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    • tetramisole Accessibility and physico chemical features of c

    2022-07-06

    Accessibility and physico-chemical features of cysteine residues define their redox-reactivity and the 3-dimensional structure of GSNOR allows to identifying such surface-exposed, redox-sensitive cysteine residues. GSNOR crystal structures are available from human (Protein Data Bank code: 1MP0), tomato (4DL9) [15] and Arabidopsis (PDB code: 3UKO, 4JJI, 4GL4). In general, the structure of GSNOR is similar to that of other members of the alcohol dehydrogenase family. The GSNOR proteins are homodimers and both active sites open on roughly the same face of the protein. The Arabidopsis GSNOR has neither intermolecular nor intramolecular redox-sensitive disulfide bridges (Fig. 2). The structure consists of both alpha helices and beta sheets. The subunits are held together by a beta sheet that runs throughout the protein consisting of residues from both subunits in equal proportion. Each subunit binds two zinc ions - one might play an important role for the protein structure (structural Zn2+) and the other one is located in the active site (catalytic Zn2+).
    Tissue-specific and subcellular location of GSNOR Tissue-specific and subcellular location of GSNOR was analyzed in Pisum sativum, Helianthus annuus, Brassica oleracea, and different Lactuca species using specific antibodies or in transgenic Arabidopsis plants producing GSNOR-GFP [20], [26], [31], [38], [39]. GSNOR-GFP was detected in roots, leaves and floral structures. Especially the apical meristem and root tip exhibited very intense fluorescence, whereas distribution in all root cell types with diffuse cytosolic and nuclear localization was observed. Moreover, petal vascular tissue signals demonstrate high presence of GSNOR-GFP. GSNOR-GFP could also be detected in tetramisole filaments, ovary, stigma, and petals of stage 14 flowers and was particularly enriched in pollen and seeds [20]. Interestingly, nuclear localization of GSNOR was also observed in yeast cells [40]. Furthermore, GSNOR was detected in vascular tissue and epidermal cells of transversal leaf sections using anti-GSNOR antibodies in combination with a fluorescent dye conjugated secondary antibody [26]. In Helianthus annuus GSNOR was detected in hypocotyl cortex cells and mainly in vascular tissue [31], [38]. A immunogold-labelling approach with anti-GSNOR antibodies revealed presence of GSNOR in different subcellular compartments of Pisum sativum leaf cells, including chloroplasts, cytosol, mitochondria, and peroxisomes [39]. The overall presence of GSNOR correlates with gene expression data in public databases (eFP Browser). The fact that subcellular location of GSNOR overlaps with subcellular loci of ROS and ·No production (e. g. peroxisomes, mitochondria, chloroplast) [41], [42] fulfills an important criteria for redox-based regulation of GSNOR. However, ROS and ·No are both detected in the cytoplasm and can easily enter the nucleus, where they can also interact with GSNOR.
    Redox-based post-translational modification of GSNOR S-Nitrosylation and oxidative modification of GSNOR has been demonstrated on recombinant proteins by mass spectrometry after treatment with nitroso-donors or H2O2, respectively [36], [43]. S-Nitrosoglutathione (GSNO) is considered as principal nitroso reservoir due to its chemical stability and GSNO levels are fine-tuned by GSNOR. Interestingly, GSNOR activity is sensitive to S-nitrosylating agents and the activity restores under reducing conditions, suggesting catalytic impairment was due to S-nitrosylation [6], [43]. The not Zn2+ chelating cysteine residues Cys10, Cys271 and Cys370 of Arabidopsis are targets for S-nitrosylation (Fig. 2), whereas modification of Cys370 seems to promote S-nitrosylation of Cys10 and Cys271 by inducing conformational changes that alters the solvent accessibility and electrostatic environment of these cysteine residues. In detail, S-nitrosylation of GSNOR slightly changes the solvent accessibility of amino acids from the substrate binding site and/or the dimer interface. Mass spectrometric analysis confirmed the presence of monomeric and dimeric S-nitrosylated GSNOR, while unmodified GSNOR exists as dimers. Consequences of S-nitrosylation of AtGSNOR are slight structural alterations resulting in reduced specific activity. Guerra et al. [43] propose that reversible inactivation of GSNOR during ·No burst allows a cell to clear reactive nitrogen following perception of a ·No signal by dynamic regulation of GSNOR through modification of conserved cysteine residues. Moreover, treatment of AtGSNOR with peroxynitrite - known as tyrosine nitrating agent – modifies this enzyme and inhibits its activity [36]. However, since western blot analysis using anti-nitrotyrosine specific antibodies did not show any nitration, the peroxynitrite-dependent loss of GSNOR activity is probably due to oxidation of cysteine residue(s) [36]. Such ·No-dependent modifications of GSNOR might be indirectly connected to ROS, since ROS are inducing ·No production in many physiological processes and stress responses.