RESUMO
Nitrogen remains an important macronutrient in plant root growth due to its application in amino acid production, in addition to its more elusive role in cellular signalling through nitric oxide (NO). NO is widely accepted as an important signalling oxidative radical across all organisms, leading to its study in a wide range of biological pathways. Along with its more stable NO donor, S-nitrosoglutathione (GSNO), formed by NO non-enzymatically in the presence of glutathione (GSH), NO is a redox-active molecule capable of mediating target protein cysteine thiols through the post translational modification, S-nitrosation. S-nitrosoglutathione reductase (GSNOR) thereby acts as a mediator to pathways regulated by NO due to its activity in the irreversible reduction of GSNO to oxidized glutathione (GSSG) and ammonia. GSNOR is thought to be pleiotropic and often acts by mediating the cellular environment in response to stress conditions. Under optimal conditions its activity leads to growth by transcriptional upregulation of the nitrate transporter, NRT2.1, and through its interaction with phytohormones like auxin and strigolactones associated with root development. However, in response to highly nitrosative and oxidative conditions its activity is often downregulated, possibly through an S-nitrosation site on GSNOR at cys271, Though GSNOR knockout mutated plants often display a stunted growth phenotype in all structures, they also tend to exhibit a pre-induced protective effect against oxidative stressors, as well as an improved immune response associated with NO accumulation in roots.
RESUMO
Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill-Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen-deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.
RESUMO
This study describes the theoretical basis and the methods for the facile synthesis and characterization of four fluorogenic probes, N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG), N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2) and N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2). In addition, the study describes the methodology for the application of these reagents for measuring and imaging of free thiols on cell surfaces as well as their use as pseudo substrates for the thiol reductase and S-nitrosothioldenitrosylase activities of protein disulfide isomerase (PDI) and S-nitrosothiol reductase activity of S-nitrosoglutathione reductase (GSNOR) in vitro and on live cells in culture.