Role of thioredoxin isoforms in plant immunity

Abstract

Post-translational protein modifications (PTM) are key mechanisms to increase proteomic complexity and functional diversity to regulate crucial cellular processes precisely. The plant immune system comprises complex signalling networks that are governed by a variety of PTMs. For example, plant immunity is activated by a burst of nitric oxide (NO), which can covalently alter cysteine thiols within target proteins to form S-nitrosothiols (SNOs) via a redox-based process known as S-nitrosylation. Another key PTM involved in plant immunity is denitrosylation, an essential mechanism involving removing NO from Cys thiol side chains of target proteins to confer protection from nitrosative stress. Some proteins are constitutively S-nitrosylated to aid signal transduction and become denitrosylated in resting cells. Denitrosylation is equally as important as S-nitrosylation for utilising post-translational modifications in cellular signalling. Work presented in this thesis reveals that, both recombinant GSNOR1 and GSNOR1-FLAG can be S-nitrosylated which appeared to inhibit its enzymatic activity. Protein-protein-interaction screening shows that GSNOR1 interacts with three Arabidopsis thioredoxin h isoforms. We also identified an interaction between human Trx1 with human GSNOR1 in Y2H. GSNOR1 presumably require to physically interact with TRX h isoforms to drive conversion of the Cys based S-nitrosothiol (S-NO) to thiol (S-H). We investigated the consequences of the interaction with biotin switch assay. Here we show that AtTRXh3, AtTRXh4 and AtTRXh5 can reverse protein-SNO modifications and their efficiency to act as GSNOR1-SNO reductase was different. Only a few enzymes with denitrosylation activity have been discovered so far, notably TRXh5 in Arabidopsis and TRX1 in humans (Kneeshaw et al., 2014; Wu et al., 2011). The GSNOR1-SNO reductase capacity of AtTRXh3 and AtTRXh4 establishes these enzymes as denitrosylases and suggests a previously unreported means by which GSNOR1 might regain its GSNO scavenging activity. AtTRXh5 is the most effective denitrosylase of GSNOR1 as it successfully denitrosylated both recombinant AtGSNOR1 and FLAG-GSNOR1. This modification helped to regain GSNOR1 activity in vitro. AtTRXh5 mediates the denitrosylation mechanism mainly in two ways. In one mechanism AtTRXh5 forms a mixed disulphide bond with the target protein and require both active site cysteines, whereas the other mechanism involves the direct transfer of NO molecule from the S-nitrosylated protein to any active site cysteines of TRX. Previous data indicated that AtTRXh5 preferably denitrosylate protein SNO in trans-denitrosylation mechanism. We investigated the possible mechanism AtTRXh5 exhibits to denitrosylate AtGSNOR1-SNO by mutating the active site cysteines individually and together. In our findings, mutation of active site cysteines compromised the denitrosylation activity of AtTRXh5 and disrupted the AtTRXh5- AtGSNOR1 interaction. The results demonstrated that AtTRXh5 might denitrosylate AtGSNOR1-SNO by forming a mixed disulphide rather that trans-denitrosylation. Moreover, our data indicates that how AtTRXh5 reverses SNO modifications differs and AtTRXh5 probably discriminates between protein-SNOs. Further work in this study revealed that during immunity, absence of TRX displayed excessive accumulation of the GSNOR1-SNO which illustrates the importance of TRX mediated denitrosylation in regulating GSNOR1 and eventually in immunity. All these findings collectively reveal that TRX isoforms may be an important regulator of GSNOR1 S-nitrosylation during immunity.