Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells, and to date have been shown to contain approximately 60 identified enzymes involved in various metabolic pathways, including the oxidation of a variety of lipids. These lipids include very long-chain fatty acids, methyl branched fatty acids, prostaglandins, bile-acid precursors and xenobiotics that are either β-oxidized or α-oxidized in peroxisomes. The recent identification of several acyl-CoA thioesterases and acyltransferases in peroxisomes has revealed their various functions in acting as auxiliary enzymes in α- and β-oxidation in this organelle. To date, 9 functional acyl-CoA thioesterases and acyltransferases have been identified in mouse and 4 functional acyl-CoA thioesterases and acyltransferases in human, thus these enzymes make up a substantial portion of peroxisomal proteins. This review will therefore focus on new and emerging roles for these enzymes in assisting with the oxidation of various lipids, amidation of lipids for excretion from peroxisomes, and in controlling coenzyme A levels in peroxisomes.     

Leptin: an essential regulator of lipid metabolism

This paper reviews the general mechanisms by which leptin acts as a regulator of lipid reserves through changes in food intake, energy expenditure and fuel selection, with an emphasis on its direct effects on cellular lipid metabolism. Briefly, when leptin levels increase, food consumption decreases via modulation of hypothalamic neuropeptides. As well, normal decreases in energy expenditures (e.g. with diurnal cycles or reduced caloric intake) do not occur. This is probably caused by an increase in mitochondrial proton leak mediated by leptin via increases in sympathetic nervous system stimulation and thyroid hormone release. The decrease in caloric input coupled with relatively higher energy expenditure, therefore, leads to negative energy balance. Leptin also changes the fuel source from which ATP is generated. Fuel preference switches from carbohydrate (glucose) to lipid (fatty acids). This effect arises through stimulation of triacylglycerol catabolism by leptin. In vitro studies show that leptin is a potent stimulator of lipolysis and fatty acid oxidation in adipocytes and other cell types. Consequently, leptin is also a regulator of cellular triacylglycerol content. Hormonal regulation of leptin, as well as its role in fasting and seasonal weight gain and energy expenditure are also briefly discussed.     

Review. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator

SUR1 is an ATP-binding cassette (ABC) transporter with a novel function. In contrast to other ABC proteins, it serves as the regulatory subunit of an ion channel. The ATP-sensitive (KATP) channel is an octameric complex of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits, and it links cell metabolism to electrical activity in many cell types. ATPase activity at the nucleotide-binding domains of SUR results in an increase in KATP channel open probability. Conversely, ATP binding to Kir6.2 closes the channel. Metabolic regulation is achieved by the balance between these two opposing effects. Precisely how SUR1 talks to Kir6.2 remains unclear, but recent studies have identified some residues and domains that are involved in both physical and functional interactions between the two proteins. The importance of these interactions is exemplified by the fact that impaired regulation of Kir6.2 by SUR1 results in human disease, with loss-of-function SUR1 mutations causing congenital hyperinsulinism and gain-of-function SUR1 mutations leading to neonatal diabetes. This paper reviews recent data on the regulation of Kir6.2 by SUR1 and considers the molecular mechanisms by which SUR1 mutations produce disease.     

Characterization of the major allergen of plum as a lipid transfer protein

Background: Allergy to Prunoideae fruit (plum, peach, cherry and apricot) is one of the most frequent food allergies in southern Europe. All these fruits cross-react in vivo and in vitro, as they share their major allergen, a 9 kD lipid transfer protein (LTP). Objective: The aim of the study was the identification and molecular characterization of the major allergen of plum. Methods: The IgE pattern of reactivity to plums was investigated by SDS–PAGE and immunoblotting with the sera of 23 patients. The identified major allergen was purified by HPLC, using a cationic-exchange column followed by gel-filtration. Further characterization was achieved by periodic-Schiff stain, isoelectrofocusing and N-terminal amino acid sequencing. Results and conclusions: The major allergen of plum is a 9 kD lipid transfer protein, not glycosylated and with a basic character (pI>9), highly homologous to the major allergen of peach.     

Identification of PTE2, a human peroxisomal long-chain acyl-CoA thioesterase

Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes.     

Amyloid beta as a regulator of lipid homeostasis

The beta-amyloid peptide (A beta) is widely considered to be the molecule that causes Alzheimer's disease (AD). Besides this pathological function of A beta, recently published data reveal that A beta also has an essential physiological role in lipid homeostasis. Cholesterol increases A beta production, and conversely A beta production causes a decrease in cholesterol synthesis. The latter appears to be mediated by the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), a key enzyme in cholesterol synthesis, in an action similar to that of statins. Moreover, A beta regulates sphingolipid metabolism by directly activating sphingomyelinases (SMases). This review summarizes the molecular basis for the known physiological functions of A beta and amyloid precursor protein (APP), the roles of A beta and APP in lipid homeostasis and the medical implications of addressing lipid homeostasis in respect to AD. This knowledge might provide new insights for current and future therapeutic approaches to AD.     

Cardiotrophin-1 is a key regulator of glucose and lipid metabolism

Cardiotrophin-1 (CT-1) is a member of the gp130 family of cytokines. We observed that ct-1(-/-) mice develop mature-onset obesity, insulin resistance, and hypercholesterolemia despite reduced calorie intake. Decreased energy expenditure preceded and accompanied the development of obesity. Acute treatment with rCT-1 decreased blood glucose in an insulin-independent manner and increased insulin-stimulated AKT phosphorylation in muscle. These changes were associated with stimulation of fatty acid oxidation, an effect that was absent in AMPKα2(-/-) mice. Chronic rCT-1 treatment reduced food intake, enhanced energy expenditure, and induced white adipose tissue remodeling characterized by upregulation of genes implicated in the control of lipolysis, fatty acid oxidation, and mitochondrial biogenesis and genes typifying brown fat phenotype. Moreover, rCT-1 reduced body weight and corrected insulin resistance in ob/ob and in high-fat-fed obese mice. We conclude that CT-1 is a master regulator of fat and glucose metabolism with potential applications for treatment of obesity and insulin resistance.     

Follistatin as a potent regulator of bone metabolism

Follistatin is a monomeric glycoprotein, distributed in a wide range of tissues. Recent work has demonstrated that this protein is a pluripotential molecule that has no structural similarity but is functionally associated with members of the transforming growth factor (TGF)-β superfamily, which indicates its wide range of action. Members of the TGF-β superfamily, especially activins and bone morphogenetic proteins are involved in bone metabolism. They play an important role in bone physiology, influencing bone growth, turnover, bone formation and cartilage induction. As follistatin is considered to be the antagonist of the TGF-β superfamily members, it plays an important role in bone metabolism and development.     

Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5′ and 3′ ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1–C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V _max and apparent K _M values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV–Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.     

Chloroplastic thioredoxin m functions as a major regulator of Calvin cycle enzymes during photosynthesis in vivo

Thioredoxins (Trxs) regulate the activity of various chloroplastic proteins in a light‐dependent manner. Five types of Trxs function in different physiological processes in the chloroplast of Arabidopsis thaliana. Previous in vitro experiments have suggested that the f‐type Trx (Trx f) is the main redox regulator of chloroplast enzymes, including Calvin cycle enzymes. To investigate the in vivo contribution of each Trx isoform to the redox regulatory system, we first quantified the protein concentration of each Trx isoform in the chloroplast stroma. The m‐type Trx (Trx m), which consists of four isoforms, was the most abundant type. Next, we analyzed several Arabidopsis Trx‐m‐deficient mutants to elucidate the physiological role of Trx m in vivo. Deficiency of Trx m impaired plant growth and decreased the CO₂ assimilation rate. We also determined the redox state of Trx target enzymes to examine their photo‐reduction, which is essential for enzyme activation. In the Trx‐m‐deficient mutants, the reduction level of fructose‐1,6‐bisphosphatase and sedoheptulose‐1,7‐bisphosphatase was lower than that in the wild type. Inconsistently with the historical view, our in vivo study suggested that Trx m plays a more important role than Trx f in the activation of Calvin cycle enzymes.     

Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.

Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).     

Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide ((.)NO(2)), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO(-/-)), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stress in vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F(2)-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F(2)-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO(-/-) mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO(-/-) group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and (.)NO(2), respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO(-/-) samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of (.)NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo.