Abstract
Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein.
Highlights
The interaction of peroxynitrite (ONOO−) and its derivatives with tyrosine residues leads to the formation of 3-nitrotyrosine (3-NT) [1,2]
The influence of chemical nitration procedures on purified wildtype alpha synuclein (ASYN) and an ASYN mutant, in which all four endogenous tyrosines were substituted by serine or phenylalanine (S39F125S133F136) was tested by the application of authentic ONOO−, the ONOO−-generating compound Sin-1, or tetranitromethane (Fig. 1A)
To obtain an overview on the effect of chemical tyrosine nitration on the overall protein, full length ASYN was analyzed by linear ion trap mass spectrometry (MS)
Summary
The interaction of peroxynitrite (ONOO−) and its derivatives with tyrosine residues leads to the formation of 3-nitrotyrosine (3-NT) [1,2]. Almost all of these investigations are based on chemical nitration procedures, involving authentic peroxynitrite, peroxynitrite-generating compounds (such as Sin-1), or tetranitromethane [2,10,11]. Such chemical nitration procedures usually result in complex patterns of nitrated and non-nitrated residues. They cause oxidative modifications of other amino acids, as well as covalent di-tyrosine crosslinks [12]. The multiple parallel reactions limit the establishment of causal correlations between the nitration of a given tyrosine residue
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