Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration

Abstract

Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and that is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-Hand metalbinding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. NMR spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-Hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. In order to develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF Hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity (K d = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched on nitration. The greater terbium affinity upon tyrosine nitration resulted in high dynamic range and sensitivity to substoichiometric nitration. An improved approach was also developed to the synthesis of peptides containing nitrotyrosine, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

The Evolution of Nitric Oxide Function: From Reactivity in the Prebiotic Earth to Examples of Biological Roles and Therapeutic Applications

“…Thus, tyrosine nitration of specific proteins is reversible and may contribute to signal transduction. A small protein motif (14–18 amino acids) responsive to tyrosine nitration has been developed by Urmey et al [ 138 ].…”

Hydrogenases and the Role of Molecular Hydrogen in Plants

“…In a similar way, it might be possible to predict if such proteins are tyrosine nitrated. A nitration motif has been suggested [ 41 ] with the sequence: -DKDADGWISPA Y AK- where the target tyrosine is underlined and bold. However, even though the HYDA1 sequence has nine tyrosine residues, there is no similarity in their surrounding sequences to the nitration sequence given here.…”

“…All three sequences (Human, Arabidopsis , and Artemisia : Figure 1 ) have eight conserved tyrosine residues. It is possible that they may be used for nitro-tyrosine formation, but there is no evidence of the conserved region used by Urmey and Zondlo [ 41 ]. In a similar way, looking for the cysteine residues which may be glutathionylated [ 43 ], there is no evidence of this sequence being in the hydrogenase amino acid sequences investigated here ( Figure 1 ).…”

Lysosome-Targeting Iridium(III) Probe with Near-Infrared Emission for the Visualization of NO/O2•- Crosstalk via In Vivo Peroxynitrite Imaging

“…Nitric oxide (NO) and superoxide anions (O 2 •‑ ) are two noteworthy reactive species implicated in various physiological and pathological processes. , Recently, increased research in the area has revealed that cellular NO and O 2 •‑ have a close relationship. , Furthermore, the interaction (“crosstalk”) between them may form a new redox signaling molecule peroxynitrite (ONOO – ) without enzymatic catalysis in a diffusion-controlled reaction ( k = 6.7 × 10 9 M –1 s –1 ). , Early studies focused on the proclivity of ONOO – to cause cell damage due to its high oxidative and nitrosative activity toward biomolecules such as proteins, nucleic acids, and lipids . Recently ONOO – has also been shown to modulate cell signal transduction via nitrating tyrosine residues as well as involvement in immunogenic response against pathogen invasion. − ONOO – can mediate protein nitration to modulate enzymatic activity and protein stability, , raise immunogenicity of modified proteins, induce the interaction between proteins by mimicking phosphotyrosine, , and crosstalk with tyrosine kinases and phosphatases via alteration of their substrate structures. , Additionally, misregulated nitration is unfavorable and has been related to liver injury, Alzheimer’s disease, arthritis, cancer, inflammatory disease, and other disorders. , Nevertheless, the role of ONOO – is still controversial and research on the specific and intricate relationship between NO and O 2 •‑ is slow owing to the elusive nature of ONOO – under physiological conditions due to its extremely short lifetime (<10 ms), nanomolar homeostasis concentration, and high chemical reactivity. , In this context, it is of great interest to investigate NO/O 2 •‑ crosstalk in vivo through ONOO – -responsive molecular tools in order to fully investigate the physiological and pathological mechanisms involved.…”

“…7 Recently ONOO − has also been shown to modulate cell signal transduction via nitrating tyrosine residues as well as involvement in immunogenic response against pathogen invasion. 8−10 ONOO − can mediate protein nitration to modulate enzymatic activity and protein stability, 11,12 raise immunogenicity of modified proteins, 13 induce the interaction between proteins by mimicking phosphotyrosine, 14,15 and crosstalk with tyrosine kinases and phosphatases via alteration of their substrate structures. 16,17 Additionally, misregulated nitration is unfavorable and has been related to liver injury, Alzheimer's disease, arthritis, cancer, inflammatory disease, and other disorders.…”