GstO2 (CG6673) is an Omega-class cytosolic glutathione S-transferase of Drosophila melanogaster. It has the thioredoxin-like GST N-terminal domain with an active-site cysteine (Cys31) and the all-alpha GST C-terminal domain, and is expressed as two alternatively spliced isoforms, A and B, that differ in their N-terminal region including the active-site motif. Like other Omega GSTs it has little activity toward CDNB but high glutathione-dependent thiol transferase and dehydroascorbate reductase (DHAR) activity; isoform B carries most of the DHAR activity and loss of the gene lowers whole-fly DHAR activity, perturbs the ascorbate/dehydroascorbate ratio and sensitises flies to paraquat. Isoform A catalyses S-glutathionylation of proteins, notably the mitochondrial F1F0-ATP synthase beta subunit, which supports ATP synthase activity and assembly; its overexpression suppresses parkin and PINK1 mutant phenotypes and neurotoxicity in fly TDP-43, TAF15 and FUS proteinopathy models. The protein is found in the cytosol and mitochondria.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004364 glutathione transferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that GstO2 is a glutathione transferase, placed at the Omega-class GST node (PTN000883725) seeded by fly and human GSTO experimental annotations, including GstO2 itself. Reason: GstO2 is a canonical Omega-class GST with the GST N- and C-terminal domains and the Omega active-site cysteine (Cys31 in isoform A). Direct assays show GSH-dependent transferase/thiol transferase chemistry and GSH-dependent protein S-glutathionylation. The IBA is consistent with the target's own experimental data. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0004364 glutathione transferase activity | IDA PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: Recombinant CG6673A and CG6673B were assayed with CDNB, thiol transferase (HEDS) and DHA substrates; activity toward CDNB was low but present, thiol transferase activity relatively high. Reason: Direct enzymatic assay of the purified gene product shows the GSH-dependent transferase/thiol-transferase activity typical of Omega GSTs. Low CDNB activity does not undermine the term, which covers GSH conjugation generally. Supporting Evidence: PMID:16712527 As a result, we identified five candidates, the products of CG6776, CG6781, CG6673A, CG6673B and CG6662 PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA |
| GO:0004364 glutathione transferase activity | IDA PMID:22082028 A preliminary characterization of the cytosolic glutathione ... | ACCEPT | Summary: Survey of the Drosophila cytosolic GST proteome in which recombinant GSTs, including the GstO2 isoforms, were expressed and kinetically characterised. Reason: Only the abstract is cached; the curator saw the full text with per-enzyme activities. The activity agrees with independent assays (PMID:16712527, PMID:22219196), so the annotation is accepted. Supporting Evidence: PMID:22082028 The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA |
| GO:0004364 glutathione transferase activity | IDA PMID:24533905 Structural modelling and molecular characterization of omega... | ACCEPT | Summary: Biochemical characterisation of recombinant GstO2A and GstO2B (pH/temperature optima, native mass, kinetics). Reason: Abstract-only cache; it reports characterisation of both recombinant isoforms. The GST activity is consistent with other direct assays, so we defer to the FlyBase curator. Supporting Evidence: PMID:24533905 In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. PMID:24533905 The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. |
| GO:0004364 glutathione transferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro GST omega (IPR005442) / EC 2.5.1.18 mapping to glutathione transferase activity. Reason: The family assignment is correct and the activity is experimentally confirmed for this gene product; consistent with the IDA rows. Supporting Evidence: file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA |
| GO:0004734 pyrimidodiazepine synthase activity | IDA NOT PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: NOT annotation: recombinant CG6673A/B lack pyrimidodiazepine (PDA) synthase activity, which is restricted to the sepia product CG6781. Reason: The paper explicitly tested all five Omega GST candidates for PDA synthesis and only CG6781 was active; the negative result for CG6673 is direct and informative given the close paralogy. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:16712527 Interestingly, only the product of CG6781 had this activity |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference of cytoplasmic location for Omega-class GSTs. Reason: The location claim is correct but general. GstO2 already carries direct (IDA) annotations to two more specific parts of the cytoplasm, cytosol and mitochondrion (PMID:24533905), and its best-characterised protein target in vivo is the mitochondrial ATP synthase beta subunit. Replace with those two terms. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN005192534 SUPPORTS TRANSFER The inherited cytoplasm claim holds for GstO2; the row is only less specific than the gene's own IDA cytosol and mitochondrion annotations. Proposed replacements: cytosol mitochondrion Supporting Evidence: PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | MODIFY | Summary: InterPro GST omega (IPR005442) mapping to cytoplasm. Reason: The location claim is correct but general. GstO2 already carries direct (IDA) annotations to two more specific parts of the cytoplasm, cytosol and mitochondrion (PMID:24533905), and its best-characterised protein target in vivo is the mitochondrial ATP synthase beta subunit. Replace with those two terms. Proposed replacements: cytosol mitochondrion Supporting Evidence: PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | MODIFY | Summary: Transfer of cytoplasm location from rat GSTO1 (RGD:70952). Reason: The location claim is correct but general. GstO2 already carries direct (IDA) annotations to two more specific parts of the cytoplasm, cytosol and mitochondrion (PMID:24533905), and its best-characterised protein target in vivo is the mitochondrial ATP synthase beta subunit. Replace with those two terms. Proposed replacements: cytosol mitochondrion Supporting Evidence: PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0005739 mitochondrion | IDA PMID:24533905 Structural modelling and molecular characterization of omega... | ACCEPT | Summary: Direct localisation of GstO2 to mitochondria (FlyBase IDA from Kim & Yim 2014). Reason: Full text is not cached, so the assay (likely fractionation or immunostaining) cannot be checked for ER contamination; we defer to the curator. The claim is biologically coherent: isoform A directly glutathionylates the mitochondrial F1F0-ATP synthase beta subunit and restores ATP synthase activity in parkin mutants. Kept at the organelle root; no sub-compartment is asserted because only one line of evidence (the ATP synthase target) points to the matrix side. Supporting Evidence: PMID:24533905 In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH |
| GO:0005829 cytosol | IDA PMID:24533905 Structural modelling and molecular characterization of omega... | ACCEPT | Summary: Direct localisation of GstO2 to the cytosol (FlyBase IDA from Kim & Yim 2014). Reason: Omega GSTs are soluble cytosolic enzymes with no signal peptide or transmembrane segment; the cytosolic pool is where DHA reduction and ascorbate recycling would occur. Abstract-only cache, deferring to the curator. Supporting Evidence: PMID:24533905 In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. file:DROME/GstO2/GstO2-uniprot.txt DR InterPro; IPR005442; GST_omega. |
| GO:0006749 glutathione metabolic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Omega GSTs participate in glutathione metabolism. Reason: GstO2 uses GSH as cosubstrate and oxidises it to GSSG during DHA reduction and forms glutathione-protein mixed disulfides, so it does take part in glutathione metabolism. The process is broad and not the specific biological role, so it is kept as non-core. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH |
| GO:0006749 glutathione metabolic process | IDA PMID:22082028 A preliminary characterization of the cytosolic glutathione ... | KEEP AS NON CORE | Summary: Glutathione metabolic process inferred from in vitro GST assays in the Drosophila GST proteome survey. Reason: Consistent with the IBA row; GSH-consuming enzyme activity supports participation in glutathione metabolism, but this is a generic consequence of the MF rather than its specific role. Abstract-only cache. Supporting Evidence: PMID:22082028 The diverse kinetic properties as well as physiological substrate specificity of the DmGSTs are such that each individual enzyme displayed a unique character PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of dehydroascorbate reductase activity at the Omega GST node; donors include fly and human GSTOs and GstO2 itself. Reason: DHA reductase activity is directly shown for both GstO2 isoforms and the null mutant loses most whole-fly DHAR activity; the IBA agrees with target-specific evidence. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:22219196 The DHAR activity in DmGSTO1null flies was dramatically decreased |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IDA PMID:16712527 Identification and characteristics of the structural gene fo... | ACCEPT | Summary: Recombinant CG6673A and CG6673B reduce dehydroascorbate in a GSH-dependent assay. Reason: Direct assay of the purified protein. Isoform B is markedly more active than isoform A, but both have the activity, and the null mutant shows that GstO2 accounts for most DHAR activity in the fly. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:22219196 We have shown that DmGSTO1B has much higher DHAR activity than DmGSTO1A in vitro PMID:22219196 The DHAR activity in DmGSTO1null flies was dramatically decreased file:DROME/GstO2/GstO2-deep-research-manual.md Isoform B has much higher DHAR activity than isoform A |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IDA PMID:24533905 Structural modelling and molecular characterization of omega... | ACCEPT | Summary: Kinetic parameters for DHA reduction were determined for recombinant GstO2A and GstO2B. Reason: Direct kinetic characterisation of the activity (abstract-only cache, but the abstract states the measurement). Supporting Evidence: PMID:24533905 The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IEA GO_REF:0000120 | ACCEPT | Summary: ARBA/EC 1.8.5.1 mapping to glutathione dehydrogenase (ascorbate) activity. Reason: The electronic prediction is correct for this gene product; independent experimental support comes from direct assays (PMID:16712527, PMID:24533905) rather than from the ARBA rule. Supporting Evidence: PMID:16712527 All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA PMID:24533905 The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. |
| GO:0045824 negative regulation of innate immune response | HMP PMID:19520911 Genome-wide RNAi screen identifies genes involved in intesti... | MARK AS OVER ANNOTATED | Summary: From a genome-wide in vivo RNAi survival screen after oral Serratia marcescens infection; FlyBase presumably scored GstO2 as a hit whose knockdown increased survival. Reason: GstO2 is not discussed in the main text; the result is a single high-throughput survival phenotype from supplementary tables. Altered survival after infection does not show that GstO2 regulates the innate immune response (it could act through redox or epithelial homeostasis), and no follow-up exists. The same screen also yielded a positive defense-response annotation, which is hard to reconcile with this one. Supporting Evidence: PMID:19520911 We identified 95 genes (10.7% of the total hits) that confer resistance to S. marcescens infections when silenced |
| GO:0050829 defense response to Gram-negative bacterium | HMP PMID:19520911 Genome-wide RNAi screen identifies genes involved in intesti... | MARK AS OVER ANNOTATED | Summary: From the same genome-wide RNAi survival screen after oral Serratia marcescens infection. Reason: A screen-level survival phenotype with no gene-specific follow-up and no mechanistic link to antibacterial defense; it sits alongside a negative-regulation annotation from the same paper. GstO2's characterised roles are redox enzymology (DHA reduction, protein glutathionylation), so this process is at most indirect. Supporting Evidence: PMID:19520911 We identified 95 genes (10.7% of the total hits) that confer resistance to S. marcescens infections when silenced |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: Inferred from the DHAR MF via inter-ontology links. Reason: DHA reductase recycles ascorbate, an antioxidant, and the null mutant is paraquat-sensitive, so a role in cellular antioxidant defense is reasonable, but it is an indirect consequence of ascorbate recycling rather than the direct detoxification of an oxidant by GstO2. Supporting Evidence: PMID:22219196 These results indicate that DmGSTO1B has a protective role in response to paraquat-induced stress and plays an important role in the in vivo conversion of DHA to AsA. PMID:22219196 The DHAR activity in DmGSTO1null flies was dramatically decreased |
| GO:0010731 protein glutathionylation | IDA PMID:22219196 Glutathione s-transferase omega 1 activity is sufficient to ... | NEW | Summary: Isoform A (DmGSTO1A, the Q9VSL5 product) directly glutathionylates the mitochondrial ATP synthase beta subunit in vitro in a GSH- and dose-dependent manner; the C31A active-site mutant fails to increase glutathionylation in vivo. Reason: GstO2 itself catalyses the step (transfer of glutathione onto a protein cysteine), with an active-site cysteine requirement, so it performs the work of the process rather than merely being required for it. This is the best-supported physiological role of isoform A and is not represented in GOA. Isoform B does not glutathionylate this target. Supporting Evidence: PMID:22219196 We also confirmed that DmGSTO1A can directly glutathionylate the ATP synthase Ξ² subunit in a dose-dependent manner in the presence of GSH PMID:22219196 We constructed a catalytically inactive form of DmGSTO1A, DmGSTO1AC31A, in which cysteine 31 was mutated to alanine. PMID:22219196 DmGSTO1A expression increased the level of glutathionylation of this protein, whereas DmGSTO1AC31A did not. |
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Download this section (compressed HTML)Q: Does GstO2 isoform A directly catalyse deglutathionylation of FUS Cys447 (as reported in vivo, PMID:35320731), and if so should protein-glutathione oxidoreductase (glutathione) activity (GO:0141049) be annotated alongside its glutathionylating activity on ATP synthase beta?
Q: Which sub-mitochondrial compartment holds GstO2 isoform A, and is the mitochondrial pool imported or peripherally associated, given that the protein lacks an obvious presequence?
Q: Are the opposing immune annotations from the Cronin et al. 2009 RNAi screen (negative regulation of innate immune response and defense response to Gram-negative bacterium) based on the same RNAi line, and were they reproduced?
Experiment: Fractionate adult thorax mitochondria (with ER and cytosol markers) and use protease protection and isoform-specific antibodies to localise GstO2A and GstO2B.
Hypothesis: Isoform A, but not B, is present in the mitochondrial matrix where it glutathionylates ATP synthase beta.
Experiment: Measure glutathionylation and deglutathionylation kinetics of recombinant GstO2A, GstO2B and the C31A mutant on purified ATP synthase beta and FUS peptides containing Cys447.
Hypothesis: The direction of the GstO2 thiol-disulfide exchange depends on substrate and GSH/GSSG ratio, and the isoform N-terminal motifs (CPFS vs CPYS) set substrate preference.
Glutathione S transferase O2 (GstO2) encodes a protein involved in glutathione metabolism.
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