GLOD4 (glyoxalase domain-containing protein 4, formerly C17orf25) is a 313-residue metalloprotein of the vicinal oxygen chelate (VOC) superfamily, the structural fold shared by glyoxalase I, glyoxalase II and related enzymes. Despite the fold and the gene name, no glyoxalase or other metabolic activity has been demonstrated for it and it carries no EC number. Its first assigned catalytic activity, reported in 2026, is an unusual one: GLOD4 accelerates the decomposition of peroxynitrite and uses it to nitrate tyrosine residues on a small set of protein substrates, principally alpha-synuclein and beta-synuclein, with protein phosphatase PPM1B and polyglutamine-binding protein PQBP1 nitrated in vitro. The reaction is selective for peroxynitrite as cosubstrate - nitric oxide, NO-derived oxidants and hydrogen peroxide plus nitrite do not substitute - and depends on a zinc site formed by His8 and Glu70 together with Cys254, mutation of which abolishes activity. Cells and mice lacking GLOD4 show large, gene-dose-dependent reductions in alpha-synuclein tyrosine nitration, both at baseline and after methamphetamine challenge, although knockout mice are viable and outwardly normal to 18 months. The protein is found in both cytosol and mitochondria, with more in the cytosol, and is expressed in neurons, astrocytes and microglia as well as in erythrocytes. Because tyrosine nitration of alpha-synuclein promotes its aggregation and is a feature of Parkinson's disease pathology, enzymatic rather than purely chemical nitration is of pathological interest; the enzymatic-nitration category itself rests on this one study.
Definition: Catalysis of the peroxynitrite-dependent addition of a nitro group to position 3 of the phenolic ring of a tyrosine residue in a protein substrate, producing peptidyl-3-nitro-L- tyrosine.
Justification: GO currently has no term, in any branch, for protein nitration. Querying the ontology for "nitration" returns nothing, and every candidate that exists says something else: GO:0017014 protein nitrosylation covers addition of a nitric oxide group (S-nitrosylation of cysteine), which is different chemistry on a different residue and is specifically excluded for GLOD4 because nitric oxide and NO-derived oxidants do not support the reaction; GO:0072541 peroxynitrite reductase activity and GO:0062213 peroxynitrite isomerase activity both denote conversion of peroxynitrite to a free anion, that is detoxification, and would invert the biological meaning of GLOD4's action even though GLOD4 does accelerate peroxynitrite decomposition; and GO:0018212 peptidyl-tyrosine modification is obsolete. Every other post-translational modification of comparable biological standing - phosphorylation, sulfation, hydroxylation of tyrosine - has both an MF and a BP term, and tyrosine nitration is a long-studied modification with an established role in neurodegeneration, inflammation and cancer. The term is proposed as a reviewer suggestion tied to a single 2026 study and should be created only if the activity is independently replicated; if it is, the term will be needed for a family rather than one protein, since the same work reports autonitration by several other vicinal-oxygen-chelate superfamily members.
Parent term: catalytic activity, acting on a protein
Supporting Evidence:
Definition: The nitration of peptidyl-tyrosine to form peptidyl-3-nitro-L-tyrosine.
Justification: The biological-process counterpart of the molecular function above, and missing for the same reason. GO has peptidyl-tyrosine phosphorylation (GO:0018108), sulfation (GO:0006478) and hydroxylation (GO:0018336) but nothing for nitration, so a protein that nitrates tyrosines can only be annotated to the generic GO:0036211 protein modification process. Tyrosine nitration is measured routinely as 3-nitrotyrosine, is implicated in Parkinson's disease through alpha-synuclein, and would be annotatable from many existing studies once a term exists - including studies of non-enzymatic nitration, which the term would also serve. The wording follows the pattern of the existing peptidyl-tyrosine modification terms and is deliberately silent on mechanism, so that it does not depend on the enzymatic-catalysis claim being upheld.
Parent term: protein modification process
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of UniProt's subcellular-location annotation, which is itself an ECO:0000305 inference drawn from a 2003 interaction study rather than a direct localisation experiment. Reason: Independently corroborated. GLOD4 is in the MitoCoP high-confidence human mitochondrial proteome, and subcellular fractionation in the 2026 study detects it in the mitochondrial fraction. The qualification worth recording is that the same fractionation puts more GLOD4 in the cytosol, and all of the characterised enzymology concerns a cytosolic substrate, so mitochondrion is a real but not exclusive location. Supporting Evidence: PMID:41628334 GLOD4 is present in both mitochondria and cytosol, with relatively higher levels in the cytosol |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: Detection in MitoCoP, a quantitative high-confidence human mitochondrial proteome built from multiple orthogonal proteomic criteria rather than a single enrichment. Reason: A well-controlled high-throughput source, consistent with the targeted fractionation in PMID:41628334. Same action as the IEA row for this term. |
| GO:0045296 cadherin binding | HDA PMID:25468996 E-cadherin interactome complexity and robustness resolved by... | MARK AS OVER ANNOTATED | Summary: Derived from BioID proximity biotinylation of the E-cadherin cytoplasmic tail, a method that reports residence within a labelling radius rather than binding. The source study itself returned 561 proteins from this one bait. Reason: This is the artefact class that produces most spurious cadherin binding annotations: abundant soluble cytosolic proteins are labelled by a membrane-tethered biotin ligase and enter GOA as cadherin binders. Nothing else links GLOD4 to adherens junctions - no targeted interaction study, no junctional localisation, and no mechanistic connection to its characterised peroxynitrite chemistry. The term is also uninformative about molecular function even where such proximity is real. Not removed, because the underlying detection is a genuine experimental observation and the curator applied the correct evidence code for it. Supporting Evidence: PMID:25468996 We used proximity biotinylation and quantitative proteomics to identify 561 proteins in the vicinity of the cytoplasmic tail of E-cadherin. |
| GO:0070062 extracellular exosome | HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres... | KEEP AS NON CORE | Summary: Detection of GLOD4 in exosomes purified from expressed prostatic secretions in urine. Reason: The detection is real, but abundant cytosolic proteins are routinely recovered in exosome proteomes and this compartment is not where the enzyme acts. Retained as a peripheral location rather than discarded. |
| GO:0140096 catalytic activity, acting on a protein | IDA PMID:41628334 Selective peroxynitrite-mediated protein nitration catalyzed... | NEW | Summary: Proposed NEW annotation, and deliberately an unspecific one. GLOD4 is reported to be an enzyme that transfers a nitro group onto tyrosine residues of protein substrates using peroxynitrite as cosubstrate. GO has no term for protein nitration in either the molecular-function or the biological-process branch, so this general term is used as an explicit placeholder for the activity until the terms filed under proposed_new_terms exist. Reason: The underlying evidence is strong for a single study - Michaelis-Menten kinetics on an alpha-synuclein peptide with kcat/Km of 1.6e7 per M per s, MS localisation of the modified tyrosines, stopped-flow demonstration that GLOD4 accelerates peroxynitrite decomposition, loss of activity in structure-guided active-site mutants, an 86% reduction of alpha-synuclein nitration in knockout cells and gene-dose-dependent reductions in knockout mice. It is nevertheless a single paper from a single laboratory with a declared patent application on the activity, the authors themselves present the enzyme class as a proposal ("tentatively refer to as nitrases"), and the catalytic mechanism and the role of the zinc are stated to be unresolved. This annotation is therefore offered as a reviewer proposal for a well-evidenced but as yet unreplicated activity, not as a settled assignment. It is placed at this general altitude on purpose: the specific alternatives in GO today would be wrong. GO:0017014 protein nitrosylation is S-nitrosylation by nitric oxide, and the paper explicitly excludes nitric oxide and NO-derived oxidants as GLOD4 cosubstrates; GO:0072541 peroxynitrite reductase activity and GO:0062213 peroxynitrite isomerase activity denote detoxification of peroxynitrite to nitrite or nitrate, which would assert the opposite biological consequence from nitrating a target protein. Supporting Evidence: PMID:41628334 Here, we showed that glyoxalase domain-containing protein 4 (GLOD4), a previously uncharacterized protein, is an enzyme that catalyzes selective protein nitration. PMID:41628334 confirmed that GLOD4 catalyzed the nitration of the tyrosine residues Y39 and Y125 of Ξ±-syn PMID:41628334 The estimated catalytic efficiency kcat/Km was 1.6 Γ 107 Mβ1 sβ1, indicating that GLOD4 is an efficient enzyme for nitrating Ξ±-synuclein. PMID:41628334 The data illustrate that GLOD4 selectively uses peroxynitrite as a cosubstrate, and not nitric oxide or nitric oxide-derived oxidants, or H2O2 combined with nitrite, to nitrate Ξ±-syn. PMID:41628334 In the GLOD4-KO line, tyrosine nitration of Ξ±-syn was reduced by 86% upon adding peroxynitrite under the same experimental conditions, indicating that GLOD4 mediates Ξ±-syn nitration in cells. |
| GO:0008270 zinc ion binding | IDA PMID:41628334 Selective peroxynitrite-mediated protein nitration catalyzed... | NEW | Summary: Proposed NEW annotation. Zinc was detected in purified GLOD4 by ICP-MS, a Zn-soaked crystal structure was analysed, and alanine substitution of the two putative metal ligands His8 and Glu70 reduced or eliminated catalytic activity. Reason: This is the most concrete and least model-dependent molecular statement the paper makes, and it is consistent with GLOD4 belonging to the vicinal oxygen chelate superfamily of metalloenzymes, whose defining feature is a paired-oxygen metal chelation site. The one caveat, stated by the authors, is that whether zinc is the catalytic metal in vivo is not settled - manganese, nickel and iron are raised as alternatives - so the binding claim is firmer than any mechanistic interpretation of it. Supporting Evidence: PMID:41628334 However, inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed that Zn2+ was present in purified GLOD4 PMID:41628334 Mutation of GLOD4 at the putative metal-binding residues H8A and E70A and its active site C254A reduced or eliminated its ability to catalyze Ξ±-syn nitration. |
| GO:0005829 cytosol | IDA PMID:41628334 Selective peroxynitrite-mediated protein nitration catalyzed... | NEW | Summary: Proposed NEW annotation. Subcellular fractionation of H4 cells, with Tomm20 and LDHA as compartment markers, detects GLOD4 in both mitochondrial and cytosolic fractions, more abundantly in the cytosol. Reason: GOA currently records only mitochondrion for this protein, which understates where it is and where it works - alpha-synuclein, its principal characterised substrate, is cytosolic, and the cellular knockout experiments read out cytosolic nitration. Adding cytosol makes the location record match the enzymology. Supporting Evidence: PMID:41628334 GLOD4 is present in both mitochondria and cytosol, with relatively higher levels in the cytosol |
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Download this section (compressed HTML)Q: Can the nitrase activity be reproduced by an independent laboratory? The entire assignment rests on one study from one company that has filed a patent application on the activity. Replication with recombinant GLOD4, the published H8A/E70A/C254A mutants and an orthogonal nitration readout is the single most valuable next step, and should precede creation of new GO terms for the activity.
Suggested experts: Harry Ischiropoulos, Irene Griswold-Prenner
Q: What is the catalytic mechanism, and is zinc the catalytic metal? The authors state that the mechanism is unresolved and raise manganese, nickel and iron as alternatives. Without a mechanism it is hard to distinguish true catalysis from a metal-dependent scaffold that positions a target tyrosine for radical chemistry.
Suggested experts: Aaron L. Lucius, Andy Jennings
Q: Does GLOD4 have any glyoxalase-family metabolic activity at all? The gene name and the UniProt family assignment are fold-based, no EC number has ever been assigned, and no systematic screen of small-molecule substrates has been published.
Q: Why is GLOD4 mitochondrial as well as cytosolic, and does it nitrate mitochondrial proteins? All characterised substrates are cytosolic, yet fractionation and the MitoCoP proteome both place a substantial pool in mitochondria.
Suggested experts: Harry Ischiropoulos
Q: What is the physiological consequence of losing selective alpha-synuclein nitration? GLOD4 knockout mice are healthy to 18 months, so the activity is either dispensable, redundant, or important only under pathological challenge.
Suggested experts: Robert P. Brendza, Mike Beckstead
Experiment: Establish turnover independently: measure multiple catalytic cycles per enzyme molecule with limiting GLOD4 and excess substrate and peroxynitrite, determine the full steady-state kinetic parameters for several substrates, and test whether the enzyme is consumed or inactivated by autonitration during the reaction. Pair this with the published active-site mutants as internal negative controls, in a laboratory with no interest in the outcome.
Hypothesis: GLOD4 is a true catalyst of tyrosine nitration rather than a peroxynitrite-binding protein that accelerates adventitious chemistry on bound substrates.
Type: independent enzymology and turnover measurement
Experiment: Swap the C-terminal tyrosine-containing region between alpha-synuclein (a substrate) and gamma-synuclein (not a substrate), and test nitration of the chimeras; in parallel, measure binding of GLOD4 to each synuclein by a label-free method in the absence of peroxynitrite. If selectivity follows the transplanted segment but binding does not, selectivity is local chemistry; if binding tracks substrate status, GLOD4 has a substrate-recognition surface worth mapping.
Hypothesis: Substrate selection by GLOD4 is determined by a docking interaction with the substrate rather than by local tyrosine chemistry.
Type: chimera and binding analysis
Experiment: Cross GLOD4 knockout mice into an alpha-synuclein preformed-fibril or A53T transgenic model and quantify seeding, spread, nitrated-synuclein burden and dopaminergic loss against GLOD4-competent littermates. A conditional, adult-onset knockout would separate developmental compensation from an ongoing requirement.
Hypothesis: GLOD4-dependent alpha-synuclein nitration modifies synuclein pathology rather than baseline physiology, which is why knockout mice appear normal.
Type: mouse genetics in a synucleinopathy model
Experiment: Screen purified GLOD4 against a panel of glyoxalase-pathway and related VOC-superfamily substrates (methylglyoxal, S-D-lactoylglutathione, hydroxyacylglutathiones, bleomycin, fosfomycin-type epoxides) with and without added Zn2+, Mn2+, Ni2+ and Fe2+, reading out by coupled assay or LC-MS. A negative result would be publishable and would firmly retire the glyoxalase framing of the gene name.
Hypothesis: GLOD4 retains an ancestral vicinal oxygen chelate metabolic activity in addition to, or instead of, protein nitration.
Type: broad enzymatic substrate screen
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