Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Inorg Chem. 2010 Feb 1;49(3):1211-9. doi: 10.1021/ic9024298.
Tristetraprolin (TTP or NUP475) is a non-classical zinc finger protein that is involved in inflammatory response. TTP regulates the production of cytokines by binding to specific mRNA sequences. TTP contains two Cys(3)His metal binding domains that can coordinate zinc, cobalt, ferric and ferrous iron. When zinc, cobalt, ferric or ferrous iron are bound, TTP peptides can bind to their cognate RNA. During inflammation there are increased levels of reactive oxygen species and iron. It has been proposed that reactive oxygen species may play a role in regulating zinc finger protein function by oxidizing cysteine thiolates that bind zinc and inactivating the protein. To elucidate the effect of the reactive oxygen species H(2)O(2) on the integrity of TTP and its ability to bind to target RNA, a simple and rapid assay using cobalt as a spectroscopic probe for zinc was developed. The oxidative susceptibility of peptides consisting of the zinc binding domains of a single zinc finger domain of TTP, TTP-D1 and the tandem zinc finger domains of TTP, TTP-2D was measured. Fe(II)-TTP-D1 and Fe(II)-TTP-2D were more rapidly oxidized by H(2)O(2) than their Zn(II) bound counterparts. Electron paramagnetic resonance (EPR) spin trapping using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) demonstrated that oxidation of ferrous iron substituted TTP-D1 and TTP-2D resulted in the formation of hydroxyl radicals via Fenton chemistry. The oxidized peptides exhibited a diminished affinity for target RNA compared to their unoxidized counterparts suggesting that oxidation of TTP inactivates the protein.
Tristetraprolin（TTP 或 NUP475）是一种参与炎症反应的非经典锌指蛋白。TTP 通过与特定的 mRNA 序列结合来调节细胞因子的产生。TTP 包含两个 Cys(3)His 金属结合域，可协调锌、钴、铁和亚铁。当锌、钴、铁或亚铁结合时，TTP 肽可以与其同源 RNA 结合。在炎症过程中，活性氧和铁的水平会增加。有人提出，活性氧可能通过氧化结合锌的半胱氨酸硫醇来调节锌指蛋白的功能，并使蛋白质失活。为了阐明活性氧 H(2)O(2)对 TTP 完整性及其与靶 RNA 结合能力的影响，开发了一种使用钴作为锌光谱探针的简单快速测定法。测量了由 TTP 的单个锌指域的锌结合域 TTP-D1 和 TTP 的串联锌指域 TTP-2D 组成的肽的氧化易感性。与其 Zn(II) 结合的对应物相比，Fe(II)-TTP-D1 和 Fe(II)-TTP-2D 被 H(2)O(2)更快地氧化。使用 2-乙氧基羰基-2-甲基-3,4-二氢-2H-吡咯-1-氧化物（EMPO）的电子顺磁共振（EPR）自旋捕获实验表明，铁取代 TTP-D1 和 TTP-2D 的氧化导致通过 Fenton 化学形成羟基自由基。与未氧化的对应物相比，氧化的肽对靶 RNA 的亲和力降低，表明 TTP 的氧化使蛋白质失活。
