Cadmium Exchange with Zinc in the Non-Classical Zinc Finger Protein Tristetraprolin

Abstract

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger protein that regulates inflammation. TTP targets AU-rich RNA sequences of cytokine mRNAs forming a TTP/mRNA complex. This complex is then degraded, switching off the inflammatory response. Cadmium, a known carcinogen, triggers proinflammatory effects, and there is evidence that Cd increases TTP expression in cells, suggesting that Zn-TTP may be a target for cadmium toxicity. We sought to determine whether Cd exchanges with Zn in the TTP active site and measure the effect of RNA binding on this exchange. A construct of TTP that contains the two CCCH domains (TTP-2D) was employed to investigate these interactions. A spin-filter ICP-MS experiment to quantify the metal that is bound to the ZF after metal exchange was performed, and it was determined that Cd exchanges with Zn in Zn₂-TTP-2D and that Zn exchanges with Cd in Cd₂-TTP-2D. A native ESI-MS experiment to identify the metal-ZF complexes formed after metal exchange was performed, and M-TTP-2D complexes with singular and double metal exchange were observed. Metal exchange was measured in both the absence and presence of TTP's partner RNA, with retention of RNA binding. These data show that Cd can exchange with Zn in TTP without affecting function.

Figure 1.

Cartoon of the effect of zinc binding to a ZF protein on structure. In the absence of zinc (left) the protein is unstructured, in the presence of zinc (right) the protein adopts secondary structure. ZF structure shown here is of Tis11d, a CCCH type ZF. (PDB = 1RGO, PyMOL).

Figure 2.

Schematic of the competitive metal binding TTP-2D spin filter method.

Figure 3.

Change in the native nanoelectrospray mass spectrum of Zn₂-TTP-2D (A) following the addition of 1 and 2 molar equivalents of Cd(OAc)₂·2H₂O to Zn₂-TTP-2D (B to C) measured on a Waters Synapt-G2S mass spectrometer.

Figure 4.

Comparison of the observed isotopic distribution pattern (red) of 6+ charged Zn₂-TTP-2D (yellow inset), 6+ charged Cd₁Zn₁TTP-2D (green inset), and 6+ charged Cd₂-TTP-2D (purple inset) ions with the simulated theoretical isotopic distribution (black) obtained with enviPat Web 2.4.

Figure 5.

Change in the native nanoelectrospray mass spectrum of Cd₂-TTP-2D (A) following the addition of 1 and 2 mol equiv of Zn(OAc)₂·2H₂O to Cd₂-TTP-2D (B to C) measured on a Waters Synapt-G2S mass spectrometer.

Figure 6.

Plot of the change in anisotropy upon the addition of Zn₂-TTP-2D (red) to the RNA oligonucleotide UUUAUUUAUUU-F (F = fluorescein) followed by (B) the addition of CdCl₂. FA experiments were performed in 200 mM HEPES, 100 mM NaCl, at pH 7.5 via ISS multifrequency phase fluorometer (n = 3).

Figure 7.

Change in the nanoelectrospray mass spectrum of Zn₂-TTP-2D/RNA complex (A) following the addition of 1 and 2 mol equiv of Cd(OAc)₂·2H₂Oto Zn₂-TTP-2D/RNA complex (B to C) measured on a Waters Synapt-G2S mass spectrometer.