We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2 ؊/؊ mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2 ؊/؊ mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2 ؊/؊ mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7-to 9-day-old PEX2 ؊/؊ mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2 ؊/؊ mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2 ؊/؊ mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2 ؊/؊ mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2 ؊/؊ mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisomedeficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.
Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunoXuorescence and cell permeabilization techniques. Our present Wndings clearly show and conWrm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal -oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.
Regulation of hepatic cholesterol biosynthesis, lipogenesis, and insulin signaling intersect at the transcriptional level by control of SREBP and Insig genes. We previously demonstrated that peroxisome-deficient PEX2 ؊/؊ mice activate SREBP-2 pathways but are unable to maintain normal cholesterol homeostasis. In this study, we demonstrate that oral bile acid treatment normalized hepatic and plasma cholesterol levels and hepatic cholesterol synthesis in early postnatal PEX2 mutants, but SREBP-2 and its target gene expressions remained increased. SREBP-2 pathway induction was also observed in neonatal and longer surviving PEX2 mutants, where hepatic cholesterol levels were normal. Abnormal expression patterns for SREBP-1c and Insig-2a, and novel regulation of Insig-2b, further demonstrate that peroxisome deficiency widely affects the regulation of related metabolic pathways. We have provided the first demonstration that peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, especially the integrated stress response mediated by PERK and ATF4 signaling. Our studies suggest a mechanism whereby ER stress leads to dysregulation of the endogenous sterol response mechanism and concordantly activates oxidative stress pathways. Several metabolic derangements in peroxisome-deficient PEX2 ؊/؊ liver are likely to trigger ER stress, including perturbed flux of mevalonate metabolites, altered bile acid homeostasis, changes in fatty acid levels and composition, and oxidative stress.
To date, isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) is presumed to have a cytosolic localization. However, we have recently shown that in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol, implying that all of the enzymes required for the conversion of mevalonate to farnesyl diphosphate are found in the peroxisome. To provide unequivocal evidence for the subcellular localization of IPP isomerase, in this study, we have cloned the rat and hamster homologues of IPP isomerase and identified the signal that targets this enzyme to peroxisomes. In addition, we also demonstrate that IPP isomerase is regulated at the mRNA level.The isoprenoid biosynthetic pathway is ubiquitous to all living organisms. A few of the important end products of this complex pathway include: dolichols; vitamins A, D, E, and K; steroid hormones; carotenoids; bile acids; and cholesterol (1). The enzyme isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase 1 ; EC 5.3.3.2) plays a crucial role in this pathway by catalyzing the interconversion of isopentenyl diphosphate (IPP) to its highly electrophilic isomer, dimethylallyl diphosphate (2). These two isomers are the building blocks for the successive head-to-tail condensation reactions that result in the synthesis of farnesyl diphosphate (FPP), and ultimately, cholesterol (3).Recently, it has been shown by our group and others that peroxisomes contain a number of enzymes involved in cholesterol biosynthesis that were previously thought to be cytosolic. Specifically, peroxisomes have been shown to contain acetoacetyl-CoA thiolase (4, 5), 3-hydroxy-3-methylglutaryl coenzyme A synthase (6), 3-hydroxy-3-methylglutaryl coenzyme A reductase (7-9), mevalonate kinase (10, 11), phosphomevalonate kinase (12), mevalonate diphosphate decarboxylase (12), and FPP synthase (13). Both mevalonate kinase and FPP synthase seem to be localized predominantly, if not exclusively, to peroxisomes (11, 13). To date, IPP isomerase is presumed to have a cytosolic localization (1); however, the following three observations have led us to believe that the enzyme is localized to peroxisomes: (i) in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol in equal amounts to that observed in nonpermeabilized cells, therefore suggesting that the cytosol does not contain enzymes necessary for the conversion of mevalonate to FPP (12); (ii) IPP isomerase activity in tissues from patients with peroxisome-deficient diseases (Zellweger and neonatal adrenoleukodystrophy) is 50% of that found in tissues from control patients (13); and (iii) the deduced amino acid sequence from the human isomerase cDNA, which has been recently cloned (14) and characterized (15), contains two putative peroxisomal targeting sequences.At the C-terminal end of human isomerase is a putative peroxisomal targeting sequence 1 (PTS1) consisting of YRM (single-letter amino acid notation), and at the N-terminal end is a p...
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.
In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.In mammalian cells, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1 reductase is the rate-limiting enzyme for the synthesis of mevalonic acid, the precursor of cholesterol and other non-sterol isoprenoids. We and others (1-4) have demonstrated that HMG-CoA reductase is localized in two distinct intracellular compartments, endoplasmic reticulum (ER) and peroxisomes. ER HMG-CoA reductase is a 97-kDa transmembrane glycoprotein. A short non-conserved sequence links the multiple transmembrane domain to the highly conserved catalytic domain, which extends out into the cytosol. Because of its role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. The levels of the ER enzyme are regulated by transcription (5-7), translation (8, 9), and enzyme degradation (10, 11). Another critical role for this enzyme has emerged in recent years, due to the requirement of farnesyl diphosphate and geranyl-geranyl diphosphate in isoprenylation of proteins (12). Keller et al. (1) were the first to demonstrate that in the liver HMG-CoA reductase is present not only in the ER but also within the peroxisomes. The function of the peroxisomal reductase in cholesterol/isoprenoid metabolism has yet to be defined. However, it is clear that the ER and peroxisomal HMG-CoA reductases can be regulated differently and, therefore, may play different functional roles (2, 13). The ER reductase has a diurnal cycle distinct from that of the peroxisomal reductase (13). However, the two reductases can also be regulated coordinately. Both reductase activities are induced by cholestyramine (a bile acid resin) (2). No information is available regarding the function of the peroxisomal reductase in cholesterol/ isoprenoid metabolism, nor has the structure of the peroxisomal HMG-CoA reductase been determined. Accordingly, to facilitate our studies of the f...
We recently described the identification of a novel isopentenyl diphosphate isomerase, IDI2 in humans and mice. Our current data indicate that, in humans, IDI2 is expressed only in skeletal muscle. Isoprenoids and isoprenoid-derived compounds play an essential role in all living systems. They provide a necessary function in the organization of many biological systems including membrane structure, signal transduction, and redox chemistry. Several of the important end products of the isoprenoid biosynthetic pathway include: prenylated proteins, dolichols, vitamins A, D, E, and K, steroid hormones, carotenoids, bile acids, and cholesterol (1). In addition, this complex pathway also produces farnesyl diphosphate (FPP) 2 and geranyl-geranyl diphosphate (GGPP), compounds required for the isoprenylation of various G proteins (2).All isoprenoids are derived from the 5-carbon isoprene defined by isopentenyl diphosphate (IPP) and its highly electrophilic isomer dimethylallyl diphosphate (DMAPP). The enzyme isopentenyl diphosphate isomerase (IDI1; EC 5.3.3.2) transforms unreactive IPP into its reactive isomer DMAPP by the concerted addition and abstraction of protons at C-4. These two isomers are the building blocks for the successive head-to-tail condensation reactions that result in the synthesis of geranyl diphosphate (GPP, C 10 ) FPP (C 15 ) and ultimately, non-sterol products and cholesterol (3).IDI1 first identified in Saccharomyces cerevisiae (4) has since been characterized in numerous organisms including humans. Most recently, IDI1 has been identified in hamster and rat where it was shown to localize to the peroxisome by a Pex-5p-dependent PTS1 mechanism (5).Analysis of IDI1 in S. cerevisiae revealed two catalytically active amino acids, Cys 139 and Glu 207 . Mutagenesis analysis in the yeast enzyme demonstrated that a C139S mutation resulted in a significant reduction in isomerase activity whereas a Cys to Val or Ala change at this site abolished activity completely (6).Several examples of multiple IDI isozymes have been reported in plants and algae. In Nicotiana tobacum, the two IDI isozymes are regulated at the transcriptional level under a variety of environmental conditions (7). Similar duplications exist in Cinchona robusta and the green alga Hematococcus pluvialis. Additionally, multiple isozymes of IDI have been identified in higher eukaryotes Sus domesticus and Gallus gallus. In all cases the isozymes maintain specialized functions by different expression patterns, subcellular localization, and susceptibility to inhibitors (8, 9).We previously reported a detailed phylogenetic and structural analysis of IDI2 (10). Molecular modeling suggested that IDI2 is likely to perform functionally as an isomerase despite a Ser to Cys change within the putative active site. In the current study we present the biochemical and functional characterization of IDI2 in mammals. Our data illustrate that IDI2 has a distinct tissue expression pattern, has functional isomerase activity in vivo, a unique kinetic profile, and is...
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