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Event: 927

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

KE1 : S-Glutathionylation, eNOS

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
S-Glutathionylation, eNOS

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
endothelial cell of vascular tree

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
protein glutathionylation nitric oxide synthase, endothelial increased
protein glutathionylation cysteine residue increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Hypertension KeyEvent Frazer Lowe (send email) Not under active development Under Development

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
Homo sapiens Homo sapiens Moderate NCBI
Bos taurus Bos taurus Moderate NCBI
Mus musculus Mus musculus Low NCBI
Rattus norvegicus Rattus norvegicus Low NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages High

Sex Applicability

No help message More help
Term Evidence
Unspecific High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

S-glutathionylation is a redox-dependent, reversible post-translational modification that is involved in the regulation of various regulatory, structural, and metabolic proteins (Pastore and Piemonte, 2013). Under oxidative stress, S-glutathionylation targets cysteine residues of a protein and adds glutathione through thiol-disulfide exchange with oxidized glutathione (GSSG) or reaction of oxidant-induced protein thiyl radicals with reduced glutathione (Chen et al., 2010, 2011, Schuppe et al. 1992). Endothelial nitric oxide synthase (eNOS) regulates vascular function by generating nitric oxide which is involved in endothelium-dependent relaxation, and control of blood pressure and vascular tone. It has been shown that cysteine residues are important for the maintenance of normal eNOS function. Under oxidative stress, S-glutathionylation of eNOS was induced by GSSG at residue sites Cys 689 and Cys 908, resulting in a decrease in eNOS activity and an increase in superoxide generation, also known as eNOS uncoupling. Furthermore, eNOS S-glutathionylation was shown to be abundant in the vessel walls of spontaneously hypertension rats (SHRs), in contrast to non-hypertensive rats.   SHRs demonstrated impaired endothelium-dependent vasodilation, which was reversible upon administration of the reducing agent, dithiothreitol (Chen et al. 2010).  Similarly in human aortic endothial cells, exposure to ultrafine particles caused a decrease in NO production in a dose-depedent manner.  This was shown to be prevented upon over-expression of glutaredoxin-1, which inhibits eNOS S-glutathionylation (Du et al. 2013).

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

There are four general approaches to detect protein S-glutathionylation (Pastore and Piemonte, 2013).

  1. Quantification of Total S-Glutathionylated Proteins: Use sample lysis or homogenization in non-reducing buffer containing N-ethylmaleimide to eliminate thiols, followed by protein precipitation, reduction of gluthionyl-protein adducts, and derivatization of protein thiols or free glutathione with fluorescence probes. Fluorescence can be measured by fluorometric analysis with or without prior HPLC separation. This method allows for quantification of glutathionylated proteins but cannot detect glutathione adducts on specific proteins.
  2. Labeling of Glutathione: Use 35S-cysteine radiolabeling or biotin labeling to detect glutathione adducts on S-thiolated proteins.
  3. Use of Anti-Glutathione Antibodies: Use commercially available anti-glutathione to detect glutathionylated proteins by Western blots, immunoprecipitation or immunocytolocalization. This method is useful for analysis of individual proteins like eNOS but not for large-scale detection of glutathionylated proteins.
  4. Top-Down Proteomic Approach: Use liquid chromatography-coupled mass spectrometry to identify S-glutathionylated proteins on whole protein extract from cells without using labeling or anti-glutathione antibody.

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

S-glutathionylation of eNOS has been demonstrated in humans, cows, mice and rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).

Evidence for Perturbation by Stressor

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

Chen CA, Lin CH, Druhan LJ, Wang TY, Chen YR, Zweier JL.  Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling.  J Biol Chem. 2011 286(33):29098-107.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem. Biophys. Res. Commun. 436, 462–466.

Pastore, A., and Piemonte, F. (2013). Protein glutathionylation in cardiovascular diseases. Int. J. Mol. Sci. 14, 20845–20876.

Schuppe I, Moldéus P, and Cotgreave IA. Protein-specific S-thiolation in human endothelial cells during oxidative stress. (1992) Biochem. Pharmacol. 44: 1757–1764.