Janis E. Shackelford

Localization of the pre-squalene segment of the isoprenoid biosynthetic pathway in mammalian peroxisomes

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 immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm 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 β-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.

Cloning and Subcellular Localization of Hamster and Rat Isopentenyl Diphosphate Dimethylallyl Diphosphate Isomerase A PTS1 MOTIF TARGETS THE ENZYME TO PEROXISOMES

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.

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model.

ABSTRACT 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 peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.

Peroxisome Deficiency Causes a Complex Phenotype because of Hepatic SREBP/Insig Dysregulation Associated with Endoplasmic Reticulum Stress

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.

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken☆

Abstract The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.

Characterization of UT2 Cells THE INDUCTION OF PEROXISOMAL 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE

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.

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.

IDI2, a Second Isopentenyl Diphosphate Isomerase in Mammals

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. Expression constructs of human IDI2 in Saccharomyces cerevisiae can complement isomerase function in an idi1-deficient yeast strain. Furthermore, IDI2 has the ability to catalyze the isomerization of [14C]IPP to [14C]DMAPP. Enzyme kinetic analysis of partially purified IDI2 demonstrate the novel isozyme has a maximal relative specific activity of 1.2 × 10-1 ± 0.3 μmol min-1 mg-1 at pH 8.0 with a KIPPm value of 22.8 μm IPP. Both isozymes, IDI1 and IDI2 are localized to the peroxisome by a PTS1-dependent pathway. Finally, our data suggest that IDI2 is regulated independently from IDI1, by a mechanism that may involve PPARα.

STUDIES ON THE REGULATION OF PROTEIN CONCENTRATIONS IN “RED” AND “WHITE” SKELETAL MUSCLES*

Publisher Summary This chapter presents the studies on the regulation of protein concentrations in red and white skeletal muscles. The structural and functional characteristics of animal cells are largely determined by the assortment of proteins they contain. As is the case with other types of animal cells, much of the fate of a muscle cell must be predetermined by events occurring during the process of cellular differentiation, and many of the biochemical changes that occur during embryogenesis in vivo have been observed when cultures of myogenic cells are allowed to differentiate in vitro. Myosin and actin occupy the same intracellular compartment, the myofibril. A final consideration on compartmentalization of the proteins concerns the possibility that certain muscle enzymes, especially those operating in the glycolytic pathway, may interact with each other to form a multienzyme complex at or near the myofibril.

Similarities in properties, content, and relative rates of synthesis of fructose-P2 aldolase in livers of fed and starved rats

The present work gives evidence that, in contrast to the situation reported by Pontremoli et al. for the rabbit (Proc. Natl. Acad. Sci. U.S.A. 76, 6323–6325, 1979; Arch. Biochem. Biophys. 203, 390–394, 1980; Proc. Natl. Acad. Sci. U.S.A., 79, 5194–5196, 1992), starvation for as long as 3 days does not cause intracellular covalent modification and inactivation of fructose-P2 aldolase molecules in rat liver cells. This conclusion is based on our observations that liver aldolase molecules isolated from fed and starved rats in the presence of proteolytic inhibitors were not distinguished on the basis of specific catalytic activity, electrophoretic mobility, subunit molecular weight, NH2-terminat structure, or COOH-terminal structure. Further, the approximate 40% loss in rat liver mass which occurred during the 3-day fast was not associated with appreciable changes in the content of aldolase and most other abundant cytosolic proteinsper gram of rat liver, as judged by electrophoretic analysis of 100 000-g soluble fractions of liver extracts . Finally, a 3-day fast had no appreciable effect on therelative rates of synthesis of aldolase and most other abundant cytosolic proteins in rat liver. Our findings suggest that nutrient deprivation has no preferential effect on the concentration or metabolism of aldolase in rat liver cells.