Study on a new screening method for LXRα active effect substances
TANG Zhuo-heng1,2, GAO Jun-min1, JIA Ying-ting2, HU Wen-xin2, HU Jian-ying2
1. Key Laboratory of the Three Gorges Reservoir Region's Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China; 2. Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Peking 100871, China
Abstract:To find potential pollutants that had activity effects of LXRs in complex environmental mixtures. In this study, taking the LXRα protein as an example, the pCold-TF-LXRα recombinant protein was constructed using the principle of affinity binding of nuclear receptor protein and small molecules, and the expression conditions of the recombinant protein were optimized, and a method for specifically capturing active substances of LXRα was established. The results showed the optimal expression conditions of recombinant protein when the induction temperature was 20℃ and the concentration of the inducer IPTG was 0.4mmol/L in the protein production system of LXRα. The linear regression curve measured by the spiked recovery experiment of 4gradient concentrations of LXRα agonist T0901317(0.25、2.5、25、250 μg/L) was y=0.83604x + 0.40763, R2=0.9948, proving the effectiveness of the method. Comparing the recovery rate of empty carrier protein which was the pCold-TF (6.42%) and recombinant protein (79.83%) to T0901317 (250μg/L), the method is specific. For proofing the practicality of the method, recombinant protein and a mixed standard sample of 15typical OPEs was subjected to a "capture" experiment. Among them, TPHP, BPADP, TCrP, EHDPP, TNBP, RDP, TEHP, TDCIPP were identified to have the active effect of LXRα. Lastly, the yeast two-hybrid experiment was used to verify that all of active OPEs are LXRα antagonists.
汤卓衡, 高俊敏, 贾映亭, 胡文欣, 胡建英. 具有LXRα活性效应物质的新型筛查方法研究[J]. 中国环境科学, 2020, 40(7): 3156-3164.
TANG Zhuo-heng, GAO Jun-min, JIA Ying-ting, HU Wen-xin, HU Jian-ying. Study on a new screening method for LXRα active effect substances. CHINA ENVIRONMENTAL SCIENCECE, 2020, 40(7): 3156-3164.
Daniel J P, Turley S D, Ma W Z, et al. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXRα[J]. Cell, 1998,93(5):693-704.
[2]
Wang J H, Keisala T, Solakivi T, et al. Serum cholesterol and expression of ApoAI, LXRβ and SREBP2 in vitamin D receptor knock-out mice[J]. Journal of Steroid Biochemistry and Molecular Biology, 2009,113(3):222-226.
[3]
Repa J J, Liang G, Ou J, et al. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[J]. Genes & Development, 2000,14(22):2819-2830.
[4]
Repa J J, Berge K E, Chris P, et al. Regulation of ATP-binding cassette sterol transporters ABCG5and ABCG8by the liver X receptors alpha and beta[J]. Journal of Biological Chemistry, 2002, 277(21):18793-18800.
[5]
Tontonoz P, Mangelsdorf D J. Liver X receptor signaling pathways in cardiovascular disease[J]. Molecular Endocrinology, 2003,17(6):985-993.
[6]
Guo S Y, Li L X, Yin H Y. Cholesterol homeostasis and liver X receptor (LXR) in atherosclerosis[J]. Cardiovascular & Hematological Disorders-Drug Targets, 2018,18(1):27-33.
[7]
TimothyT L, Makishima M, Repa J J, et al. Molecular Basis for Feedback Regulation of Bile Acid Synthesis by Nuclear Receptors[J]. Molecular Cell, 2000,6(3):507-515.
[8]
Im S S, Osborne T F. Liver X receptors in atherosclerosis and inflammation[J]. Circulation Research, 2011,108(8):996-1001.
[9]
Snehal U N, Wang X, Silva J S D, et al. Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo[J]. Circulation, 2006,113(1):90-97.
[10]
Bischoff E D, Daige C L, Petrowski M, et al. Non-redundant roles for LXRalpha and LXRbeta in atherosclerosis susceptibility in low density lipoprotein receptor knockout mice[J]. Journal of Lipid Research, 2010,51(5):900-906.
[11]
Dai X Y, Ou X, Hao X R, et al. The effect of T0901317on ATP-binding cassette transporter A1and Niemann-Pick type C1in apoE-/-mice[J]. J. Cardiovasc. Pharmacol., 2008,51(5):467-475.
[12]
Levin N, Bischoff E D, Daige C L, et al. Macrophage liver X receptor is required for antiatherogenic activity of LXR agonists[J]. Arterioscler Thromb Vasc Biol, 2005,25(1):135-142.
[13]
Peng D C Hiipakka R A, Reardon C A, et al. Differential anti-atherosclerotic effects in the innominate artery and aortic sinus by the liver X receptor agonist T0901317[J]. Atherosclerosis, 2009,203(1):59-66.
[14]
Naoki T, Hiroshima A, Koieyama T, et al. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice[J]. Febs Letters, 2003,536(1):6-11.
[15]
Peng D C, Hiipakka R A, Dai Q, et al. Antiatherosclerotic effects of a novel synthetic tissue-selective steroidal liver X receptor agonist in low-density lipoprotein receptor-deficient mice[J]. Journal of Pharmacology & Experimental Therapeutics, 2008,327(2):332-342.
[16]
Joseph S B, Mckilligin E, Pei L, et al. Synthetic LXR ligand inhibits the development of atherosclerosis in mice[J]. Proceedings of the National Academy of ences, 2002,99(11):7604-7609.
[17]
Hu W X, Jia Y T, Kang Q Y, et al. Screening of house dust from Chinese homes for chemicals with liver X receptors binding activities and characterization of atherosclerotic activity using an in vitro macrophage cell line and ApoE -/- Mice[J]. Environmental Health Perspectives, 2019,127(11):117003.
[18]
Burgess R M, Ho K T, Brack W, et al. Effects-directed analysis (EDA) and toxicity identification evaluation (TIE):Complementary but different approaches for diagnosing causes of environmental toxicity[J]. Environmental Toxicology & Chemistry, 2013,32(9):1935-1945.
[19]
Marklund A, Barbro A, Peter H. Screening of organophosphorus compounds and their distribution in various indoor environments[J]. Chemosphere, 2003,53(9):1137-1146.
[20]
王晓伟,刘景富,阴永光.有机磷酸酯阻燃剂污染现状与研究进展[J]. 化学进展, 2010,22(10):1983-1992. Wang X W, Liu J F, Yin Y G. The pollution status and research progress on organophosphate ester flame retardants[J]. Progress in Chemistry, 2010,22(10):1983-1992.
[21]
Niu Z G, Zhang Z Z, Li J F, et al. Threats of organophosphate esters (OPEs) in surface water to ecological system in Haihe River of China based on species sensitivity distribution model and assessment factor model[J]. Environmental Science and Pollution Research, 2019, 26(11):10854-10866.
[22]
Ding J J, Shen X L, Liu W P, et al. Occurrence and risk assessment of organophosphate esters in drinking water from Eastern China[J]. Science of The Total Environment, 2015,538959-965.
[23]
Cao D D, Guo J H, Wang Y W, et al. Organophosphate esters in sediment of the Great Lakes[J]. Environmental Science & Technology, 2017,51(3):1441-1449.
[24]
Brommer S, Harrad S, Eede N V D, et al. Concentrations of organophosphate esters and brominated flame retardants in German indoor dust samples[J]. Journal of Environmental Monitoring, 2012, 14.
[25]
刘琴,印红玲,李蝶,等.室内灰尘中有机磷酸酯的分布及其健康风险[J]. 中国环境科学, 2017,37(8):2831-2839. Liu Q, Yin H L, Li D, et al. Distribution characteristic of OPEs in indoor dust and its health risk[J]. China Environmental Science, 2017, 37(8):2831-2839.
[26]
Zhou L L, Hiltscher M, Gruber D et al. Organophosphate flame retardants (OPFRs) in indoor and outdoor air in the Rhine/Main area, Germany:comparison of concentrations and distribution profiles in different microenvironments[J]. Environmental Science and Pollution Research, 2017,24(12):10992-11005.
[27]
Tan H L, Chen D, Peng C F, et al. Novel and traditional organophosphate esters in house dust from South China:Association with hand wipes and exposure estimation[J]. Environmental Science & Technology, 2018,52(19):11017-11026.
[28]
Wingfield P T, Palmer I, Liang S M. Folding and purification of insoluble (inclusion body) proteins from Escherichia coli[J]. Curr Protoc Protein Sci, 2001:785-6.
[29]
Schein C H, Noteborn M H M. Formation of Soluble Recombinant Proteins in Escherichia Coli is Favored by Lower Growth Temperature[J]. Nature Biotechnology, 1988,6(3):291-294.
[30]
Germ N L R, Ceccarelli E A. Recombinant protein expression in Escherichia coli:advances and challenges[J]. Frontiers in Microbiology, 2014,5(172):172.
[31]
Moore J T, Uppal A, Maley F, et al. Overcoming Inclusion Body Formation in a High-Level Expression System[J]. Protein Expression and Purification, 1993,4(2):160-163.
[32]
Martina M, Forsten E, Noack S, et al. Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures[J]. Microbial Cell Factories, 2017,16(1):220.
[33]
Nakashima N, Tomohiro T. A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35degrees C[J]. Biotechnology & Bioengineering, 2010,86(2):136-148.
[34]
Donovan R S, Robinson C W, Glick B R. Review:Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J]. Journal of Industrial Microbiology, 1996,16(3):145-154.
[35]
Berrow N S, Büssow K, Coutard B, et al. Recombinant protein expression and solubility screening in Escherichia coli:a comparative study[J]. Acta Crystallographica, 2010,62(10):1218-1226.
[36]
夏铸.可溶性全长hPPARγ重组蛋白的制备及其活性研究[D]. 重庆:重庆医科大学, 2012. Xia Z. Study on preparation and activity of soluble full length human PPARγ recombinant protin[D]. Chongqing:Chongqing Medical University, 2012
[37]
Zhu J F, Yi X J, Liu W H, et al. Immobilized fusion protein affinity chromatography combined with HPLC-ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products[J]. Talanta, 2016,165508.
[38]
Morris P J, Daniel M C, Ann H, et al. Organophosphorus flame retardants inhibit specific liver carboxylesterases and cause serum hypertriglyceridemia[J]. Acs Chemical Biology, 2014,9(5):1097-1103.
[39]
Hao Z L, Zhang Z J, Lu D Z, et al. The organophosphorus flame retardants impaired intracellular lipid metabolic function in human hepatocellular cells[J]. Chemical Research in Toxicology, 2019.
[40]
Ballesteros-Gómez A, Brandsma S H, Boer J D, et al. Analysis of two alternative organophosphorus flame retardants in electronic and plastic consumer products:Resorcinol bis-(diphenylphosphate) (PBDPP) and bisphenol A bis (diphenylphosphate) (BPA-BDPP)[J]. Chemosphere, 2014,11610-14.
[41]
Katerina K, Xu F C, Padilla-Sanchez J A, et al. Legacy and alternative flame retardants in Norwegian and UK indoor environment:Implications of human exposure via dust ingestion[J]. Environment International, 2017,10248-56.
[42]
Christina C, Poma G, Besis A, et al. Legacy and emerging organophosphοrus flame retardants in car dust from Greece:Implications for human exposure[J]. Chemosphere, 2018,196231-239.