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Event: 1863

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

Inhibition of Proline and Asparagine Hydroxylation Leads to Stabilization of HIF1-Alpha

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
Inhibition Hydroxylase, HIF-1Alpha Stabilization

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
eukaryotic cell

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 hydroxylation decreased

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
Inhibition of Hydroxylase leads to Breast Cancer MolecularInitiatingEvent Arthur Author (send email) Under development: Not open for comment. Do not cite

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 High NCBI
mammals mammals High 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

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed Low

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

Hydroxylation often refers to the addition of a hydroxyl group (-OH) into an organic compound. This can be done synthetically and biologically. Hydroxylation reactions can be facilitated by enzymes called hydroxylases in a biological context. Alcohols can be formed from C-H and an oxygen atom (Huang and Groves, 2017). Other reactions add OH groups to unsaturated substrates (Kolb et al., 1994). The hydroxy groups would be added across the double bond alkene by hydrogen peroxide. In biology, cytochrome P450 is the main hydroxylation agent. However, other known hydroxylating agents include flavins, alpha-ketoglutarate-dependent hydroxylases and diiron hydroxylases (Lehninger et al., 2008).

A picture containing text, clock, gauge

Description automatically generated

Figure 1: Oxygen rebound mechanism explaining iron-catalyzed hydroxylation’s (Huang and Groves, 2017).

Hydroxylation is very important in degrading organism compounds in the air, converting lipophilic compounds into hydrophilic compounds and play a critical role in drug activity (Cerniglia, 1992). Hydroxylation can also be categorized as a post-translational modification for proteins. Most proteins are hydroxylated via 2-oxoglutarate-dependent dioxygenase (Zurlo et al., 2016). Protein hydroxylation can occur on multiple amino acids including lysine, asparagine and aspartate; however, most human protein hydroxylation occurs on proline residues. This is because roughly 30% of the proteins in humans are made up of collagen, which contains hydroxyproline at every 3rd residue in the amino acid sequence (Raju, 2019). Hydroxylation of proteins often require iron, molecular oxygen and alpha-ketoglutarate to perform oxidation. Inhibition of hydroxylation of amino acid residues can have many negative effects.

As mentioned above, protein hydroxylation requires iron, molecular oxygen and alpha-ketoglutarate to function. Some heavy metals have the ability to inhibit this event. Some known divalent heavy metals such as Cobalt, have previously been seen to show these effects (Yuan et al., 2003).

Hydroxylation can also be categorized as a post-translational modification for proteins. Most proteins are hydroxylated via 2-oxoglutarate-dependent dioxygenase (Zurlo et al., 2016). Protein hydroxylation can occur on multiple amino acids including lysine, asparagine and aspartate; however, most human protein hydroxylation occurs on proline residues. This is because roughly 30% of the proteins in humans are made up of collagen, which contains hydroxyproline at every 3rd residue in the amino acid sequence (Raju, 2019). Hydroxylation of proteins often require iron, molecular oxygen and alpha-ketoglutarate to perform oxidation.

In normal conditions, the proposed mechanism displaying Fe (II)- and 2-oxoglutarate-dependent dioxygenase-catalyzed reactions hold true.

Figure 2: Proposed mechanism of Fe (II)- and 2-oxoglutarate-dependent dioxygenase-catalyzed reactions. Amino acid side chain blue, Asparagine black, 2-oxoglutarate co-substrate dark green, succinate and CO2 light green & O2 red (Ozer & Bruick, 2007).

Figure 3: Hydroxylation of other proteins following proposed mechanism of Fe (II)- and 2-oxoglutarate-dependent dioxygenase-catalyzed reactions (Ozer and Bruick, 2007).

As shown above for proper hydroxylation of the asparagine residue (black), requires Fe (II), and 2-oxoglutarate. Amino acids with similar mechanisms for hydroxylation would include proline, aspartic acid, lysine and tryptophan. Many proteins with these amino acids are capable of undergoing this iron-mediated hydroxylation. Some proteins of particular interest are Hypoxia Inducible Factor (HIF) proteins.

Studies have shown that iron-mediated hydroxylation occurs through HIF specific proline hydroxylases (Epstein et al., 2001). It has also been suggested that hydroxylation through this mechanism requires an iron-binding centre for enzymatic activity (Yuan et al., 2003). When introducing divalent heavy metals, this hydroxylation mechanism can be inhibited. Heavy metals such as Cobalt, Cadmium and Zinc, with +2 oxidation states are capable of competing with iron for its binding centre (Yuan et al., 2003).

Figure 4: Proposed mechanism for Hydroxylation of proline 564 (Yuan et al., 2003).

Similar studies have been done with known iron chelators such as Dimethyloxaloylglycine (DMOG) & Desferrioxamine (DFO). DMOG has an extremely similar structure compared to 2-oxloglutarate. The methylation of two oxygens on this structure inhibits the binding of Fe to the compound (Duscher et al., 2017). This has been possibly due to an increase steric hindrance. The coordination of Fe does not occur; therefore, the hydroxylation of HIF cannot pursue. In the case of DFO, Fe’s binding sites are eliminated. DFO is an iron chelator which binds to iron in six different locations (Duscher et al., 2017). When DFO is present most iron is bound, therefore is eliminated from the mechanism.

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). ?

To determine that divalent metals, such as cobalt lead to the inhibition of hydroxylation, matrix-assisted laser desorption/ionization (MALDI) can be done. Cobalt can bind directly to the oxygen-dependent degradation (ODD) domain, which inhibits hydroxylation of the amino acid residues (Yuan et al., 2003). MALDI data displays that cobalt binds directly to a non-hydroxylated proline residue. In mass spectrometry, MALDI is an ionization technique by which laser energy to create ions from larger molecules (Karas and Krüger, 2003). Generally, hydroxylation of proline, lysine or asparagine involves the replacement of a hydrogem atom with a hydroxyl group (Chicooree et al., 2015). The overall mass of this transition is approximately 16 Da. This change can be measured to identify whether a amino acid has undergone hydroxylation.

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

HIF is a transcriptional complex which plays a crucial role in mammalian oxygen homeostasis (Epstein et al., 2001). Studies have defined prolyl hydroxylation as a key regulatory event targets subunits for proteasomal destruction.

HIFs key mammalian transcription factors that increase protein stability and intrinsic transcriptional potency (Lando et al., 2002). This is due to the absence of proline and asparagine hydroxylation.

HIF ensures proper adaptation throughout development and into adulthood (Dann et al., 2002). Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

Exposure to divalent metals is mostly seen in occupational exposure. This includes smelters, mining activities, hazardous waste sites, and even natural sources (Kaczmarek et al., 2009). Burning fossil fuel is one of the sources of environmental exposure of general population. It results in the emission of residual oil fly ash, which contains metal ions, such as Fe(II/III), Mn(II), Ni(II), V(V), Ca(II), Mg(II), and Zn(II) (Kaczmarek et al., 2009). These metals can be absorbed through inhalation.

As mentioned above, protein hydroxylation requires iron, molecular oxygen and alpha-ketoglutarate to function. Some heavy divalent metals have the ability to inhibit this event. Some known divalent heavy metals such as Cobalt, or Nickle have previously been seen to show these effects (Yuan et al., 2003, Salnikow et al., 2004).

The hydroxylases, for proline 402/564 and asparagine 803, are iron-containing enzymes. Because divalent metal ions resemble iron, it has been suggested that these metals could replace the iron in the enzymes (Davidson et al., 2005). It has also been suggested that ligands in the hydroxylase active centre; Histidine and asparagine carboxyl groups, are weak iron binders (Schofield and Ratcliffe, 2004). Again, introducing other divalent metals could outcompete for the iron site. This would result in inhibition of hydroxylation.

Figure 5: Proposed mechanism for Hydroxylation of proline 564 (Yuan et al., 2003).

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

Cerniglia, C.E. (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3, 351–368.

Chicooree, N., Unwin, R. D. and Griffiths, J. R. (2015). The application of targeted mass spectrometry-based strategies to the detection and localization of post-translational modifications: PTM ANALYSIS USING TARGETED MASS SPECTROMETRY. Mass Spec Rev 34, 595–626.

Dann, C. E., Bruick, R. K. and Deisenhofer, J. (2002). Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway. Proc Natl Acad Sci USA 99, 15351.

Davidson, T., Chen, H., Garrick, M. D., D'Angelo, G., & Costa, M. (2005). Soluble nickel interferes with cellular iron homeostasis. Molecular and cellular biochemistry279(1-2), 157–162.

Duscher, D., Januszyk, M., Maan, Z. N., Whittam, A. J., Hu, M. S., Walmsley, G. G., Dong, Y., Khong, S. M., Longaker, M. T. and Gurtner, G. C. (2017). Comparison of the Hydroxylase Inhibitor DMOG and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing. Plast Reconstr Surg 139, 695e-706e.

Epstein, A. C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O’Rourke, J., Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. I., Dhanda, A., et al. (2001). C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation. Cell 107, 43–54.

Huang, X. and Groves, J. T. (2017). Beyond ferryl-mediated hydroxylation: 40 years of the rebound mechanism and C–H activation. J Biol Inorg Chem 22, 185–207.

Kaczmarek, M., Cachau, R. E., Topol, I. A., Kasprzak, K. S., Ghio, A., & Salnikow, K. (2009). Metal ions-stimulated iron oxidation in hydroxylases facilitates stabilization of HIF-1 alpha protein. Toxicological sciences : an official journal of the Society of Toxicology107(2), 394–403.

Karas, M. and Krüger, R. (2003). Ion Formation in MALDI: The Cluster Ionization Mechanism. Chem. Rev. 103, 427–440.

Kolb, H., Vannieuwenhze, M., and Sharpless, K. (1994). Catalytic Asymmetric Dihydroxylation. Chem. Rev. 94, (8), 2483–2547.

Lando, D., Peet, D. J., Whelan, D. A., Gorman, J. J. and Whitelaw, M. L. (2002). Asparagine Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch. Science 295, 858.

Lehninger, A. L., Nelson, D. L. and Cox, M. M. (2008). Lehninger principles of biochemistry. 5th ed. New York: W.H. Freeman.

Ozer, A. and Bruick, R. K. (2007). Non-heme dioxygenases: cellular sensors and regulators jelly rolled into one? Nat. Chem. Biol. 3, 144–153.

Salnikow, K., Donald, S. P., Bruick, R. K., Zhitkovich, A., Phang, J. M., & Kasprzak, K. S. (2004). Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress. The Journal of biological chemistry. 279(39), 40337–40344.

Schofield, C. J., & Ratcliffe, P. J. (2004). Oxygen sensing by HIF hydroxylases. Nature reviews. Molecular cell biology5(5), 343–354.

Raju, T.S. (2019). Hydroxylation of Proteins. In Co and Post-Translational Modifications of Therapeutic Antibodies and Proteins, T.S. Raju (Ed.)

Yuan, Y., Hilliard, G., Ferguson, T. and Millhorn, D. E. (2003). Cobalt Inhibits the Interaction between Hypoxia-inducible Factor-α and von Hippel-Lindau Protein by Direct Binding to Hypoxia-inducible Factor-α. Journal of Biological Chemistry 278, 15911–15916.

Zurlo, G., Guo, J., Takada, M., Wei, W. and Zhang, Q. (2016). New Insights into Protein Hydroxylation and Its Important Role in Human Diseases. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1866, 208–220.