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Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase

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Abstract

A mammalian family of lipid hydrolases, designated “patatin-like phospholipase domain containing (PNPLA)” recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

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Abbreviations

DMEM:

Dulbecco’s modified Eagle’s medium

EDTA:

Ethylenediaminetetraacetic acid

ECL:

Enhanced chemiluminescence

GFP:

Green fluorescence protein

ER:

Endoplasmic reticulum

NEST:

NTE esterase activity domain

NP40:

Nonidet P-40

NTE:

Neuropathy target esterase

NRE:

NTE-related esterase

NREC:

The catalytic domain of NRE

NREN:

The N-terminal domain of NRE

OPIDN:

OP-induced delayed neuropathy

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PK:

Proteinase K

PNPLA:

Patatin-like phospholipase domain containing

PV:

Phenyl valerate

RT–PCR:

Reverse transcription–PCR

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TM:

Transmembrane

TX:

Triton X-100

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30870298, 30600329) and the Natural Science Foundation Project of CQ CSTC (2007BB5443), and by the Science and Technology Project from Chongqing Municipal Education Committee (KJ070510).

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Authors and Affiliations

  1. Key Laboratory of Molecular Biology, College of Bio-information, Chongqing University of Posts and Telecommunications, Nan’an District, 400065, Chongqing, People’s Republic of China

    Ping-an Chang & Wen-zhen Qin

  2. Laboratory of Molecular Toxicology, Institute of Zoology, Chinese Academy of Sciences, 100080, Beijing, People’s Republic of China

    Ping-an Chang, Ding-xin Long & Yi-jun Wu

  3. Department of Neurosurgery, The First Hospital of Xiamen Affiliated to the Fujian Medical University, 361001, Xiamen, People’s Republic of China

    Zhan-xiang Wang

  4. School of Public Health, University of South China, 421001, Hengyang, People’s Republic of China

    Ding-xin Long

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  1. Ping-an Chang
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Correspondence to Ping-an Chang.

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Ping-an Chang and Zhan-xiang Wang equally contributed to this work.

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Chang, Pa., Wang, Zx., Long, Dx. et al. Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase. Mol Cell Biochem 339, 181–190 (2010). https://doi.org/10.1007/s11010-009-0382-0

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  • DOI: https://doi.org/10.1007/s11010-009-0382-0

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