Cassandra Obie

Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata

Rhizomelic chondrodysplasia punctata (RCDP) is a rare autosomal recessive phenotype that comprises complementation group 11 of the peroxisome biogenesis disorders (PBD). PEX7, a candidate gene for RCDP identified in yeast, encodes the receptor for peroxisomal matrix proteins with the type-2 peroxisome targeting signal (PTS2). By homology probing we identified human and murine PEX7 genes and found that expression of either corrects the PTS2-import defect characteristic of RCDP cells. In a collection of 36 RCDP probands, we found two inactivating PEX7 mutations: one, L292ter, was present in 26 of the probands, all with a severe phenotype; the second, A218V, was present in three probands, including two with a milder phenotype. A third mutation, G217R, whose functional significance is yet to be determined, was present in five probands, all compound heterozygotes with L292ter. We conclude that PEX7 is responsible for RCDP (PBD CG11) and suggest a founder effect may explain the high frequency of L292ter.

Expression of PEX11β Mediates Peroxisome Proliferation in the Absence of Extracellular Stimuli

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11α and PEX11β. Overexpression of the human PEX11β gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11β induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11β from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11α also induced peroxisome proliferation but at a much lower frequency than PEX11β in our experimental system. The patterns ofPEX11α and PEX11β expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11β mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11α mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11β in the constitutive control of peroxisome abundance and suggest that PEX11α may regulate peroxisome abundance in response to extracellular stimuli.

Mutations in the gene encoding 3β-hydroxysteroid-Δ8,Δ7-isomerase cause X-linked dominant Conradi-Hunermann syndrome

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported,. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 ( ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3β-hydroxysteroid-Δ8,Δ 7-isomerase (sterol-Δ8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-Δ8-isomerase ( EBP) has been identified and mapped to Xp11.22–p11.23 (Refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-Δ8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-Δ8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.

Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188Δ, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.

Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hunermann syndrome

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported,. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 ( ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3β-hydroxysteroid-Δ8,Δ 7-isomerase (sterol-Δ8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-Δ8-isomerase ( EBP) has been identified and mapped to Xp11.22–p11.23 (Refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-Δ8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-Δ8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

Molecular Enzymology of Mammalian Δ1-Pyrroline-5-carboxylate Synthase ALTERNATIVE SPLICE DONOR UTILIZATION GENERATES ISOFORMS WITH DIFFERENT SENSITIVITY TO ORNITHINE INHIBITION

Δ1-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Δ1-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine. We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by “exon sliding,” a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the γ-glutamyl kinase active site of P5CS. The long form predominates in all tissues examined except gut. We also isolated the corresponding long and short murine P5CS transcripts. To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in γ-glutamyl kinase- and γ-glutamyl phosphate reductase-deficient strains ofSaccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms. We found that both confer P5CS activity and that the short isoform is inhibited by l-ornithine with a K i of ∼0.25 mm. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by l-ornithine.

Alternative Splicing Suggests Extended Function of PEX26 in Peroxisome Biogenesis

Matsumoto and colleagues recently identified PEX26 as the gene responsible for complementation group 8 of the peroxisome biogenesis disorders and showed that it encodes an integral peroxisomal membrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminus that interacts with the PEX1/PEX6 heterodimer through direct binding to the latter. They proposed that PEX26 functions as the peroxisomal docking factor for the PEX1/PEX6 heterodimer. Here, we identify new PEX26 disease alleles, localize the PEX6-binding domain to the N-terminal half of the protein (aa 29-174), and show that, at the cellular level, PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins. Also, we find that PEX26 undergoes alternative splicing to produce several splice forms--including one, PEX26- delta ex5, that maintains frame and encodes an isoform lacking the transmembrane domain of full-length PEX26 (PEX26-FL). Despite its cytosolic location, PEX26- delta ex5 rescues peroxisome biogenesis in PEX26-deficient cells as efficiently as does PEX26-FL. To test our observation that a peroxisomal location is not required for PEX26 function, we made a chimeric protein (PEX26-Mito) with PEX26 as its N-terminus and the targeting segment of a mitochondrial outer membrane protein (OMP25) at its C-terminus. We found PEX26-Mito localized to the mitochondria and directed all detectable PEX6 and a fraction of PEX1 to this extraperoxisomal location; yet PEX26-Mito retains the full ability to rescue peroxisome biogenesis in PEX26-deficient cells. On the basis of these observations, we suggest that a peroxisomal localization of PEX26 and PEX6 is not required for their function and that the interaction of PEX6 with PEX1 is dynamic. This model predicts that, once activated in an extraperoxisomal location, PEX1 moves to the peroxisome and completes the function of the PEX1/6 heterodimer.

Human cell transformation by Simian Virus 40: Biologic features of cloned lines

Abstract Nine lines of doubly cloned Simian Virus 40 transformed human fibroblasts have been isolated. Precrisis in vitro characteristics of growth rate, saturation density, T antigen expression, growth in methylcellulose suspension, protease production and infectious center formation were widely divergent, although distinct for individual lines. Lines with higher T antigen expression had higher growth rates and saturation densities but grew less well in methylcellulose. Chromosome counts in most proliferating lines were in the 38–48 range; one line had 67, whereas another, poorly growing line showed 22. No distinguishing chromosomal abnormalities were present. This biologic heterogeneity suggests distinct molecular differences underlying transformation in the lines and emphasizes the importance of considering various factors as indicative of the transformed state.

Restoration of peroxisome biogenesis in a peroxisome-deficient mammalian cell line by expression of either the 35 kDa or the 70 kDa peroxisomal membrane proteins

Peroxisomes are single-membrane-bound organelles present in nearly all eukaryotic cells. Their size and number vary depending on the type of cell and its physiological state. The organelle matrix contains more than 40 enzymes that catalyse a variety of anabolic and catabolic reactions. Both the 35kDa (PMP35) and 70kDa (PMP70) peroxisomal membrane proteins are found only in the peroxisomal membrane and appear to be required for peroxisome assembly. PMP70 is a member of the ATP-binding cassette transporter family, while PMP35 has a zinc-finger domain (Gartner et al 1992; Shimozawa et al 1992). Peroxisomal biogenesis involves import of newly synthesized membrane and matrix proteins into preexisting peroxisomes followed by growth and fission of the organelle