Characterization of peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT2 cells: sterol biosynthesis, phosphorylation, degradation, and statin inhibition.

We have previously identified a CHO cell line (UT2 cells) that expresses only one 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein which is localized exclusively in peroxisomes [Engfelt, H.W., Shackelford, J.E., Aboushadi, N., Jessani, N., Masuda, K., Paton, V.G., Keller, G.A., and Krisans, S.K. (1997) J. Biol. Chem. 272, 24579-24587]. In this study, we utilized the UT2 cells to determine the properties of the peroxisomal reductase independent of the endoplasmic reticulum (ER) HMG-CoA reductase. We demonstrated major differences between the two proteins. The peroxisomal reductase is not the rate-limiting enzyme for cholesterol biosynthesis in UT2 cells. The peroxisomal reductase protein is not phosphorylated, and its activity is not altered in the presence of inhibitors of cellular phosphatases. Its rate of degradation is not accelerated in response to mevalonate. Finally, the degradation process is not blocked by N-acetyl-Leu-Leu-norleucinal (ALLN). Furthermore, the peroxisomal HMG-CoA reductase is significantly more resistant to inhibition by statins. Taken together, the data support the conclusion that the peroxisomal reductase is functionally and structurally different from the ER HMG-CoA reductase.

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.

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.

Metabolism of farnesol: phosphorylation of farnesol by rat liver microsomal and peroxisomal fractions.

In this study we provide evidence for the first time that rat liver microsomal and peroxisomal fractions are able to phosphorylate free farnesol to its diphosphate ester in a CTP dependent manner. The farnesyl diphosphate (FPP) kinase activity is decreased in whole liver homogenates obtained from rats treated with cholesterol and unchanged in homogenates obtained from rats treated with cholestyramine. In contrast, farnesyl pyrophosphatase (FPPase) activity, an enzyme which specifically hydrolyzes FPP to farnesol is only found in the microsomal fraction and is unaffected by treatment of rats with cholesterol or cholestyramine. In addition, we also demonstrate that farnesol can be oxidized to a prenyl aldehyde, presumably by an alcohol dehydrogenase (ADH), and that this activity resides in the mitochondrial and peroxisomal fractions.

Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders.

We reported recently that mevalonate kinase (EC 2.7.1.36; ATP:mevalonate 5-phosphotransferase) that was isolated from rat liver and believed to be a cytosolic protein was localized in rat liver peroxisomes. In addition, we found that the mevalonate kinase monoclonal antibody used in the study also reacted with several other proteins present in the mitochondrial and cytosolic fractions. These findings raised the prospect of the presence of several isoenzymes of mevalonate kinase localized in different compartments of the cell. In the current study we produced four new polyclonal antibodies against different epitopes of mevalonate kinase to investigate the subcellular localization of the protein by several different approaches: (i) by analytical subcellular fractionation and immunoblotting of mevalonate kinase in the isolated subcellular fractions with the monospecific antibodies; (ii) by immunocryoelectron microscopy techniques; and (iii) by expressing the cDNA encoding mevalonate kinase in mammalian cells. The data obtained demonstrate that there is only one mevalonate kinase protein that is predominantly localized in peroxisomes. We also illustrate that the protein is targeted to and imported into peroxisomes. In addition, we show that in cells and tissues obtained from patients with peroxisomal deficiency diseases mevalonate kinase protein and its activity are severely reduced.

Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence.

In the present study we investigated the subcellular localization of squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21). Squalene synthase catalyzes the formation of squalene from trans-farnesyl diphosphate in two distinct steps and is the first committed enzyme for the biosynthesis of cholesterol. Recently, a truncated form of the enzyme from rat hepatocytes was purified, and monospecific antibodies for squalene synthase were produced. This enabled the subcellular localization of squalene synthase by three different methods: (i) analytical subcellular fractionation and measurements of enzyme activities; (ii) immunodeterminations of squalene synthase in the isolated subcellular fractions with a monospecific antibody; and (iii) immunoelectron microscopy. All three methods gave consistent results. The data clearly illustrate that squalene synthase enzymatic activity and squalene synthase are exclusively localized in the endoplasmic reticulum. In rat hepatic peroxisomes we were not able to detect any squalene synthase. In addition, we also demonstrated that squalene synthase in the microsomal fraction is dramatically regulated by a number of hypolipidemic drugs and dietary treatments.

Mevalonate kinase is localized in rat liver peroxisomes.

Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.

Synthesis and secretion of serum gelsolin by smooth muscle tissue.

Gelsolin is one of many actin binding proteins which regulate the structure of intracellular microfilaments. A secretory form of gelsolin, a protein also known as "actin depolymerizing factor" or "brevin," is present in animal sera. In the present studies, we: demonstrate that a 90-kDa secretory protein produced by chicken gizzard smooth muscle is serum gelsolin; show that chicken serum gelsolin, as compared with its mammalian counterparts, lacks 26 amino acid residues at its NH2-terminal end; show that gizzard smooth muscle devotes on the order of 100 times more of its total protein synthetic effort (about 1% of the total) to the production of serum gelsolin than does liver, a previously speculated major source of this protein; and give evidence that rat tissues which are rich in smooth muscle cells (blood vessels, uterine muscle) also produce serum gelsolin. Our work suggests that, in vivo, smooth muscle-containing tissues may be major producers of the serum form of this actin binding protein.

Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.

We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.

Synthesis of apolipoprotein A1 in skeletal muscles of normal and dystrophic chickens.

We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A1 (apo-A1) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following surgical denervation (Shackelford, J. E., and Lebherz, H. G. (1983) J. Biol. Chem. 258, 7175-7180; 14829-14833). In the present work we investigate the effect of avian muscular dystrophy on the synthesis of apo-A1 in chicken muscles. The relative rate of synthesis of apo-A1 and levels of apo-A1 RNA in mature dystrophic breast (fast-twitch) muscle were about 6-fold higher than normal, while synthesis of apo-A1 in breast muscles derived from 2-day-old dystrophic chicks was close to normal. These observations suggest that the elevated apo-A1 synthetic rate in mature dystrophic breast muscle results from a failure of the diseased tissue to "shut down" apo-A1 synthesis to the normal level during postembryonic maturation. Apo-A1 synthesis in the "slow-twitch" lateral adductor muscle of dystrophic chickens was found to be normal. Our work is discussed in terms of the apparent similarities between the effects of surgical denervation and muscular dystrophy on the protein synthetic programs expressed by chicken skeletal muscles.

Regulation of apolipoprotein A1 synthesis in avian muscles.

Until recently, liver and intestinal mucosa were believed to be the sole sites of synthesis of apolipoprotein A1 (Apo-A1), the major protein component of serum high density lipoprotein particles. We recently showed (Shackelford, J.E., and Lebherz, H.G. (1983) J. Biol. Chem. 258, 7175-7180) that chick breast muscle also synthesizes and secretes Apo-A1 but does so at high rates only around the time of hatching. In the present work, we investigate the regulation of synthesis of Apo-A1 in chicken muscles. 1) The primary translation product encoded for by muscle Apo-A1 mRNA is about 2600 daltons larger than the mature serum protein which is consistent with the idea that, like its mammalian liver counterpart, chick muscle Apo-A1 mRNA codes for an NH2-terminal extension (prepro segment) which is 24 amino acids long. 2) The developmentally regulated rise and fall in muscle Apo-A1 synthesis which occurs around the time of hatching is associated with a large accumulation followed by depletion of Apo-A1 mRNA molecules during this period. 3) Reinitiation of Apo-A1 synthesis to high levels in mature breast muscle occurred in vivo following surgical denervation and in vitro by maintaining breast muscle explants for several days in defined culture media. 4) Cardiac, but not smooth, muscles also synthesize and secrete Apo-A1 at high levels around the time of hatching. These and other observations are discussed in terms of possible regulatory "signals" which may control Apo-A1 synthesis in avian muscles.

Synthesis and secretion of apolipoprotein A1 by chick breast muscle.

The present work shows that chick breast muscles synthesize and secrete a protein which is very similar to chicken serum apolipoprotein A1 (Apo-A1), the major protein constituent of serum "high density" lipoprotein particles. This conclusion is based on the following observations. 1) When chick breast muscle explants were incubated in the presence of radioactive amino acids, a labeled protein of the same size as serum Apo-A1 (Mr approximately equal to 27,000) accumulated in the incubation media; 2) the muscle-derived secretory protein and serum Apo-A1 generated the same size distribution of peptide fragments following digestion with Staphylococcus aureus V8 protease; and 3) antibodies raised against serum Apo-A1 specifically precipitated the radioactive muscle secretory protein. The newly secreted muscle-derived Apo-A1 was associated with lipid, as judged by its "flotation" behavior during centrifugation of the labeled incubation media in the presence of 0.2 g/ml of sodium bromide; this observation suggests that muscle explants secreted Apo-A1 molecules as part of lipoprotein particles or that these Apo-A1 molecules became associated with lipid shortly after their secretion. The present work, together with the very recent report by Blue et al. (Blue, M.L., Ostapchuk, P., Gordon, J.S., and Williams, D.L. (1982) J. Biol. Chem. 257, 11151-11159) demonstrate that avian tissues other than liver and intestinal mucosa synthesize and secrete Apo-A1. Results of short term amino acid incorporation experiments showed that chick breast muscles synthesize Apo-A1 at high rates only during the terminal stages of embryonic development and early stages of postembryonic maturation. Around the time of hatching, the relative rate of synthesis of Apo-A1 by chick breast muscle was found to be higher than in liver, a documented major site of synthesis of this apolipoprotein. Possible physiological implications of the present work will be considered.

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, 1982), 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-terminal 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 proteins per 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 the relative 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.

Effect of denervation on the levels and rates of synthesis of specific enzymes in "fast-twitch" (breast) muscle fibers of the chicken.

It has been well documented that neural information, or the consequences of it, is required for the full phenotypic expression of different skeletal muscle fiber types. In the present work, we investigate the effect of removal of neural information, via surgical denervation, on the levels and rates of synthesis of several enzyme in mature breast ("fast-twitch") "white" muscle fibers of the chicken. Denervation of these muscles resulted in reductions in the concentrations of several glycolytic enzymes to new steady state levels which were only about 50% of normal, and these decreases in enzyme levels were completed within 2 weeks after severing the nerves. In contrast, denervation for as long as 6 weeks did not have a significant effect on the levels of creatine-P kinase molecules in this muscle type. The decreased level of the skeletal muscle-specific aldolase A4 isoenzyme in denervated breast muscle fibers was associated with a 2- to 3-fold reduction in the relative rate of synthesis of this enzyme following denervation. As expected, denervation had no appreciable effect on the relative rate of synthesis of the muscle-specific MM isoenzyme of creatine-P kinase in this muscle. Our results show that neural information, or the consequences of it, is required to maintain the levels and rates of synthesis of glycolytic enzymes but not of creatine-P kinase in mature fast-twitch muscle fibers. We suggest that denervation results in a partial "dedifferentiation" of these fibers.