Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate.

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.     

昆虫神经毒性酯酶活性的测定

首次报道了针对昆虫建立的神经毒性酯酶(NTE)活性检测方法以及据此法所测得的棉铃虫幼虫的NTE活性。将测定脊椎动物NTE活性的方法改进并微量化以适用于无脊椎动物昆虫体内NTE活性测定。对于棉铃虫幼虫,该法测得其头部、中肠和脂肪体等3个部位的NTE活性分别为5.30,1.40和14.50nmolminmgprotein。     

神经病靶标酯酶调控结构域结合蛋白筛选及其在COS-7细胞中的表达

本研究采用酵母双杂交系统探寻与神经病靶标酯酶（NTE）调控结构域相互作用的蛋白因子,揭示与NTE信号转导相关的可能机制。通过构建含有NTE调控结构域的诱饵蛋白载体筛选胎脑文库,并将筛选得到的阳性克隆在酵母中进行了验证,随后在哺乳动物细胞中表达了该蛋白。生物信息学分析显示：该阳性克隆为前列腺素受体结合蛋白54（ARA54）,具有泛素连接酶活性,提示细胞可能存在依赖于细胞周期的NTE活性调节机制,为阐明NTE生理功能创造了条件[动物学报51（5）：840—844,2005]。     

G protein beta2 subunit interacts directly with neuropathy target esterase and regulates its activity

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.     

Identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase

Neuropathy target esterase (NTE) is a member of the family of patatin domain-containing proteins and exhibits phospholipase activity in brain and cultured cells. NTE was originally identified as target enzyme for organophosphorus compounds that cause a delayed paralyzing syndrome with degeneration of nerve axons. Here we show that the structurally related murine protein NTE-related esterase (NRE) is a potent lysophospholipase. The enzyme efficiently hydrolyzes sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid. No lipase activity was observed when triacylglycerols, cholesteryl esters, retinyl esters, phosphatidylcholine, or monoacylglycerol were used as substrates. Although NTE is predominantly expressed in the nervous system, we found the highest NRE mRNA levels in testes, skeletal muscle, cardiac muscle, and adipose tissue. Induction of NRE mRNA concentrations in these tissues during fasting suggested a nutritional regulation of enzyme expression and, in accordance with this observation, insulin reduced NRE mRNA levels in a dose-dependent manner in 3T3-L1 adipocytes. A green fluorescent protein-NRE fusion protein colocalized to the endoplasmic reticulum and lipid droplets. Thus, NRE is a previously unrecognized ER- and lipid droplet-associated lysophospholipase. Regulation of enzyme expression by the nutritional status and insulin suggests a role of NRE in the catabolism of lipid precursors and/or mediators that affect energy metabolism in mammals.     

The synthesis of tritiated ribosylzeatin with high specific activity

Ribosylzeatin tritiated on the purine ring with a specific activity of 829 × 10¹⁰ Bq (22 mCi) per millimole was easily prepared by allylic oxidation of N⁶-(Δ²-isopentenyl)[2,8-³H]adenosine which had been synthesized by alkylation of commercially available [2,8-³H]adenosine. This allylic oxidation gave mainly the trans-isomer which was obtained free of the cis-isomer and with a radiochemical purity of 99.8% by a one-step purification using reversed-phase HPLC. This simple procedure yields ribosylzeatin, the most common naturally occurring cytokinin, labelled with tritium at near maximum specific activity.     

Human neuropathy target esterase catalyzes hydrolysis of membrane lipids

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A(2) (iPLA(2)) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA(2) inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V(max) approximately 0.01 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of (14)C-labeled products from two differentially labeled (14)C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V(max) approximately 20 micromol/min/mg and K(m) approximately 0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V(max) approximately 1 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.     

Degradation of neuropathy target esterase by the macroautophagic lysosomal pathway

AimsNeuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in humans and some sensitive animals. NTE was recently identified as a novel phospholipase B that is anchored to the cytoplasmic side of the endoplasmic reticulum. However, little is known about the degradation of NTE. In this study, we have investigated the role of the macroautophagic-lysosomal pathway in NTE degradation in neuronal and non-neuronal cells.Main methodsMacroautophagy inhibitors and activators were used to interrupt the lysosomal pathway, and NTE protein level was followed using western blotting analysis. A fluorescent microscopy assay was used to determine the co-localization of NTE and lysosomes.Key findingsWestern blotting analysis showed that the macroautophagy inhibitors 3-methyladenine and ammonium chloride increased the levels of a heterologously expressed NTE-GFP fusion protein as well as endogenous NTE. Starvation had the opposite effect. The role of macroautophagy in NTE degradation was further supported by the co-localization of exogenous NTE with lysosomes in starved COS7 cells. Furthermore, the contribution of NTE activity and protein domains to the degradation of NTE by macroautophagy was investigated, showing that both the transmembrane and regulatory domains played a role in the degradation of NTE and that the catalytic domain, and thus NTE activity, was not involved.SignificanceOur findings clearly demonstrate, for the first time, that the macroautophagy/lysosome pathway plays a role in controlling NTE quantity, providing a further understanding of the function of NTE.     

Protein domains,catalytic activity,and subcellular distribution of neuropathy target esterase in Mammalian cells

Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, has a predicted amino-terminal transmembrane helix (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Here we describe the expression, in COS cells, of green fluorescent protein-tagged constructs of NTE and mutant proteins lacking the TM or the R- or C-domains. Esterase assays and Western blots of particulate and soluble fractions indicated that neither the TM nor R-domain is essential for NTE catalytic activity but that this activity requires membrane association to which the TM, R-, and C-domains all contribute. Experiments involving proteinase treatment revealed that most of the NTE molecule is exposed on the cytoplasmic face of membranes. In cells expressing a moderate level of NTE and all cells expressing DeltaC-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels of NTE showed aberrant distribution of ER marker proteins and accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in cells expressing DeltaR-NTE or enzymatically inactive S966A-NTE but not DeltaTM-NTE. The data suggest that NTE is anchored in the ER via its TM, that its R- and C-domains also interact with the cytoplasmic face of the ER, and that overexpression of NTE causes ER aggregation via intermolecular association of its C-domains.     

Effect of over-expression of neuropathy target esterase on mammalian cell proliferation

Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, is expressed primarily in neural cells but is also detected in non-neural cells. Although NTE has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation, the function of NTE has not been defined in neural cells. In this study we have investigated the possible role of NTE in neuroblastoma cells and non-neural cells using an over-expression strategy. Over-expression of NTE in human neuroblastoma SH-SY5Y cells and monkey kidney COS7 cells led to an accumulation of NTE on the cytoplasmic surface of the endoplasmic reticulum and inhibition of cell proliferation. In particular, high levels of NTE arrested COS7 cells at G2/M stage yet was not associated with arrest at a particular phase of the cell cycle in SH-SY5Y cells. Moreover, over-expression of NTE did not induce apoptosis in two kinds of cell lines as assessed by flow cytometry. These results suggest that the role of NTE over-expression in cell proliferation is associated with different mechanisms in different cells.     

The role of cell cycle-dependent neuropathy target esterase in cell proliferation

Neuropathy target esterase (NTE) is a novel phospholipase B and plays a role in phospholipid homeostasis. Although over-expression of NTE inhibits cell division, the role of NTE in cell proliferation is still unknown. In the current study, we firstly used synchronous HeLa cells to study the expression profile of NTE during the cell cycle. NTE protein and activity are regulated during the cell cycle with highest level at G₁ and lowest at G₂/M phase. However, NTE mRNA levels are constant during the cell cycle. The role of NTE in cell proliferation was investigated by short hairpin RNA (shRNA) to suppress the expression of NTE. Knockdown of NTE significant down-regulated of NTE expression and reduced the glycerophosphocholine level. However, suppression of NTE did not affect phosphatidylcholine content or cell cycle progression. In addition, NTE was demonstrated to be degraded by the ubiquitin-proteasome pathway. These results suggested for the first time that NTE is a cell cycle-dependent protein, but is not essential for cell proliferation, and the ubiquitin-mediated proteolysis may be involved in the regulation of NTE during the cell cycle.     

Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate

1. It was proposed [Johnson (1974) J. Neurochem.23, 785-789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with (3)H and (32)P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37 degrees C occurred with a half-life of about 2-4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred (3)H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.     

Synthesis of tritiated abscisic acid of high specific activity

Stable abscisic acid (RS)-[³H] was synthesized at a specific activity of 21 Ci/mmol using a basic alumina catalyzed proton exchange of 1-hydroxy-4-keto-α-ionone with T₂O followed by a Wittig reaction. Abscisic acid -[³H] of specific activity 102 mCi/mmol was synthesized after carrying out a base catalyzed tritium exchange in solution.     

Phosphatidylcholine synthesis and its catabolism by yeast neuropathy target esterase 1

Phosphatidylcholine (PtdCho) is the major phospholipid component of eukaryotic membranes and deciphering the molecular mechanisms regulating PtdCho homeostasis is necessary to fully understand many pathophysiological situations where PtdCho metabolism is altered. This concept is illustrated in this review by summarizing recent evidence on Nte1p, a yeast endoplasmic reticulum resident phospholipase B that deacylates PtdCho producing intracellular glycerophosphocholine. The mammalian and Drosophila homologues, neuropathy target esterase and swiss cheese, respectively, have been implicated in normal brain development with increased intracytoplasmic vesicularization and multilayered membrane stacks as cytological signatures of their absence. Consistent with a role in lipid and membrane homeostasis, Nte1p-mediated PtdCho deacylation is strongly affected by Sec14p, a component of the yeast secretory machinery characterized by its ability to interface between lipid metabolism and vesicular trafficking. The preference of Nte1p toward PtdCho produced through the CDP-choline pathway and the downstream production of choline by the Gde1p glycerophosphodiesterase for resynthesis of PtdCho by the CDP-choline pathway are also highlighted.     

Recovery of neuropathy target esterase activity after inhibition with mipafox and O-hexyl O-2,5-dichlorophenyl phosphoramidate in bovine chromaffin cell cultures

Neuropathy target esterase (NTE) is a membrane protein present in various tissues whose physiological function has been recently suggested to be the maintenance of phosphatidylcholine homeostasis. Inhibition and further modification of NTE by certain organophosphorus compounds (OPs) were related to the induction of the "organophosphorus induced delayed neuropathy". Bovine chromaffin cells were cultured at 75,000cells/well in 96-well plates and exposed to 25microM mipafox or 3microM O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) for 60min. Inhibitors were removed by washing cells three times with Krebs solution. Then NTE activity was assayed at 0, 24, 48 and 120h after exposure using the Biomek 1000 workstation. Immediately after mipafox treatment NTE activity represented 3% of the control (6.7+/-1.9mU/10(6) cells). At 24, 48 and 120h after removing inhibitor, recorded activities were 33%, 42% and 111% of their respective controls (5.7+/-3.1; 5.7+/-1.9; 5.4+/-0.0mU/10(6) cells, respectively). Treatment with HDCP also displayed a time-dependent pattern of NTE recovery. As NTE inhibited by phosphoramidates is not reactivated in homogenized tissues, these results confirm a time-dependent regeneration of NTE after inhibition by neuropathic OPs.