Absence of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis.

To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on functional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for human Zellweger syndrome. We found that all enzymes measured, including putative peroxisomal enzymes, are at least as active in the peroxisome-deficient Zellweger mice as in control mice, indicating that mislocalization of enzymes to the cytosol does not lead to decreased activity or degradation. Prompted by these results, we re-examined this aspect in human subjects by specific enzyme activity measurements and immunoblotting with highly specific antisera. Our results show that the previously reported deficiencies of mevalonate kinase and phosphomevalonate kinase activity in livers from human Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol.Our data provide an explanation for the conflicting findings in the literature and show that great care should be taken in the interpretation of data obtained in postmortem material.     

PEX11 beta deficiency is lethal and impairs neuronal migration but does not abrogate peroxisome function

Zellweger syndrome is a lethal neurological disorder characterized by severe defects in peroxisomal protein import. The resulting defects in peroxisome metabolism and the accumulation of peroxisomal substrates are thought to cause the other Zellweger syndrome phenotypes, including neuronal migration defects, hypotonia, a developmental delay, and neonatal lethality. These phenotypes are also manifested in mouse models of Zellweger syndrome generated by disruption of the PEX5 or PEX2 gene. Here we show that mice lacking peroxisomal membrane protein PEX11 beta display several pathologic features shared by these mouse models of Zellweger syndrome, including neuronal migration defects, enhanced neuronal apoptosis, a developmental delay, hypotonia, and neonatal lethality. However, PEX11 beta deficiency differs significantly from Zellweger syndrome and Zellweger syndrome mice in that it is not characterized by a detectable defect in peroxisomal protein import and displays only mild defects in peroxisomal fatty acid beta-oxidation and peroxisomal ether lipid biosynthesis. These results demonstrate that the neurological pathologic features of Zellweger syndrome can occur without peroxisomal enzyme mislocalization and challenge current models of Zellweger syndrome pathogenesis.     

Nonspecific lipid transfer protein (sterol carrier protein-2) defective in patients with deficient peroxisomes

The biosynthesis and intracellular localization of nonspecific lipid transfer protein (nsLTP) in control human subjects and in patients with peroxisome-deficient disorders were investigated. The molecular mass of human nsLTP was indistinguishable from that of rat nsLTP (13 kDa) by immunoblot analysis. Intracellular localization was identical with that of catalase, a marker enzyme of peroxisomal matrix, by a double immunofluorescence study. The nsLTP was deficient in liver tissues or fibroblasts from patients with peroxisome-deficient disorders such as Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Pulse-chase experiments showed that nsLTP was synthesized as a large precursor in both the control and Zellweger fibroblasts. However, the processing to the 13 kDa mature protein was disturbed and the degradation was rapid in Zellweger fibroblasts. After somatic cell fusion using Zellweger fibroblasts from different genetic groups, the processing was normalized. These results suggest that the biosynthesis and localization of human nsLTP are similar to those of rat nsLTP and that the defect of nsLTP in peroxisome-deficient disorders is a phenomenon secondary to an abnormal transport mechanism of peroxisomal proteins. The defect of nsLTP may play an important role in metabolic disturbances in bile acid synthesis and steroidogenesis in peroxisome-deficient disorders.     

Pex13 inactivation in the mouse disrupts peroxisome biogenesis and leads to a Zellweger syndrome phenotype

Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13(-/-) mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.     

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice

Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2(-/-) mice on a hybrid Swiss Webster×129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2(-/-) mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2(-/-) mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2(-/-) livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2(-/-) livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2(-/-) mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2(-/-) mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.     

Overexpression of estrogen receptor alpha increases hepatic cholesterogenesis, leading to biliary hypersecretion in mice

We explored whether there is an "estrogen-ERalpha-SREBP-2" (for estrogen-estrogen receptor subtype alpha-sterol-regulatory element binding protein-2) pathway for regulating hepatic cholesterol biosynthesis in ovariectomized AKR mice treated with 17beta-estradial (E2) at 6 microg/day or E2 plus the antiestrogenic agent ICI 182,780 at 125 microg/day and on chow or fed a high-cholesterol (1%) diet for 14 days. To monitor changes in cholesterol biosynthesis and newly synthesized cholesterol secreted into bile, incorporation into digitonin-precipitable sterols in mice treated with 25 mCi of [3H]water was measured in extracts of liver and extrahepatic organs 1 h later and in hepatic biles 6 h later. ERalpha upregulated SREBP-2, with resulting activation of SREBP-2-responsive genes in the cholesterol biosynthetic pathway. The E2-treated mice continued to synthesize cholesterol in spite of its excess availability from high dietary cholesterol, which reflects a loss in controlling the negative feedback regulation of cholesterol synthesis. These alterations augmented biliary cholesterol secretion and enhanced the lithogenicity of bile. However, these lithogenic effects of E2 were fully blocked by ICI 182,780. We conclude that during estrogen treatment, more newly synthesized cholesterol determined by the estrogen-ERalpha-SREBP-2 pathway is secreted into bile, leading to biliary cholesterol hypersecretion. These studies provide insights into therapeutic approaches to cholesterol gallstones in high-risk subjects, especially those exposed to high levels of estrogen.     

OCTN3 is a mammalian peroxisomal membrane carnitine transporter

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.     

PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation

The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.     

Targeted Deletion of the PEX2 Peroxisome Assembly Gene in Mice Provides a Model for Zellweger Syndrome, a Human Neuronal Migration Disorder

Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting.

Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar–lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.

     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Partial phenotypic suppression of a peroxisome-deficient animal cell mutant treated with aminoglycoside G418.

Certain enzymes normally associated with peroxisomes, such as the dihydroxyacetone phosphate (DHAP) acyltransferase involved in plasmalogen biosynthesis, are present at low levels in peroxisome-deficient mutants of Chinese hamster ovary (CHO) cells. We now show that the aminoglycoside G418 increases the residual DHAP acyltransferase in mutant ZR-82 by 60-fold. This is accompanied by a dose- and time-dependent restoration of the plasmalogen content. G418 treatment of ZR-82 also increases residual peroxisomal beta-oxidation activity by 3.8-fold. G418 does not affect wild-type CHO cells (CHO-K1) or a different peroxisome-deficient mutant, ZR-78.1. The effects of G418 on ZR-82 are transient, since plasmalogens and DHAP-acyltransferase decline to basal levels 5 days after G418 withdrawal. Other aminoglycosides and lysosomotropic agents do not alter plasmalogen levels in ZR-82. The subcellular distribution of catalase (an enzyme of the peroxisomal matrix which is present in normal amounts in peroxisome-deficient mutants but is mislocalized in the cytosol) is unaffected by G418 treatment of ZR-82, demonstrating that G418 does not restore peroxisomes. Localization of catalase by immunofluorescence microscopy confirms a total absence of intact peroxisomes in ZR-82, either before or after exposure to G418. This study is the first to demonstrate that some peroxisome-deficient mutants can be induced to accumulate functional DHAP acyltransferase and other peroxisomal enzymes, usually missing in the absence of peroxisomes. G418 may have some therapeutic value in selected patients with inborn errors of peroxisome assembly, such as Zellweger syndrome.     

Genomic organization and characterization of human PEX2 encoding a 35-kDa peroxisomal membrane protein

Peroxins are proteins involved in peroxisome biogenesis and are encoded by PEX genes. The human PEX2 gene encodes a 35-kDa peroxisomal integral membrane protein which is a member of the zinc finger protein family. Mutations in the PEX2 gene are the primary defect in a subset of patients with Zellweger syndrome and related peroxisome biogenesis disorders. The role of zinc finger proteins in peroxisome assembly and function is poorly understood. Here we report the cloning and characterisation of the human PEX2 structural gene. PEX2 was assigned to human chromosome 8q13-q21 and its murine homologue to mouse chromosome 3. The gene is approximately 17.5 kb in length, and contains four exons. The entire coding sequence is included in one exon, exon 4. The 5'-flanking region has features of housekeeping genes (GC enrichment, two Sp1 sites) and tissue-specific, inducible genes (two CCAAT boxes). In more than 1.5 kb of 5'-flanking sequences we did not identify consensus peroxisomal proliferator responsive elements (PPRE).     

Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).     

PEX11 promotes peroxisome division independently of peroxisome metabolism

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.     

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.