Protein Tyrosine Nitration in Plant Nitric Oxide Signaling

Abstract

Nitric oxide (NO), which is ubiquitously present in living organisms, regulates many developmental and stress-activated processes in plants. Regulatory effects exerted by NO lies mostly in its chemical reactivity as a free radical. Proteins are main targets of NO action as several amino acids can undergo NO-related post-translational modifications (PTMs) that include mainly S-nitrosylation of cysteine, and nitration of tyrosine and tryptophan. This review is focused on the role of protein tyrosine nitration on NO signaling, making emphasis on the production of NO and peroxynitrite, which is the main physiological nitrating agent; the main metabolic and signaling pathways targeted by protein nitration; and the past, present, and future of methodological and strategic approaches to study this PTM. Available information on identification of nitrated plant proteins, the corresponding nitration sites, and the functional effects on the modified proteins will be summarized. However, due to the low proportion of in vivo nitrated peptides and their inherent instability, the identification of nitration sites by proteomic analyses is a difficult task. Artificial nitration procedures are likely not the best strategy for nitration site identification due to the lack of specificity. An alternative to get artificial site-specific nitration comes from the application of genetic code expansion technologies based on the use of orthogonal aminoacyl-tRNA synthetase/tRNA pairs engineered for specific noncanonical amino acids. This strategy permits the programmable site-specific installation of genetically encoded 3-nitrotyrosine sites in proteins expressed in Escherichia coli, thus allowing the study of the effects of specific site nitration on protein structure and function.

Keywords: 3-nitro-tyrosine; nitration; nitric oxide; post-translational modification; sensing; signaling.

Figure 1

Nitric oxide (NO) production and sensing pathways in mammals and plants. Arginyl transferase (ATE), group VII Ethylene Response Factor (ERFVII), Cyclic guanosine monophosphate (cGMP), guanosine triphosphate (GTP), guanylate cyclase (GC), heme NO/oxygen motif (H-NOX), methionine aminopeptidase (MAP), nitrate reductase (NR), nitrite reductase (NiR), NO synthase (NOS), oxidized cysteine ERFVII (oxC-ERFVII), peroxynitrite (ONOO⁻), plant cysteine oxidase (PCO), E3 ubiquitin ligase Proteolysis6 (PRT6), arginylated cysteine ERFVII (RC-ERFVII), and unknown NO sensor (X). NO- and peroxinitrite-triggered inactivation are shown by blunt-ended red dotted lines. Still uncertain involvement of NOS in NO production is shown with green dotted arrow.

Figure 2

Functional interaction between NO and ABA signaling through NO-related post-translational modifications (PTMs). Aspartate aminotransferase (AAT), ABA-insensitive 5 (ABI5), glutamine synthetase (GS), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), nitrate reductase (NR), nitrite reductase (NiR), type 2C protein phosphatase (PP2C), Pyrabactin resistant ABA receptor (PYR), PYR-like (PYL), SAP and MIZ1 domain-containing ligase1 (SIZ1), sucrose non-fermenting1-related protein kinase 2 (SnRK2), small ubiquitin-like modifier (SUMO), nitrosothiol (SNO), and ubiquitin (UBQ). Dotted arrows and blun-ended lines represent activation and inactivation, respectively.

Figure 3

Multiple post-translational modifications potentially alter function/activity, localization, and stability of target proteins. Phosphorylation (P), methionine sulfoxide (Met-SO), Cys oxidation to sulphonic, sulphenic, or sulphinic group (SO_nH), nitrosoCys (Cys-NO), nitroTyr (Tyr-NO₂), Ubiquitin (Ubq), and polyubiquitinated lysines (Lys-Ubq-Ubq…-Ubq).

Figure 4

Antioxidant enzymes are targets of Tyr nitration. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) are all targets of Tyr nitration and all but GR inactivated by this PTM (blunt ended red dotted lines).

Figure 5

Genetic code expansion methodologies for site-specific incorporation of non-canonical amino acids. Aminoacyl-tRNA synthetase (aaRNAs), transfer RNA (tRNA), and amber stop codon (UAG). This methodology is based on the specificity of aaRNAs/tRNA pairs in such a way that non-canonical amino acid (red circle) and orthogonal tRNA are not substrates of endogenous aaRNAs and vice versa orthogonal aaRNAs do not use canonical amino acids (yellow and orange circles) and endogenous tRNAs. This method generates a site-specific modified protein by incorporation of a non-canonical amino acid, which may be 3-nitroTyr, thus becoming a potentially useful tool to study Tyr nitration or any other PTM specific effects on target proteins.