated with Pex10p from CHO-K1 cells, where Pex5p was not associated with the Pex12p-Pex10p complex. This observation suggested that Pex5p does not bind to, or only transiently interacts with, Pex10p and Pex12p when Pex10p and Pex12p are in the oligomeric complex in peroxisome membranes. Hence, the RING finger peroxins are most likely to be involved in Pex5p-mediated matrix protein import into peroxisomes.

Asunto(s)

Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Secuencia Conservada , Cricetinae , Análisis Mutacional de ADN , Fibroblastos , Humanos , Proteínas de la Membrana/química , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Peroxinas , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Estructura Terciaria de Proteína , Ratas , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Técnicas del Sistema de Dos Híbridos

RESUMEN

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed approximately 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Peroxisomas/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Proteínas Portadoras/genética , Clonación Molecular , Cricetinae , ADN Complementario , Proteínas Fúngicas , Humanos , Líquido Intracelular/metabolismo , Membranas Intracelulares/metabolismo , Cinética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peroxinas , Peroxisomas/metabolismo , Ratas

RESUMEN

Fifty-six partial toes were transferred to reconstruct fingertip deficits. The transfers from the big toe mainly consisted of 3 trimmed big toetips, 3 vascularized nail grafts, 3 onychocutaneous flaps, 19 thin osteo-onychocutaneous flaps, and 2 hemipulp flaps. The transfers from the second toe mainly consisted of 8 trimmed second toetips, 5 reduced second toes, and 9 whole distal phalanges. The average values of postoperative sensory recovery of the osteo-onychocutaneous flaps including the vascularized nail grafts were 3.1 (Semmes-Weinstein test) and 6.3 mm (moving two-point discrimination) at 2.6 years after the transfer; those of the thin osteo-onychocutaneous flaps were 3.1 and 7.2 mm at 2.0 years after surgery; those of the trimmed big toe tip transfers were 3.61 and 6.5 mm at 1.8 years after surgery; and those of the trimmed second toetip transfers were 3.37 and 6.3 mm at 2.6 years after transfer. Those of the distal phalanx of the second toe were 3.41 and 7.9 mm at 1.2 years after surgery, and those of the reduced second toe were 3.2 and 6.7 mm at 10.6 months after surgery.

Asunto(s)

Amputación Traumática/cirugía , Traumatismos de los Dedos/cirugía , Microcirugia , Colgajos Quirúrgicos , Dedos del Pie/trasplante , Adulto , Amputación Traumática/fisiopatología , Femenino , Traumatismos de los Dedos/fisiopatología , Estudios de Seguimiento , Humanos , Masculino , Nervios Periféricos/fisiopatología , Nervios Periféricos/trasplante , Complicaciones Posoperatorias/fisiopatología , Células Receptoras Sensoriales/fisiopatología , Colgajos Quirúrgicos/irrigación sanguínea , Colgajos Quirúrgicos/inervación , Dedos del Pie/inervación , Tacto/fisiología

Asunto(s)

Trastorno Peroxisomal/genética , Peroxisomas , Animales , Clonación Molecular , Cricetinae , Humanos , Mutación , Endopeptidasa Neutra Reguladora de Fosfato PHEX , Peroxisomas/fisiología , Proteínas/genética , Saccharomyces cerevisiae/genética , Transducción de Señal

RESUMEN

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same as CG-I). Moreover, we recently found that Pex5p is involved in transport of not only PTS1- but also PTS2-protein, distinct from yeast Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.

Asunto(s)

Proteínas de la Membrana/genética , Trastorno Peroxisomal/genética , Peroxisomas/genética , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Mutación , Trastorno Peroxisomal/patología , Peroxisomas/ultraestructura

RESUMEN

We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.

Asunto(s)

Microcuerpos/genética , Microcuerpos/metabolismo , Mutación , Trastorno Peroxisomal/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Animales , Células CHO , Cricetinae , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Factor 2 de la Biogénesis del Peroxisoma , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Ratas , Receptores Citoplasmáticos y Nucleares/genética

RESUMEN

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Asunto(s)

Proteínas Portadoras , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Proteínas Represoras , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Microcuerpos/metabolismo , Datos de Secuencia Molecular , Peroxinas , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/secundario , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Neoplasias del Yeyuno/secundario , Neoplasias del Yeyuno/cirugía , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Neoplasias Encefálicas/secundario , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.

Asunto(s)

Proteínas de la Membrana/genética , Microcuerpos/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Animales , Células CHO , Catalasa/metabolismo , Fusión Celular , Línea Celular Transformada , Cricetinae , Fibroblastos/fisiología , Prueba de Complementación Genética , Humanos , Mamíferos , Proteínas de la Membrana/biosíntesis , Mutagénesis , Factor 2 de la Biogénesis del Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Pirenos , Ratas , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Transfección , Rayos Ultravioleta , Síndrome de Zellweger/genética , Síndrome de Zellweger/fisiopatología

RESUMEN

We cloned a human PEX11 cDNA by expressed sequence tag homology search using yeast Candida boidinii PEX11, followed by screening of human liver cDNA library. PEX11 encoded a peroxisomal protein Pex11p comprising 247 amino acids, with two transmembrane segments and a dilysine motif at the C-terminus. Pex11p comigrated in SDS-PAGE with a 28-kDa peroxisomal integral membrane protein (PMP28) isolated from the liver of clofibrate-treated rats and was crossreactive to anti-PMP28 antibody, thereby indicating PEX11 to encode PMP28. Pex11p exposes both N- and C-terminal parts to the cytosol. PEX11 was not responsible for ten complementation groups of human peroxisome deficiency disorders.

Asunto(s)

Clofibrato/farmacología , Proteínas Fúngicas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/genética , Microcuerpos/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , Cartilla de ADN , ADN Complementario , Proteínas Fúngicas/inmunología , Humanos , Masculino , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Peroxinas , Ratas , Ratas Endogámicas F344 , Homología de Secuencia de Aminoácido

RESUMEN

Peroxisome biogenesis disorders (PBD), such as Zellweger syndrome, are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as a malfunction of the peroxisomes, where at least 10 genotypes have been reported. We have isolated a human PEX10 cDNA (HsPEX10) by an expressed sequence tag homology search on a human DNA database using yeast PEX10 from Hansenula polymorpha, followed by screening of a human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex10p) comprising 326 amino acids, with two putative transmembrane segments and a C3HC4zinc finger RING motif. Both the N- and C-terminal regions of Pex10p are exposed to the cytosol, as assessed by an expression study of epitope-tagged Pex10p. HsPEX10 expression morphologically and biochemically restored peroxisome biogenesis in fibroblasts from Zellweger patients of complementation group B in Japan (complementation group VII in the USA). One patient (PBDB-01) possessed a homozygous, inactivating mutation, a 2 bp deletion immediately upstream of the RING motif, which resulted in a frameshift, altering 65 amino acids from the normal. This implies that the C-terminal part, including the RING finger, is required for biological function of Pex10p. PEX10 cDNA derived from patient PBDB-01 was defective in peroxisome-restoring activity when expressed in patient fibroblasts. These results demonstrate that mutation in PEX10 is the genetic cause of complementation group B PBD.

Asunto(s)

Mutación , Proteínas/genética , Síndrome de Zellweger/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Proteínas Fúngicas/genética , Expresión Génica , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Endopeptidasa Neutra Reguladora de Fosfato PHEX , Pichia/genética , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

Asunto(s)

Proteínas de la Membrana/genética , Mutación , Síndrome de Zellweger/genética , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular , Línea Celular Transformada , Clonación Molecular , Cricetinae , Citosol , ADN Complementario , Fibroblastos , Humanos , Microcuerpos/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Trastorno Peroxisomal/veterinaria , Ratas , Homología de Secuencia de Aminoácido

RESUMEN

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy (NALD), are autosomal recessive diseases caused by defects in peroxisome assembly, for which at least 10 complementation groups have been reported. We have isolated a human PEX1 cDNA (HsPEX1) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary (CHO) cell line, ZP107, transformed with peroxisome targeting signal type 1-tagged "enhanced" green fluorescent protein. This cDNA encodes a hydrophilic protein (Pex1p) comprising 1,283 amino acids, with high homology to the AAA-type ATPase family. A stable transformant of ZP107 with HsPEX1 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX1 expression restored peroxisomal protein import in fibroblasts from three patients with ZS and NALD of complementation group I (CG-I), which is the highest-incidence PBD. A CG-I ZS patient (PBDE-04) possessed compound heterozygous, inactivating mutations: a missense point mutation resulting in Leu-664 --> Pro and a deletion of the sequence from Gly-634 to His-690 presumably caused by missplicing (splice site mutation). Both PBDE-04 PEX1 cDNAs were defective in peroxisome-restoring activity when expressed in the patient fibroblasts as well as in ZP107 cells. These results demonstrate that PEX1 is the causative gene for CG-I peroxisomal disorders.

Asunto(s)

Proteínas de la Membrana/biosíntesis , Síndrome de Zellweger/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular , Clonación de Organismos , Cricetinae , Fibroblastos , Prueba de Complementación Genética , Humanos , Cinética , Proteínas de la Membrana/genética , Microcuerpos/fisiología , Datos de Secuencia Molecular , Pichia/genética , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Piel/metabolismo , Transfección

RESUMEN

Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation of peroxisome deficient CHO cell mutants and (2) homology searches for human dbEST, based on yeast genes involved in peroxisome biogenesis (PEX genes). We report here the first identification of a new complementation group which is genetically different from peroxisome deficient CHO mutants. There were no complementations by the human PEX 13 gene. The nature of the related gene is being investigated.

Asunto(s)

Prueba de Complementación Genética , Proteínas de la Membrana/genética , Trastorno Peroxisomal/genética , Animales , Células CHO , Catalasa/inmunología , Mapeo Cromosómico , Clonación Molecular , Cricetinae , ADN Complementario/genética , Fibroblastos , Técnica del Anticuerpo Fluorescente , Humanos , Lactante , Masculino , Fenotipo , Transfección/genética

RESUMEN

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Asunto(s)

Microcuerpos/genética , Receptores Citoplasmáticos y Nucleares/genética , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Proteínas Fúngicas , Regulación de la Expresión Génica , Humanos , Microcuerpos/metabolismo , Datos de Secuencia Molecular , Mutación , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Alineación de Secuencia

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microcuerpos/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO/metabolismo , Catalasa/metabolismo , Clonación Molecular , Cricetinae , Humanos , Hígado/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Microcuerpos/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Ratas , Fracciones Subcelulares , Síndrome de Zellweger/genética

RESUMEN

We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/ UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.

Asunto(s)

Microcuerpos/enzimología , Microcuerpos/ultraestructura , Mutación , Acetil-CoA C-Aciltransferasa/biosíntesis , Acil-CoA Oxidasa , Animales , Células CHO , Catalasa/análisis , Fusión Celular , Cricetinae , Citosol/enzimología , Digitonina/farmacología , Fibroblastos , Prueba de Complementación Genética , Humanos , Ácidos Láuricos/farmacología , Mamíferos , Proteínas de la Membrana/genética , Mutagénesis , Oxidorreductasas/biosíntesis , Factor 2 de la Biogénesis del Peroxisoma , Rayos Ultravioleta , Síndrome de Zellweger/metabolismo

RESUMEN

We made use of the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III.

Asunto(s)

Adenosina Trifosfatasas/genética , Microcuerpos/genética , Mutación , Trastorno Peroxisomal/genética , Síndrome de Zellweger/genética , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Células CHO , Cricetinae , Resistencia a Medicamentos , Prueba de Complementación Genética , Humanos , Ácidos Láuricos/farmacología , Trastorno Peroxisomal/clasificación , Síndrome de Zellweger/clasificación