gst-44 (F13A7.10) encodes an omega-class glutathione S-transferase (GSTO), one of five GSTO genes in C. elegans and closest to GSTO-1 and GSTO-2. It has a thioredoxin-like GST N-terminal domain carrying the conserved active-site cysteine (Cys35) and an all-alpha GST C-terminal domain. Like other omega-class enzymes it works as a glutathione-dependent reductase rather than a conjugating GST. Recombinant GST-44 reduces dehydroascorbate (DHAR activity) and monomethylarsonate (MMA-V) using glutathione, has thioltransferase (glutaredoxin-like) and cumene hydroperoxide peroxidase activity, and shows no conjugating activity toward CDNB or ethacrynic acid. GST-44 is a cytoplasmic protein, also seen weakly in nuclei, of the intestine and the excretory canal cell. Arsenite induces it strongly in the excretory system through SKN-1/Nrf2 and PHA-4/FoxA; pathogens, oxidants and dimethyl fumarate also induce it. Deletion or RNAi knockdown makes worms sensitive to arsenite and arsenate, and this sensitivity is specific among the GSTO paralogs. Deletion mutants also have a shorter lifespan and are modestly sensitive to juglone and to reductive ER stress, which points to a main role in arsenic detoxification and redox defence.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000302 response to reactive oxygen species | IMP PMID:23593298 The glutathione reductase GSR-1 determines stress tolerance ... | MODIFY | Summary: WormBase IMP from a small RNAi screen of 67 glutathione-metabolism genes (Luersen et al. 2013). In that screen gst-44(RNAi) cut survival on 3 mM arsenite to 15%, but survival on the pro-oxidant juglone stayed at control level (90.0 +/- 3.3% vs 90.8 +/- 7.4%). Reason: The full text is cached and read. The only gst-44 phenotype the cited paper reports is arsenite sensitivity; knockdown did not change tolerance of the ROS generator juglone, and the authors say the role of gst-44 "remains unclear". The phenotype supports "response to arsenic-containing substance" (GO:0046685), not a response to reactive oxygen species. Both are biological-process terms, so this is a lateral correction within one aspect. A later study (PMID:40186509) found modest juglone sensitivity in the gst-44 deletion mutant, but that is a different reference and does not rescue this row. A source defect is recorded. Proposed replacements: response to arsenic-containing substance Supporting Evidence: PMID:23593298 Furthermore, knockdown of an alpha-class related GST (gst-32), an omega-class GST (gst-44) and an extracellular glutathione peroxidase (C11E4.1) less profoundly affected the survival rate under arsenite stress PMID:23593298 Among the 67 GSH-metabolism genes tested only C46F11.2 was found to be absolutely essential for juglone tolerance PMID:23593298 However, the role of gst-44 remains unclear. |
| GO:0004364 glutathione transferase activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the omega-class GST node PTN000883725. The node is seeded by Drosophila GSTOs, human GSTO1 and GSTO2, and C. elegans GSTO-1. Reason: GO:0004364 means GSH conjugation to an electrophile (RX + GSH, EC 2.5.1.18). GST-44 is a genuine omega-class enzyme: it keeps the active-site Cys35 and the G-site. Omega-class enzymes, however, use that cysteine for glutathione-dependent redox chemistry (DHAR, thioltransferase, arsonate reduction), and their conjugating activity is weak or absent. Purified GST-44 showed no conjugation of the two standard GST substrates, CDNB and ethacrynic acid. Its measured activities are captured by GO:0045174 and GO:0050610. The conjugation term is therefore an over-annotation for this gene. It is not refuted outright, because only two substrates were tested and the negative data are "not shown". Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE GRANULARITY MISMATCH Sources checked: PANTHER:PTN000883725 · GST omega-class node (PTHR43968) SUPPORTS SOURCE BUT NOT TARGET GST-44 is correctly placed inside the omega clade; that much is right. The trouble is the term placed on the node. "Glutathione transferase activity" rests on the weak CDNB conjugation of some seeds, such as human GSTO1, and it does not hold for GST-44, which showed no conjugation. The clade's shared activity is the glutathione-dependent reductase chemistry (GO:0045174), which this review accepts. WB:WBGene00016204 · C. elegans gsto-1 (C29E4.7) SUPPORTS SOURCE BUT NOT TARGET Worm paralog seed; its own activity data do not transfer conjugation activity to GST-44. Supporting Evidence: PMID:40186509 For the typical GST substrates 1‐chloro‐2,4‐dinitrobenzene (CDNB) and ethacrynic acid, no GSH‐conjugating activity could be shown (data not shown). PMID:40186509 Due to a cysteine at the active center, GSTOs do not demonstrate activity with typical GST substrates such as CDNB or ethacrynic acid but rather exhibit activity with substrates typically associated with glutaredoxins or thioredoxins file:worm/gst-44/gst-44-deep-research-manual.md My reading of the sequence: the catalytic-cysteine motif S-M-R-F-C-P is present with Cys35 |
| GO:0004364 glutathione transferase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: Combined electronic annotation from the UniRule omega-class rule, ARBA and EC 2.5.1.18. Reason: This row makes the same claim as the IBA row and gets the same action. EC 2.5.1.18 comes from the generic GST-omega UniRule. Recombinant GST-44 showed no GSH conjugation with CDNB or ethacrynic acid; its activities are the omega-class reductase reactions. Supporting Evidence: PMID:40186509 For the typical GST substrates 1‐chloro‐2,4‐dinitrobenzene (CDNB) and ethacrynic acid, no GSH‐conjugating activity could be shown (data not shown). |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for cytoplasm from a deep GST node seeded by human GSTO1, E. coli SspA and a Drosophila GSTO. Reason: GST-44 is a soluble protein with no signal peptide or transmembrane segment. A translational GST-44::GFP reporter is mainly cytoplasmic in intestinal cells and in the excretory canal cell, which confirms the inherited cytoplasmic location. The weaker nuclear signal is not annotated. Supporting Evidence: PMID:40186509 In adult worms, a robust signal is observed in the cytoplasm of the anterior and posterior intestinal cells, accompanied by a weaker signal in the intestinal nuclei and in the excretory canal cell. |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the GST omega signature IPR005442. Reason: This row agrees with the IBA row and with the cytoplasmic GST-44::GFP signal in worms. Supporting Evidence: PMID:40186509 In adult worms, a robust signal is observed in the cytoplasm of the anterior and posterior intestinal cells |
| GO:0006749 glutathione metabolic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from the omega-class node, seeded by Drosophila GSTOs. Reason: GST-44 oxidises GSH to GSSG as the electron donor in each reaction it catalyses (DHA, MMA(V) and peroxide reduction). It therefore acts on glutathione and takes part in glutathione turnover. The term is a broad metabolic context, and the gene's specific roles are ascorbate recycling, arsenical reduction and redox defence, so the row is kept as non-core. Supporting Evidence: PMID:40186509 Furthermore, the enzyme was capable of using GSH as an electron donor to reduce dehydroascorbate (DHA) (6.35 ± 0.15 μmol·min−1·mg−1) and monomethylarsenate (MMAv) (0.635 ± 0.13 μmol·min−1·mg−1). |
| GO:0006749 glutathione metabolic process | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule-based electronic annotation, the same claim as the IBA row. Reason: Kept as non-core for the reasons given on the IBA row. Supporting Evidence: PMID:40186509 Furthermore, the enzyme was capable of using GSH as an electron donor to reduce dehydroascorbate (DHA) |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA for dehydroascorbate reductase (DHAR) activity, the hallmark activity of the omega clade. Reason: DHAR is the signature catalytic activity of the GST omega clade, and the inheritance is confirmed on GST-44 itself: recombinant GST-44 reduces dehydroascorbate with GSH as the electron donor (6.35 umol/min/mg), its highest measured activity. Supporting Evidence: PMID:40186509 Furthermore, the enzyme was capable of using GSH as an electron donor to reduce dehydroascorbate (DHA) (6.35 ± 0.15 μmol·min−1·mg−1) |
| GO:0045174 glutathione dehydrogenase (ascorbate) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation (EC 1.8.5.1), the same claim as the IBA row. Reason: Confirmed for GST-44 by direct enzyme assay. Supporting Evidence: PMID:40186509 Furthermore, the enzyme was capable of using GSH as an electron donor to reduce dehydroascorbate (DHA) (6.35 ± 0.15 μmol·min−1·mg−1) |
| GO:0050610 methylarsonate reductase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic annotation (EC 1.20.4.2) from the GST omega UniRule. Reason: Recombinant GST-44 reduces monomethylarsonate (MMA-V) with GSH as the electron donor (0.635 umol/min/mg), which is exactly the EC 1.20.4.2 reaction. This activity probably underlies the arsenic hypersensitivity of gst-44 mutants. The paper's abstract calls it "arsenate reductase", but the substrate assayed was MMA(V). Supporting Evidence: PMID:40186509 and monomethylarsenate (MMAv) (0.635 ± 0.13 μmol·min−1·mg−1) |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000108 | KEEP AS NON CORE | Summary: Inter-ontology inference from GO:0045174 DHAR activity. Reason: DHAR by itself regenerates ascorbate rather than detoxifying an oxidant, so the inference chain is indirect. The conclusion does hold for GST-44 on other evidence. The enzyme reduces cumene hydroperoxide (peroxidase activity) and protects E. coli against it, and the deletion mutant is sensitive to the superoxide generator juglone. Oxidant detoxification is a real but secondary role, after ascorbate recycling and arsenical reduction. Supporting Evidence: PMID:40186509 Notably, GST‐44 exhibited tenfold higher glutathione peroxidase activity toward cumene hydroperoxide than the activity observed for GSTO‐1. PMID:40186509 E. coli strains overexpressing GST‐44 displayed enhanced resistance to prolonged oxidative stress. |
| GO:0004601 peroxidase activity | IDA PMID:40186509 Unraveling the functional dynamics of Caenorhabditis elegans... | NEW | Summary: Recombinant GST-44 reduces cumene hydroperoxide using GSH (0.37 umol/min/mg). Reason: This activity was measured directly on the purified protein, about ten-fold higher than C. elegans GSTO-1. The substrate is an organic hydroperoxide, so the generic peroxidase term is used instead of GO:0004602 glutathione peroxidase activity, which is defined on H2O2. The activity is modest, so it is non-core. It is neither an ancestor nor a descendant of GO:0045174 or GO:0050610. Supporting Evidence: PMID:40186509 we assessed the peroxidase activity of the recombinant protein and found a specific activity (0.37 ± 0.11 μmol·min−1·mg−1) PMID:40186509 To evaluate the glutathione peroxidase activity of GST‐44, cumene hydroperoxide was employed as a substrate |
| GO:0071722 detoxification of arsenic-containing substance | IMP PMID:40186509 Unraveling the functional dynamics of Caenorhabditis elegans... | NEW | Summary: gst-44(tm6133) deletion mutants survive both arsenite and arsenate poorly, and the gsto-1, gsto-2 and gsto-3 deletions do not. GST-44 catalyses GSH-dependent reduction of an arsenical (MMA-V), and arsenite induces it in the excretory system through SKN-1 and PHA-4. Reason: Process term chosen after applying the participation test. GST-44 performs a catalytic step in arsenical biotransformation itself: it reduces MMA(V), which matches the accepted GO:0050610. The mutant phenotype is specific to gst-44 among the four GSTO paralogs tested. Human GSTO1 and GSTO2, the comparators in the same role, are the reference arsenic-reducing omega GSTs. The evidence is therefore catalysis plus specific loss of function, not necessity evidence alone. Whether the methylarsenical reduction detoxifies arsenic in worms (in mammals MMA(III) is more toxic) is noted as a question. Supporting Evidence: PMID:40186509 While the GST‐44 showed a significantly reduced survival rate under arsenic, this was not observed in the deletion mutants gsto‐1(eva301) III, gsto‐2(eva302) III, and gsto‐3(eva303) III (Fig. 9B). PMID:40186509 Our findings highlight the essential role of GST‐44 in mediating arsenic detoxification |
Loading supporting content…
Download this section (compressed HTML)Q: Does GST-44 reduce inorganic arsenate (AsV) to arsenite directly, as the paper's "arsenate reductase" wording implies, or only methylated arsenicals (MMA-V)? And does C. elegans methylate arsenic, so that MMA(V) is a physiological substrate?
Q: Why is gst-44, and not gsto-1, gsto-2 or gsto-3, required for arsenic tolerance? Is it the expression in the excretory canal cell, or a substrate preference?
Q: Should the PAINT omega-class node PTN000883725 carry GO:0004364 glutathione transferase activity, given that several omega members (including GST-44) show no conjugation with CDNB or ethacrynic acid?
Experiment: Test purified GST-44 with inorganic arsenate, MMA(V) and DMA(V) and identify the products by HPLC-ICP-MS. Compare with a Cys35Ser (active-site) mutant.
Hypothesis: GST-44 reduces pentavalent arsenicals through its active-site cysteine, and Cys35Ser abolishes the activity.
Experiment: Rescue gst-44(tm6133) arsenic sensitivity with excretory-cell-specific versus intestine-specific expression of wild-type or Cys35Ser GST-44.
Hypothesis: Catalytically active GST-44 in the excretory system is sufficient to restore arsenic tolerance.
Loading supporting content…
Download this section (compressed HTML)gst-44 encodes a putative omega-class glutathione transferase (GST; EC 2.5.1.18) which, like its paralog GSTO-1, might have thiol oxidoreductase and dehydroascorbate reductase activity; GST-44 is expressed in excretory cell cytoplasm; other GST-44 paralogs include C02D5.3 and K10F12.4; GST-44 has no obvious function in mass RNAi assays.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)