Cholesterol ester hydrolase inhibitors reduce the production of synaptotoxic amyloid-β oligomers

The production of amyloid-β (Aβ) is the key factor driving pathogenesis in Alzheimer's disease (AD). Increasing concentrations of Aβ within the brain cause synapse degeneration and the dementia that is characteristic of AD. Here the factors that affect the release of disease-relevant forms Aβ were studied in a cell model. 7PA2 cells expressing the human amyloid precursor protein released soluble Aβ oligomers that caused synapse damage in cultured neurons. Supernatants from 7PA2 cells treated with the cholesterol synthesis inhibitor squalestatin contained similar concentrations of Aβ₄₂ to control cells but did not cause synapse damage in neuronal cultures. These supernatants contained reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers. Treatment of 7PA2 cells with platelet-activating factor (PAF) antagonists had similar effects; it reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. PAF activated cholesterol ester hydrolases (CEH), enzymes that released cholesterol from stores of cholesterol esters. Inhibition of CEH also reduced concentrations of Aβ₄₂ oligomers and increased concentrations of Aβ₄₂ monomers in cell supernatants. The Aβ monomers produced by treated cells protected neurons against Aβ oligomer-induced synapse damage. These studies indicate that pharmacological manipulation of cells can alter the ratio of Aβ monomer:oligomer released and consequently their effects on synapses.     

Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr−/− mice

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR^−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.     

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.     

Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23?%, 17?% and 64?% increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Glycoside hydrolase family 31 Escherichia coli α-xylosidase

Escherichia coli YicI is a retaining α-xylosidase, which strictly recognizes the α-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli α-xylosidase (YicI). Detection of distinguishing features between α-xylosidases and α-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an α-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, α-d-Xylp-(1→6)-d-Manp, α-d-Xylp-(1→6)-d-Fruf, and α-d-Xylp-(1→3)-d-Frup, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal α-glucosidases.     

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 2. Pronase-catalysed hydrolysis of tryptophan methyl ester

The influence of pH, temperature, EDTA and metal ions on the rate of hydrolysis of methyl esters of d- and l-tryptophan by pronase from Streptomyces griseus has been studied. pH, Ca²⁺ and Co²⁺ ions have different effects on the hydrolysis of d- and l-substrates, which permits the enantioselectivity of the reaction to be regulated. Maximum enantioselectivity was observed at pH 7 in the presence of 10^?3–10^?2m Co²⁺. The data obtained are compared with the literature data for the hydrolysis of other low molecular weight substrates by pronase. It is suggested that leucine aminopeptidase makes a considerable contribution to the observed esterase activity of pronase.     

Microbial production of optically active β-phenylalanine ethyl ester through stereoselective hydrolysis of racemic β-phenylalanine ethyl ester

The ability to produce (R)- or (S)-β-phenylalanine ethyl ester (3-amino-3-phenylpropionic acid ethyl ester, BPAE) from racemic BPAE through stereoselective hydrolysis was screened for in BPAE-assimilating microorganisms. Sphingobacterium sp. 238C5 and Arthrobacter sp. 219D2 were found to be potential catalysts for (R)- and (S)-BPAE production, respectively. On a 24-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Sphingobacterium sp. 238C5 as the catalyst, 1.15% (w/v) (R)-BPAE (60 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 46%. On a 48-h reaction, with 2.5% (w/v) racemic BPAE (130 mM) as the substrate and wet cells of Arthrobacter sp. 219D2 as the catalyst, 0.87% (w/v) (S)-BPAE (45 mM) with enantiomeric purity of 99% e.e. was obtained, the molar yield as to racemic BPAE being 35%. The enzyme stereoselectively hydrolyzing (S)-BPAE was purified to homogeneity from the cell-free extract of Sphingobacterium sp. 238C5. The enzyme was a monomeric protein with a molecular mass of about 42,000. The enzyme catalyzed hydrolysis of β-phenylalanine esters, while the common aliphatic and aromatic carboxylate esters were not catalyzed.     

Amino acid ester synthesis by immobilized α-chymotrypsin

Summary A screening of immobilized -chymotrypsin preparations suitable for the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and up to 99% ethanol was carried out. -Chymotrypsin adsorbed to Sepharose LH-20 or covalently bound to Sepharose 4B (tresyl chloride activation) was found to be an efficient catalyst. A column packed with immobilized enzyme retained 60% of its initial activity after 6 days of operation in a cyclohexane-ethanol medium.     

Chalcone oxides—potent selective inhibitors of cytosolic epoxide hydrolase

The systematic screening of over 150 compounds for inhibitory activity on mammalian cytosolic epoxide hydrolase led to identification of chalcone oxide (trans-1-benzoyl-2-phenyloxirane) as an optimal inhibitory structure. Important structural features for inhibition include two hydrophobic moieties preferably orientating in a trans manner from an electrophilic center such as an activated olefin or epoxide, with the epoxide giving maximal activity. Synthesis of chalcone oxide derivatives bearing a single p-substituent on either phenyl ring has led to very potent inhibitors of the enzyme, the best being 4-phenylchalcone oxide (50% inhibition at 6.4 × 10^?8m). Multiple factorial analysis on the inhibition data for the two series of chalcone oxides prepared (phenyl or benzoyl substituted) revealed both the essentialness of hydrophobic interactions and the apparent nonequivalence of the two hydrophobic sites involved in the inhibitory process. Steric factors were considerably less crucial while electronic effects were unimportant in the compounds examined. The chalcone oxides were either inactive or only weak inhibitors of the other major epoxide-metabolizing enzymes in mouse liver, cytosolic glutathione S-transferase, and microsomal epoxide hydrolase. The nature of the inhibition of cytosolic epoxide hydrolase by chalcone oxides was further investigated through steady-state kinetic analysis and the use of amino acid modifiers. Chalcone oxides give a slowly reversible mixed-noncompetitive inhibition. They may interact covalently with a cysteine residue possibly essential to the catalytic action of cytosolic epoxide hydrolase, and may indeed be alternative substrates with very low turnover. The cytosolic and microsomal epoxide hydrolases can be clearly distinguished by these inhibitors, further indicating different catalytic mechanisms.     

Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.     

Enzymatic preparation of D-β-acetylthioisobutyric acid and cetraxate hydrochloride using a stereo- and/or regioselective hydrolase, 3,4-dihydrocoumarin hydrolase from Acinetobacter calcoaceticus

3,4-Dihydrocoumarin hydrolase (DCH) from Acinetobacter calcoaceticus F46, which was previously found on screening for aromatic lactone-hydrolyzing enzymes, catalyzes the hydrolysis of several linear esters. The substrate specificity of the enzyme toward linear esters was quite characteristic, i.e., (1) it was specific toward methyl esters, (2) it recognized the configuration at the 2-position, and (3) it hydrolyzed diesters to monoesters. DCH hydrolyzed the methyl esters of beta-acetylthioisobutyrate and cetraxate. The products of these reactions were identified as D-beta-acetylthioisobutyrate and cetraxate, respectively, i.e., the hydrolysis reactions catalyzed by DCH were stereo- and/or regioselective. With recombinant Escherichia coli cells expressing the DCH gene as a catalyst, stereospecific hydrolysis of methyl beta-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate proceeded efficiently.     

Acetyl-coenzyme A hydrolase,an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase

1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.     

L-Dopa ester synthesis catalyzed by α-chymotrypsin in organic solvents

Summary Esters of N-unprotected amino acids have been shown to be suitable substrates for -chymotrypsin-catalyzed reactions in organic solvents. Based on this observation, a wide range of L-Dopa esters were synthesized via enzymic transesterification, and up to 50% yields were obtained using various alcohols as acyl acceptors.     

Crystal structures of glycoside hydrolase family 51 α-L-arabinofuranosidase from Thermotoga maritima

α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-β-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8-2.3 ? resolution to determine the architecture of the substrate binding pocket. Subsite -1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.     

New ester derivatives of dehydroepiandrosterone as 5α-reductase inhibitors

The aim of this study was to synthesize different ester derivatives of dehydroepiandrosterone with therapeutic potential as antiandrogens.The biological effect of these steroids was demonstrated in in vivo as well as in vitro experiments. In the in vivo experiments, we measured the activity of seven steroids on the weight of the prostate and seminal vesicles of gonadectomized hamsters treated with testosterone. For the in vitro studies, we determined the IC₅₀ values by measuring the concentration of the steroidal derivatives that inhibits 50% of the activity of 5α-reductase present in human prostate and also its binding capacity to the androgen receptors (AR) obtained from rat’s prostate cytosol. The results from these experiments indicated that compounds 7 5α,6β-dibromo-3β-propanoyloxyandrostan-17-one, 8 5α,6β-dibromo-3β-butanoyloxyandrostan-17-one and 9 5α,6β-dibromo-3β-(3′-oxapentanoyloxy)-androstan-17-one, significantly decreased the weight of the prostate and seminal vesicles as compared to testosterone treated animals; this reduction of the weight of these glands was comparable to that produced by Finasteride 11. On the other hand, compounds 4 3β-acetoxyandrost-5-en-17-one, 5 3β-hexanoyloxyandrost-5-en-17-one 6 3β-(3′-oxapentanoyloxy)-androst-5-en-17-one, 7 and 12 dehydroepiandrosterone, (commercially available) inhibited the enzyme 5α-reductase. Compounds 4, 5, 6, 8 and 9 (IC₅₀ values of 5.2 ± 1.2, 0.049 ± 0.002, 6.4 ± 1.1, 0.10 ± 0.045, and 6.8 ± 0.9 nM, respectively) exhibited the highest inhibitory activity. However, none of these compounds binds to the AR.     

Characterization of a glycoside hydrolase family 42 ��-galactosidase from Deinococcus geothermalis

A putative recombinant β-galactosidase from Deinococcus geothermalis was purified as a single 79 kDa band of 42 U activity/mg using His-Trap affinity chromatography. The molecular mass of the native enzyme was a 158 kDa dimer. The catalytic residues E151 and E325 of β-galactosidase from D. geothermalis were conserved in all aligned GH family 42 β-galactosidases, indicating that this enzyme is also a GH family 42 β-galactosidase. Maximal activity of the enzyme was at pH 6.5 and 60°C. It has a unique hydrolytic activity for p-nitrophenyl(pNP)-β-D-galactopyranoside (k (cat)/K (m) = 69 s(-1) mM(-1)), pNP-β-D-fucopyranoside (13), oNP-β-D-galactopyranoside (9.5), oNP-β-D-fucopyranoside (2.6), lactose (0.97), and pNP-α-L-arabinopyranoside (0.78), whereas no activity, or less than 2% of the pNP-β-D-galactopyranoside activity, for other pNP- and oNP-glycosides.     

A novel ubiquitin carboxy1 terminal hydrolase is involved in toad oocyte maturation

P28，a 28kD protein from toad (Bufo bufo gargarizans)oocytes,was identified by using P13^suc1-agarose affinity chromatography.Sequence homology analysis of the full-length cDNA of P28(Gene Bank accession number:AF 314091)indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70%positives to encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1,and contains homological functional domains of UCH family.Anti-p28 monoclonal antibody,on injecting into the oocytes,could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner.The recombinant protein P28 showed similar SDS/PAGE behaviors to the native one,and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases(UCHs).The results in this paper reveal that a novel protein,p28 ,exists in the toad oocytes,is a UCH Ll homolog,was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation.     

Synthesis and structure–activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.