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Post-translational regulation of redox-sensitive glutamylcysteine ligase (GCL) - revisited

Yang, Yingxue

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Abstract

Glutathione (GSH) has been reported for its crucial roles in maintaining plant growth as well as responding to environmental stresses. The multiple functions of glutathione require a tight control of GSH levels. Glutamylcysteine ligase (GCL) catalyzes the first rate-limiting step of glutathione biosynthesis. However, the mechanism of redox-dependent regulation on GCL is still largely unknown in plants. Previous findings have demonstrated that formation of an intramolecular disulfide bond followed by homodimerization is unique to the redox-mediated activation of plant GCL. To address whether the disulfide bond formation is sufficient for GCL activation or the subsequent homodimerization is a necessary step, we generated recombinant mutated GCLs unable to form dimers. Enzyme activity assays showed that disrupting dimer formation did not prevent redox-activation of GCL. Substrate affinities were similar among recombinant GCL variants. The dissociation constant of GCL was estimated by FPLC analysis to be less than 10-6 M; additionally, the GCL concentration in plastids was estimated to be approximately 5 mM. Therefore, the GCL dimer is likely to occur in vivo. Taken together, this study reveals that GCL activation relies primarily on intramolecular disulfide bridge whereas dimerization has little contribution. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated. Mitogen-activated protein kinase (MAPK) cascades mediate signal transduction of diverse extracellular stimuli including pathogen attack and oxidative stress. Arabidopsis MAPK3 and MAPK6 can be deactivated by MAPK phosphatase2 (MKP2) which may be involved in oxidative stress-related responses. Therefore, the MKP2-inducible transgenic Arabidopsis lines were used to explore the redox dependency of MKP2 regulation. After induction, an attenuated MKP2 accumulation was observed under sustained oxidative stress conditions. MKP2 may act as a potential target for plants to perceive oxidative stress and enhance MAPK signaling. It is conceivable that the post-translational modification of MKP2 exerts such regulation.

Item Type: Dissertation
Supervisor: Rausch, Prof. Dr. Thomas
Date of thesis defense: 12 September 2018
Date Deposited: 05 Oct 2018 10:36
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 580 Botanical sciences
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