Cysteine oxidation enhanced by iron in tristetraprolin, a zinc finger peptide.
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.
Lee
Seung Jae
SJ
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA.
Michel
Sarah L J
SL
D003545:Cysteine / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D010084:Oxidation-Reduction; D051816:Tristetraprolin / Q000737:chemistry*
D051816:Tristetraprolin; D007501:Iron; D003545:Cysteine