Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants
Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants
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DOI：
10.1073/pnas.172398899
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发表时间：
2002-09-03
影响因子：
11.1
通讯作者：
Talalay, P
中科院分区：
文献类型：
--
作者：
Dinkova-Kostova, AT;Holtzclaw, WD;Talalay, P
2期蛋白的协同诱导和谷胱甘肽水平的升高可保护细胞免受亲电试剂和氧化剂的毒性及致癌作用。所有诱导剂与巯基的共价反应速率与其效力密切相关。诱导剂破坏肌动蛋白结合蛋白Keap1和转录因子Nrf2之间的胞质复合物，从而释放Nrf2迁移至细胞核，在细胞核中它激活2期基因的抗氧化反应元件（ARE）并加速其转录。我们克隆、过表达并纯化了小鼠Keap1，并在天然凝胶上证明了Keap1与Nrf2的Nrf2结构域形成复合物，以及它们被萝卜硫素和双（2 - 羟基苄叉）丙酮等诱导剂以浓度依赖的方式破坏。Keap1的25个半胱氨酸巯基中反应性最强的巯基的动力学、化学计量和反应顺序已通过从[H - 3]甲磺酸地塞米松（一种诱导剂和巯基的不可逆修饰剂）中掺入氚以及用萝卜硫素、2,2'-二吡啶基二硫化物和4,4'-二吡啶基二硫化物（巯基的滴定剂）和两种密切相关的迈克尔反应受体[双（2 - 和4 - 羟基苄叉）丙酮]进行紫外光谱测定，这两种受体的诱导效力相差100倍，并且其紫外光谱因加入巯基而褪色。当这些试剂大量过量时，Keap1几乎所有的巯基都会反应，但依次用三个连续的单当量（每个半胱氨酸残基）的二吡啶基二硫化物进行反应，显示出与准一级动力学极好的一致性、反应速度的快速连续下降，以及每个反应的半胱氨酸化学计量形成两当量的硫代吡啶酮。这一发现表明半胱氨酸巯基反应之后会快速形成蛋白质二硫键。Keap1最具反应性的残基（C - 257、C - 273、C - 288和C - 297）通过对胰蛋白酶肽段进行质谱分析来定位地塞米松修饰的半胱氨酸而得以确定。这些残基位于Keap1的BTB和Kelch重复结构域之间的中间区域，可能是2期系统诱导剂的直接传感器。
Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Nrf2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [H-3]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2'-dipyridyl disulfide and 4,4'-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C-257, C-273, C-288, and C-297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.