Protein targets of reactive electrophiles in human liver microsomes

被引:75
作者
Shin, Nah-Young [1 ]
Liu, Qinfeng [1 ]
Stamer, Sheryl L. [1 ]
Liebler, Daniel C. [1 ]
机构
[1] Vanderbilt Univ, Sch Med, Dept Biochem, Nashville, TN 37212 USA
关键词
D O I
10.1021/tx700031r
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.
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收藏
页码:859 / 867
页数:9
相关论文
共 49 条
[31]   Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells [J].
Liu, H ;
Bowes, RC ;
vandeWater, B ;
Sillence, C ;
Nagelkerke, JF ;
Stevens, JL .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (35) :21751-21759
[32]   Analysis of protein covalent modification by xenobiotics using a covert oxidatively activated tag: Raloxifene proof-of-principle study [J].
Liu, J ;
Li, Q ;
Yang, XF ;
van Breemen, RB ;
Bolton, JL ;
Thatcher, GRJ .
CHEMICAL RESEARCH IN TOXICOLOGY, 2005, 18 (09) :1485-1496
[33]   MECHANISMS OF THE FORMATION AND DISPOSITION OF REACTIVE METABOLITES THAT CAN CAUSE ACUTE LIVER-INJURY [J].
NELSON, SD .
DRUG METABOLISM REVIEWS, 1995, 27 (1-2) :147-177
[34]  
NELSON SD, 1990, ANNU REV PHARMACOL, V30, P169
[35]   Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry [J].
Qiu, YC ;
Benet, LZ ;
Burlingame, AL .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (28) :17940-17953
[36]  
SATOH H, 1985, MOL PHARMACOL, V28, P468
[37]   ER stress and the unfolded protein response [J].
Schröder, M ;
Kaufman, RJ .
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, 2005, 569 (1-2) :29-63
[38]   Identification of protein targets for mycophenolic acid acyl glucuronide in rat liver and colon tissue [J].
Shipkova, M ;
Beck, H ;
Voland, A ;
Armstrong, VW ;
Gröne, HJ ;
Oellerich, M ;
Wieland, E .
PROTEOMICS, 2004, 4 (09) :2728-2738
[39]  
Simpson RJ, 2000, ELECTROPHORESIS, V21, P1707, DOI 10.1002/(SICI)1522-2683(20000501)21:9<1707::AID-ELPS1707>3.3.CO
[40]  
2-H