palmitoyl-CoA Hydrolase Research Articles

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target

Histone deacetylase (HDAC) targeting drugs have entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules, notably for HDAC6, and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-beta-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nM potency. MBLAC2 enzymatic inhibition and knock down led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Capsicum anum L. Derived Phytochemicals against Haemophilus influenzae Causing Bronchitis

Phytochemicals from Capsicum anum L. plant extract are traditionally used to cure bronchitis. Bronchitis is caused by Haemophilus influenzae. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that myrcetin and quercetin can effectively deactivate the Palmitoyl-CoA hydrolase enzyme thereby interrupting the life cycle of Haemophilus influenza.

Mechanism of action of liver growth induced by peroxisome proliferators

Humans are ubiquitously exposed to peroxisome proliferators including hypolipidemic agents, industrial solvents and atural products. Because of this and the fact that peroxisome proliferators cause non-genotoxic hepatocarcinogenesis in rodents, it is of importance to elucidate the mechanism of action of the peroxisome proliferators in order to provide an assessment of the hazard, if any, of these compounds to humans. It is also known that the peroxisome proliferators begin their actions by inducing hepatic DNA synthesis. Thus, the aim of this thesis was to find genes that could be responsible for triggering the induction of hepatic DNA synthesis caused by peroxisome proliferators, specifically ciprofibrate. First, it was important to indicate when the induction of hepatic DNA synthesis actually happens. This was done with BrdU immunohistochemical procedures. The induction of hepatic DNA synthesis with ciprofibrate in mice was observable only after 4 days making it difficult to specify when the induction actually happened. In rats the induction of hepatic DNA synthesis was found to peak at 24 hours and this system gave the better opportunity to find the genes responsible. The difference in the timing of induced hepatic DNA synthesis betweenmice and rats implied that there could be a species difference in the mechanism of each species’ response to PPAR. With immunohistochemistry it was noticed that there was a difference in the lobular localization of hepatic DNA synthesis in the liver tissues of rats and mice dosed with different inducers, with the rat livers exhibiting periportal distribution while hepatic DNA synthesis in the mice seemed to be distributed throughout the liver tissue. The effects of ciprofibrate or cyproterone acetate on liver gene expression in rats were studied, using cDNA microarrays, transcriptome sequencing and quantitative real- time PCR. A 1- 5 hour treatment period was chosen to detect the immediate early gene response, while a 24 hour time point was chosen to elucidate the confounding effects from the hepatic DNA synthesis seen during the 24 hour stimulation. The results showed that ciprofibrate altered the expression of numerous genes including previously known PPARa agonist-responsive genes involved in processes such as PPAR signalling pathways, fatty acid metabolic pathway, cell cycle, palmitoyl-CoA hydrolase activity, lipid metabolism, inflammatory responses, and stress responses, in addition to a large number of novel candidate genes. Three novel induced genes G0s2, Ccnd1 and Scd1, (and two marker genes CYP4A1 and CYP3A1) were confirmed with quantitative real- time PCR. The G0s2, Ccnd1 and Scd1 were found to be up-regulated at the hours 1 and 3 after dosing and not 24 hours, and the G0s2 and Scd1 were specific for the ciprofibrate suggesting they were involved in a distinct PPARa pathway responsible for the hepatic DNA synthesis. The complete database of the transcriptional response provided here opens doors of opportunity for further research to identify genes responsible for the liver growth induced by peroxisome proliferators.

Gene expression profiling in the type 1 diabetes rat diaphragm.

BackgroundRespiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression.Methodology/Principal FindingsDiaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested.Conclusions/SignificanceThese data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.

A 50-kDa isoform of mouse brain acyl-CoA hydrolase: expression and molecular properties

Brain acyl-CoA hydrolase (BACH) is responsible for most of the long-chain acyl-CoA hydrolyzing activity in the brain and is localized exclusively in neurons. There are two BACH isoforms: the major isoform, a 43-kDa BACH, and a lesser isoform, a 50-kDa BACH. In our previous work [Brain Res. Mol. Brain Res. 98 (2002) 81], a possibility was raised that these BACH isoforms might be generated from a single mRNA species via a mechanism of alternative use of translation start sites. However, the results obtained in the current study indicated that the 43-kDa BACH and 50-kDa BACH are not generated from a single mRNA species, but from distinct mRNA species transcribed by alternative use of transcription start sites. The molecular properties of the 50-kDa BACH were compared to those of the 43-kDa BACH. Palmitoyl-CoA hydrolase activity and protein stability were almost the same between both BACH isoforms. In addition, both 43-kDa BACH and 50-kDa BACH that were fused to green fluorescent protein showed cytosolic distribution. These results suggest that the 50-kDa BACH plays a similar role as the 43-kDa BACH. Therefore, since the 43-kDa BACH is expressed at higher levels than 50-kDa BACH, the 43-kDa BACH should largely contribute to understanding the physiological functions of the BACH gene in neurons.

Inducible expression of long-chain acyl-CoA hydrolase gene in cell cultures.

A long-chain acyl-CoA hydrolase, BACH, is markedly distributed in the brain and localized in neurons. However, the physiological significance of BACH is unclear. To study the gene function, we expressed the mouse BACH gene in C3H 10T1/2 fibroblastic cells using a mifepristone (RU486)-inducible gene expression system. A cell clone, 10T-S6/44, was generated by stable transfection of two plasmids encoding a mifepristone-dependent transactivator and an inducible transgene product, BACH with a C-terminal MYC-tag (BACH-MYC). The transgene expression in the 10T-S6/44 cells was tightly regulated by mifepristone. Induction of BACH-MYC and an increase in palmitoyl-CoA hydrolase activity were observed in the cells treated with 3 x 10(-11) M mifepristone and reached maximal levels at a concentration of 1 x 10(-9) M for 48 h. The growth rate of cells showing the maximal induction of BACH-MYC was reduced, whereas phospholipid synthesis was unchanged. These results suggested that BACH affects specific cellular systems and functions, but not all acyl-CoA-utilizing processes.

Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization

Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.

Nitric oxide reduces the palmitoylation of rat myelin proteolipid protein by an indirect mechanism.

Brain slices from 20-day-old rats were incubated with [3H]palmitate for 2 hours in the absence or presence of the NO-donors S-nitroso-N-acetyl-penicillamine (SNAP), ethyl-2-[hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), 4-phenyl-3-furoxan carbonitrile (PFC) and sodium nitroprusside (SNP). Each of these drugs reduced the incorporation of [3H]palmitate into myelin proteolipid protein (PLP) in a concentration-dependent manner, SNP being the most active. The effect of SNAP was prevented by the NO-scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide). Furthermore, decayed-SNAP, sodium nitrite and N- nitrosopyrrolidine were inactive, suggesting that free NO and/or some of its direct oxidation products are the active molecular species. The amount of fatty acids bound to PLP and the rate of deacylation were unaffected by NO. Although NO diminished the number of thiols in brain and myelin proteins, with the formation of both nitrosothiols and disulfides, these changes did not parallel those in PLP acylation. In contrast, NO was effective at reducing the palmitoylation of brain and myelin lipids, and this effect along with that of PLP, was ascribed to a decrease in palmitoyl-CoA levels. The NO-induced reduction in acyl-CoA concentration was due to the decline in ATP levels, while the amount of [3H]palmitate incorporated into the tissue, the activity of palmitoyl-CoA ligase and palmitoyl-CoA hydrolase, and the concentration of CoASH were unaltered by the drugs. Experiments with endogenously-synthesized [18O]fatty acids confirmed that NO affects predominantly the ATP-dependent palmitoylation of PLP. In conclusion, the inhibitory action of NO on the fatty acylation of PLP is indirect and caused by energy depletion.

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

Purification, Molecular Cloning, and Genomic Organization of Human Brain Long-Chain Acyl-CoA Hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.

Molecular Cloning and Expression of Palmitoyl-protein Thioesterase 2 (PPT2), a Homolog of Lysosomal Palmitoyl-protein Thioesterase with a Distinct Substrate Specificity

Palmitoyl-protein thioesterase is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. Mutations in this enzyme were recently shown to underlie the hereditary neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis, and lipid thioesters derived from acylated proteins were found to accumulate in lymphoblasts from individuals with the disorder. In the current study, we describe the cloning and expression of a second lysosomal thioesterase, palmitoyl-protein thioesterase 2 (PPT2), that shares an 18% identity with palmitoyl-protein thioesterase. Transient expression of a PPT2 cDNA led to the production of a glycosylated lysosomal protein with palmitoyl-CoA hydrolase activity comparable with palmitoyl-protein thioesterase. However, PPT2 did not remove palmitate groups from palmitoylated proteins that are substrates for palmitoyl-protein thioesterase. In cross-correction experiments, PPT2 did not abolish the accumulation of protein-derived lipid thioesters in palmitoyl-protein thioesterase-deficient cell lines. These results indicate that PPT2 is a lysosomal thioesterase that possesses a substrate specificity that is distinct from that of palmitoyl-protein thioesterase.

Molecular Cloning and Expression of cDNAs Encoding Rat Brain and Liver Cytosolic Long-Chain Acyl-CoA Hydrolases

cDNAs encoding the long-chain acyl-CoA hydrolases (ACHs) from rat brain and liver, referred to as rBACH and rLACH1, respectively, were isolated and sequenced. The rBACH cDNA contained an open reading frame encoding a 338-amino acid polypeptide with a calculated molecular weight of 37,559, of which the deduced amino acid sequence matched partial amino acid sequences directly determined for peptides generated by tryptic digestion or CNBr cleavage of purified rBACH. The rLACH1 cDNA contained an open reading frame encoding a 343-amino acid polypeptide with a molecular weight of 38,240. When expressed inEscherichia coli,these cDNAs produced palmitoyl-CoA hydrolase activity and 44-kDa proteins with molecular masses similar to those of purified rBACH and rLACH1 (43 kDa). These expressed proteins and enzyme activity were immunoblotted and neutralized, respectively, by anti-rBACH or anti-rLACH1 antibodies. rLACH1 cDNA had 84 and 94% identity with rBACH cDNA at the nucleotide and amino acid levels, respectively. However, the 5′-end of the former cDNA which contained the N-terminal coding region of rLACH1 was entirely different from the corresponding region of rBACH cDNA, suggesting that these enzymes may be generated by alternative use of exons of the same gene. Northern blot analysis showed that ACH mRNA was expressed constitutively in the rat brain and testis, whereas its expression in the liver was inducible by treatment with the peroxisome proliferator. This study demonstrated the molecular diversity of ACH and suggested the presence of tissue-specific mechanisms to regulate the ACH gene expression.

On the effects of thia fatty acid analogues on hydrolases involved in the degradation of m etabolisable and non-m etabolisable acyl-CoA esters

1. We investigated the nature and roles of various xenobiotic acyl-CoA hydrolases in liver subcellular fractions from rat treated with sulphur-substituted (thia) fatty acids. To contribute to our understanding of factors influencing enzymes involved in the degradation of activated fatty acids, the effects on these activities of the oppositely acting thia fatty acid analogues, the peroxisome proliferating 3-thia fatty acids (tetradecylthioacetic acid and 3- dithiacarboxylic acid), which are blocked for β-oxidation, and a non-peroxisomeproliferating 4-thia fatty acid (tetradecylthiopropionic acid), which undergoes one cycle of β-oxidation, were studied. 2. The hepatic subcellular distributions of palmitoyl-CoA, tetradecylthioacetyl-CoA and tetradecylthiopropionyl-CoA hydrolase activities were similar to each other in the control and 3-thia fatty acid-treated rat. In control animals, most of these hydrolases were located in the microsomal fraction, but after treatment with the 3-thia fatty acids, the specific activities of the mitochondrial, peroxisomal, and cytosolic palmitoyl-CoA, tetradecylthioacetyl-CoA, and tetradecylthiopropionyl-CoA hydrolase activities were significantly increased. This increase in activity was seen mostly for the enzymes using tetradecylthiopropionyl-CoA and tetradecylthioacetyl-CoA as substrates. The increased mitochondrial activities for these two substrates were seen already after 1 day of treatment, whereas the peroxisomal activities increased after 3 days. No stimulation was seen after treatment with the 4-thia fatty acid analogue, tetradecylthiopropionic acid, but a decrease in peroxisomal hydrolase activities for all three substrates was observed. 3. The cellular distributions of clofibroyl-CoA, POCA-CoA, and sebacoyl-CoA hydrolase activities were different from those of the 'long-chain acyl-CoA' hydrolases mentioned above both in the normal and 3-thia fatty acid treated rat. This group of hydrolases was found in the mitochondrial, peroxisomal, and cytosolic fractions. 3-Thia fatty acid treatment increased the activities of clofibroyl-CoA and sebacoyl-CoA hydrolases in all three fractions. Clofibroyl-CoA and sebacoyl-CoA hydrolase activities were increased after 1 day of treatment. Only the cytosolic POCA-CoA hydrolase was stimulated after 3- thia fatty acid treatment after only 1 day of treatment, whereas treatment with the 4-thia fatty acid led to an increase of enzyme activity in the mitochondrial and peroxisomal fractions. 4. Based on the subcellular distributions and specific activities, we suggest that several enzymes exist which may act as regulators of intracellular acyl-CoA levels.

Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase)

A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses omicron-nitrophenyl acetate and alpha-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688-29696; Yan, Yang, and Parkinson (1995) Arch. Biochem. Biophys. 317, 222-234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.

Long-Chain Acyl-CoA Hydrolase from Rat Brain Cytosol: Purification, Characterization, and Immunohistochemical Localization

Long-chain acyl-CoA hydrolase (EC 3.1.2.2), which is found primarily in the brain in rats, catalyzes the hydrolysis of fatty acyl-CoA thioesters. We purified this enzyme, referred to as ACH, from the rat brain cytosol. The molecular masses of the native enzyme and the subunit were estimated to be 104 and 36 kDa, respectively. The enzyme showed high activity with long-chain acyl-CoAs, e.g., with maximal velocity of 262 μmol/min/mg andKmof 5.7 μMfor palmitoyl-CoA, but acyl-CoAs with carbon chain lengths of C8–18were also good substrates. The enzyme was refractory to the inhibitory effect of diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but sensitive top-chloromercuribenzoate. In the rat brain cytosol, about 90% of palmitoyl-CoA hydrolase activity was titrated by anti-ACH antibody, which accounted for over 70% of the enzyme activity found in the brain tissue. Immunoblots of the cytosol prepared from rat brain regional blocks indicated the broad distribution of ACH over the brain, with a relatively high level in the pons and medulla. Immunohistochemically, ACH was localized to neurons. In addition to various nuclei, some neuronal cells, such as mitral cells in the olfactory bulb, pyramidal cells in the cerebral cortex, and Purkinje cells in the cerebellum, were also immunostained with anti-ACH antibody. Brain cytosols prepared from ten mammalian species including human contained a single polypeptide reactive to anti-ACH antibody with molecular masses of 34–36 kDa, together with high activities of palmitoyl-CoA hydrolase. These findings suggest the physiological significance of ACH in the brain, although its precise role remains to be determined.

Different effects of fibrates on the microsomal fatty acid chain elongation and the acyl composition of phospholipids in guinea‐pigs

1. The effects in vitro and in vivo of three fibric acid derivatives, clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on some enzyme activities related to fatty acid biosynthesis, namely palmitoyl-CoA synthetase and hydrolases (microsomal and cytosolic), NADH and NADPH cytochrome c reductases and acyl-CoA elongases were investigated in guinea-pigs. 2. The three fibrates inhibited acyl-CoA elongation in vitro, irrespective of the substrate of elongation used (saturated, monounsaturated, polyunsaturated) and with an order of potency GFB > BFB > CFB. In the case of GFB, inhibition occurred at concentrations that can be reached in vivo. 3. Microsomal palmitoyl-CoA hydrolase and synthetase were also inhibited in vitro (GFB > or = BFB > CFB), whereas NADH cytochrome c reductase activity was increased by GFB. Nevertheless, the magnitude of changes were lower than those observed in elongation activities. 4. Treatment with fibrates did not produce peroxisomal proliferation in guinea-pigs, as measured by peroxisomal beta-oxidation activity and liver weight/body weight ratio. Nevertheless, fibrates provoked a reduction in plasma cholesterol and triglycerides, at least in GFB- and BFB-treated animals. 5. Fatty acid elongation was significantly modified by GFB treatment in vivo. The remaining enzyme activities studied were only slightly changed by fibrate treatment. 6. Treatment with BFB and to a lesser extent with CFB, increased the relative proportion of MUFA (palmitoleic and oleic acids) in microsomal phospholipids, whereas PUFA (mainly linoleic acid) decreased. GFB behaved differently, increasing palmitic and linoleic acids and decreasing stearic and oleic acids. The latter changes are attributable to an inhibition of elongation activity by GFB. 7. The changes observed after fibrate treatment in both rats and guinea-pigs, as they are not directly related to peroxisome proliferation, could be more reliably extrapolated to man than those observed only in rats.

27] Assay and isolation of palmitoyl-protein thioesterase from bovine brain using palmitoylated H-Ras as substrate

Publisher Summary This chapter discusses the assay and isolation procedure of palmitoyl-protein thioesterase from bovine brain using palmitoylated H-Ras as substrate. In the case of H-Ras, palmitoylation increases association of the protein with cellular membranes and is required for full transforming activity. A dynamic cycle of Ras acylation and deacylation is demonstrated in vivo, suggesting a possible regulatory pathway mediating membrane association. Using H-Ras as a substrate, an assay for an enzyme that removes the covalently bound palmitate and purified it to homogeneity from a soluble fraction of bovine brain is developed. In addition to palmitoyl-Ras thioesterase activity, the enzyme has palmitoyl-CoA hydrolase activity comparable to mammalian thioesterases I and II and Escherichia coli thioesterase I; none of the other thioesterases removes palmitate groups from H-Ras. [3H]Palmitate-labeled, histidine-tagged H-Ras substrate is prepared using the insect baculovirus system. A recombinant baculovirus is created that encodes H-Ras with an N-terminal histidine affinity tag (vHis6-HRas) to facilitate protein purification. Infected Sf9 cells are metabolically labeled with [3H]palmitate, and [3H]palmitate-labeled, histidine-tagged H-Ras is purified from detergent-solubilized cell membranes by nickel affinity chromatography.

Relationship between plasma lipids and palmitoyl‐CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates

1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities. 7. Some of the effects of fibrate treatment can be explained, at least in part, in terms of peroxisomal induction and caution should be exercised in the extrapolation of these results to species, such as man,that are insensitive to peroxisomal proliferation.