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.
ZF proteins are ubiquitous eukaryotic proteins that play important roles in gene regulation. ZFs contain small domains made up of a combination of four cysteine and histidine residues and are classified on the basis of the identity of these residues and their spacing. One emerging class of ZFs are the Cys3His (or CCCH) class of ZFs. These ZFs play key roles in regulating RNA. In this minireview, an overview of the CCCH class of ZFs, with a focus on tristetraprolin (TTP), is provided. TTP regulates inflammation by controlling cytokine mRNAs, and there is an interest in modulating TTP activity to control inflammation. Two methods to control TTP activity are to target with exogenous metals (a “metals in medicine” approach) or to target with endogenous signaling molecules. Work that has been done to target TTP with Fe, Cu, Cd, and Au as well as with H2S is reviewed. This includes attention to new methods that have been developed to monitor metal exchange with the spectroscopically silent ZnIIincluding native electro‐spray ionization mass spectrometry (ESI‐MS), spin‐filter inductively coupled plasma mass spectrometry (ICP‐MS), and cryo‐electro‐spray mass spectrometry (CSI‐MS); along with fluorescence anisotropy (FA) to follow RNA binding.
more » « less- Award ID(s):
- 1708732
- NSF-PAR ID:
- 10277989
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- European Journal of Inorganic Chemistry
- Volume:
- 2021
- Issue:
- 37
- ISSN:
- 1434-1948
- Page Range / eLocation ID:
- p. 3795-3805
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
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Abstract 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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Zinc finger (ZF) proteins are proteins that use zinc as a structural cofactor. The common feature among all ZFs is that they contain repeats of four cysteine and/or histidine residues within their primary amino acid sequence. With the explosion of genome sequencing in the early 2000s, a large number of proteins were annotated as ZFs based solely upon amino acid sequence. As these proteins began to be characterizedexperimentally, it was discovered that some of these proteins contain iron–sulfur sites either in place of or in addition to zinc. Here, we describe methods to isolate and characterize one such ZF protein, cleavage and polyadenylation specificity factor 30 (CPSF3O) with respect to its metal-loading and RNA-binding activity.more » « less
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Rationale The burgeoning concern of
N ‐nitrosamine (NAM) contamination found in various pharmaceutical compositions has increased the demand for rapid and reliable screening methods to better assess the breadth of the problem. These carcinogenic compounds are also found in food, water, and soil, and they have been used in poison‐related homicides.Methods A combination of complementary, ambient ionization methods, paper spray ionization (PSI) and filter cone spray ionization (FCSI)‐mass spectrometry (MS), was characterized towards trace‐level residue screening of select NAMs (e.g.,
N ‐nitrosodimethylamine,N ‐nitrosodiethylamine,N ‐nitrosodibutylamine) directly from complex and problematic matrices of interest, including prescription and over‐the‐counter tablets, drinking water, soil, and consumable goods. Spectral data for analyte confirmation and detection limit studies were collected using a Thermo LCQ Fleet ion trap mass spectrometer.Results PSI‐MS and FCSI‐MS readily produced mass spectral data marked by their simplicity (e.g., predominantly protonated molecular ions observed) and congruence with traditional electrospray ionization mass spectra in under 2 min. per sample. Both methods proved robust to the complex matrices tested, yielding ion signatures for target NAMs, as well as active pharmaceutical ingredients for analyzed tablets, flavorants inherent to food products, etc. Low part‐per‐million detection limits were observed but were shown dependent on sample composition.
Conclusions PSI‐MS and FCSI‐MS were successful in detecting trace‐level NAMS in complex liquid‐ and solid‐phase matrices with little to no prior preparation. This work suggests that these methodologies can provide a means for assessing problematic pharmaceutical adulterants/degradants for expedited quality control, as well as enhancing environmental stewardship efforts and forensic investigations.
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