Tristetraprolin (TTP) is a nonclassical zinc finger (ZF) protein that plays a key role in regulating inflammatory response. TTP regulates cytokines at the mRNA level by binding to AU-rich sequences present at the 3′-untranslated region, forming a complex that is then degraded. TTP contains two conserved CCCH domains with the sequence Cys-X8CysX5CysX3His that are activated to bind RNA when zinc is coordinated. During inflammation, copper levels are elevated, which is associated with increased inflammatory response. A potential target for Cu(I) during inflammation is TTP. To determine whether Cu(I) binds to TTP and how Cu(I) can affect TTP/RNA binding, two TTP constructs were prepared. One construct contained just the first CCCH domain (TTP-1D) and serves as a peptide model for a CCCH domain; the second construct contains both CCCH domains (TTP-2D) and is functional (binds RNA) when Zn(II) is coordinated. Cu(I) binding to TTP-1D was assessed via electronic absorption spectroscopy titrations, and Cu(I) binding to TTP-2D was assessed via both absorption spectroscopy and a spin filter/inductively coupled plasma mass spectrometry (ICP-MS) assay. Cu(I) binds to TTP-1D with a 1:1 stoichiometry and to TTP-2D with a 3:1 stoichiometry. The CD spectrum of Cu(I)-TTP-2D did not exhibit any secondary structure, matching that of apo-TTP-2D, while Zn(II)-TTP-2D exhibited a secondary structure. Measurement of RNA binding via fluorescence anisotropy revealed that Cu(I)-TTP-2D does not bind to the TTP-2D RNA target sequence UUUAUUUAUUU with any measurable affinity, while Zn(II)-TTP-2D binds to this site with nanomolar affinity. Similarly, addition of Cu(I) to the Zn(II)-TTP-2D/RNA complex resulted in inhibition of RNA binding. Together, these data indicate that, while Cu(I) binds to TTP-2D, it does not result in a folded or functional protein and that Cu(I) inhibits Zn(II)-TTP-2D/RNA binding.

H2S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P‐SSH). One family of proteins modified by H2S are zinc finger (ZF) proteins, which contain multiple zinc‐coordinating cysteine residues. Herein, we report the reactivity of H2S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2S was observed by low‐temperature ESI‐MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2S to form superoxide, as detected by ESI‐MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

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The zinc finger protein tristetraprolin (TTP) regulates inflammation by downregulatingc ytokinem RNAs. Misregulation resultsi na rthritis, sepsis and cancer,a nd there is an interesti nm odulating TTP activity with exogenous agents. Gold hasa nti-inflammatory properties and has recently been shown to modulate the signaling pathway that produces TTP,s uggesting that TTP may be at arget of gold. The reactivity of [Au III (terpy)Cl]Cl 2 with TTPw as investigated by UV/Vis spectroscopy,s pin-filter inductively coupled plasma mass spectrometry,X -ray absorption spectroscopy and native electrospray ionization mass spectrometry.A u III was found to replace zinc in the protein active site in the reduced Au I form, with the Au I ion coordinated to two cysteine residues in al inear geometry.T he replacement of Zn II with Au I resultsi nl oss of both secondary structure and RNA binding function. In contrast, when Zn II TTP is boundt oi ts RNA target, no replacement of Zn II with Au I is observed, even in the presence of excessA u III terpy.T his discovery of differential reactivity of gold with TTP versus TTP/RNA offers ap otentials trategy for selectivet argeting with gold complexest oc ontroli nflammation.Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.org/10.1002/chem.201904837.Scheme1.Cartoon diagram of role of TTP in TNF-a signaling. TTP (in yellow) regulates TNF-a by binding to and degrading its mRNA as part of the NF-kBs ignaling pathway.

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