Cassidy Pamela B, Edes Kornelia, Nelson Chad C, Parsawar Krishna, Fitzpatrick F A, Moos Philip J
Huntsman Cancer Institute, The Department of Medicinal Chemistry, University of Utah, L.S. Skagg's Pharmacy, Room 201, 30 S 2000 Salt Lake City, UT 84112, USA.
Carcinogenesis. 2006 Dec;27(12):2538-49. doi: 10.1093/carcin/bgl111. Epub 2006 Jun 15.
Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF7 cells.
先前的研究表明，内源性和外源性亲电试剂对硫氧还蛋白还原酶（TrxR）的共价修饰会导致肿瘤抑制蛋白p53的构象破坏。在此我们报告，亲电修饰或缺失C端催化性硒代半胱氨酸残基导致正常细胞TrxR酶活性丧失，其功能后果与人RKO结肠癌细胞中TrxR蛋白缺失所产生的后果不同。对纯化的TrxR进行了全面的动力学分析，以表征其被亲电试剂抑制的机制。此外，电喷雾质谱法证实了脂质亲电试剂对TrxR的烷基化作用，液相色谱 - 质谱/质谱法确定C端是烷基化的一个靶点。然后将亲电试剂对TrxR的修饰对p53构象、反式激活和细胞凋亡的影响，与用针对TrxR1的小干扰RNA处理细胞以耗尽TrxR蛋白的效果进行了比较和对比。我们发现，与表达正常水平TrxR的细胞相比，耗尽TrxR的细胞实际上对亲电试剂诱导的p53构象破坏和细胞凋亡不太敏感。当用缺乏催化性硒代半胱氨酸的TrxR C端突变体转染耗尽野生型TrxR的RKO细胞时，发现p53的构象紊乱，类似于用亲电试剂处理的细胞。这些结果使我们得出结论，TrxR的C端修饰对于破坏p53和诱导细胞凋亡既是必要的也是充分的。内源性脂质亲电试剂一直是我们的主要研究重点；然而，激素的代谢激活可产生内源性诱变剂，并且我们证明雌酮醌会减弱人MCF7细胞中的p53功能。
