FSH3 (YOR280C, "Family of Serine Hydrolases 3") is a 266-residue serine hydrolase of the alpha/beta-hydrolase fold (AB-hydrolase-3 / Pfam FSH1 family) in the budding yeast Saccharomyces cerevisiae. It carries a complete, canonical Ser-Asp-His catalytic triad, with the nucleophilic serine (Ser117) in a classic GXSXG "nucleophile elbow" motif, and was directly detected as a catalytically active serine hydrolase in yeast by activity-based protein profiling. Its physiological substrate and specific reaction are undetermined; UniProt assigns only the uncommitted esterase activity EC 3.1.-.- and describes it as a "serine hydrolase of unknown specificity". FSH3 is one of three paralogous S. cerevisiae FSH proteins (with FSH1 and FSH2) and is the yeast counterpart of the human candidate tumor-suppressor esterase OVCA2. It localizes to the peroxisome and to cytoplasmic pools; its expression is induced by the DNA-damage-checkpoint effector Crt1/RFX1 and by oxidative (H2O2) stress. Overexpression of FSH3 perturbs cellular lipid content (notably reducing phosphatidylcholine) and triggers a NUC1-dependent apoptotic phenotype. Single deletion causes no growth defect under standard conditions, but a double mutant lacking both FSH3 and the peroxisomal lipase Lpx1 has reduced peroxisomal fatty-acid beta-oxidation activity, implicating FSH3 (redundantly with Lpx1) in peroxisomal lipid/fatty-acid metabolism; its precise molecular contribution and physiological substrate remain unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetically inferred (IBA) nuclear localization propagated across the OVCA2/FSH family. It is not supported by the yeast-specific experimental evidence, which places FSH3 in the peroxisome (IDA), nor by any FSH3 assay. This is a weak, over-propagated location for this gene. Reason: IBA-only nuclear localization with no experimental support in yeast; the experimentally verified compartment is the peroxisome (GO:0005777, IDA). Family-wide phylogenetic propagation of a nuclear location is not substantiated for FSH3. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: UniProtKB:Q8WZ82 Β· OVCA2 (human) SUPPORTS SOURCE BUT NOT TARGET PANTHER OVCA2 subfamily (PTHR48070:SF6) seed; a nuclear location for the family is not substantiated for yeast FSH3, whose experimental compartment is the peroxisome. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetically inferred (IBA) cytoplasmic localization. FSH3 has cytoplasmic/stress-granule associations (CD-CODE stress granule cross-ref) and a soluble alpha/beta-hydrolase is consistent with a cytoplasmic pool, so this general location is plausible, but it is non-core relative to the experimentally established peroxisomal localization. Reason: General, phylogenetically inferred location that is plausible for a soluble hydrolase but is not the informative, experimentally supported compartment; retained as non-core alongside the IDA peroxisome annotation. |
| GO:0016787 hydrolase activity | IBA GO_REF:0000033 | MODIFY | Summary: Correct but uninformatively general. FSH3 has a complete, canonical Ser-Asp-His catalytic triad (Ser117 in a GXSXG motif, Asp180, His209) on an alpha/beta-hydrolase scaffold, and was detected as a catalytically active serine hydrolase by activity-based proteomics. These support the more specific mechanism-defined term serine hydrolase activity (GO:0017171). Reason: The generic hydrolase activity term should be replaced by the mechanism-specific GO:0017171 (serine hydrolase activity), which is directly supported by the intact Ser-Asp-His triad, the diagnostic GXSXG nucleophile-elbow motif, and the activity-based proteomic detection of FSH3 as an active serine hydrolase. The specific substrate/reaction remains unknown (EC 3.1.-.-), so a more specific catalytic term is not yet justified. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: UniProtKB:Q8WZ82 Β· OVCA2 (human) SUPPORTS TRANSFER The IBA hydrolase-activity transfer from the OVCA2 esterase subfamily is correct in branch but too general; the intact Ser-Asp-His triad and GXSXG motif in FSH3 support the more specific serine hydrolase activity. Proposed replacements: serine hydrolase activity Supporting Evidence: PMID:14645503 Three of the previously uncharacterized proteins are members of a eukaryotic serine hydrolase family |
| GO:0005777 peroxisome | IDA PMID:36164978 Systematic multi-level analysis of an organelle proteome rev... | ACCEPT | Summary: Experimentally determined (IDA) peroxisomal localization from a systematic peroxisome-proteome analysis in which FSH3 is one of the newly identified peroxisomal proteins. This is the informative, experimentally supported compartment for FSH3 and is retained as a core localization. Reason: Direct experimental evidence (IDA) from a systematic peroxisome-proteome study; the curator had access to the full dataset. This is the strongest localization annotation for FSH3. Supporting Evidence: PMID:36164978 Lipidomic analysis on mutants of 10 newly identified peroxisomal proteins whose molecular function in the yeast cell is putative or unknown shows that cells overexpressing FSH3 had the most significant change in all conditions compared to the control strain, with a reduction of PC |
| GO:0003674 molecular_function | ND GO_REF:0000015 | REMOVE | Summary: Root-level placeholder (ND, "no biological data") reflecting the historical absence of a curated molecular function. It is superseded by the informative serine hydrolase activity now supported by the domain/triad analysis and the IBA hydrolase annotation. Reason: ND root placeholder is superseded by informative molecular-function evidence (serine hydrolase activity); root ND annotations should not be retained once a real function is available. |
| GO:0008150 biological_process | ND GO_REF:0000015 | REMOVE | Summary: Root-level placeholder (ND) for biological process. FSH3's native biological process is genuinely unresolved (no deletion phenotype under standard conditions), so this remains a real knowledge gap rather than a curatable term. Reason: ND root placeholder; uninformative. The genuine uncertainty about FSH3's biological role is recorded in knowledge_gaps rather than as a root-term ND annotation. |
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Download this section (compressed HTML)Q: What is the natural substrate of FSH3, and does the S117A catalytic-dead mutant lose the overexpression phenotypes (apoptosis, PC reduction)?
Q: Is FSH3 functionally redundant with FSH2 (its OVCA2-subfamily paralog) and/or FSH1, and does a triple fsh deletion reveal a phenotype absent in single mutants?
Q: Does FSH3 act in peroxisomal glycerophospholipid metabolism, and is the OVCA2 human ortholog's function conserved?
Experiment: Compare wild-type FSH3, catalytic-dead FSH3(S117A), and empty-vector strains for the overexpression phenotypes (ROS accumulation, viability, PC levels). Loss of phenotype in S117A would establish that they depend on catalytic serine-hydrolase activity.
Hypothesis: FSH3 is a catalytically active serine hydrolase whose in-cell phenotypes require the Ser117 nucleophile.
Experiment: Purify recombinant FSH3 and assay activity against fluorogenic ester panels and defined lipids (including phosphatidylcholine) in vitro; combine with untargeted lipidomics/metabolomics of fsh3-delta vs wild type to identify accumulating candidate substrates.
Hypothesis: FSH3 hydrolyzes a peroxisomal glycerophospholipid or ester substrate.
Experiment: Construct single, double and triple fsh deletions and phenotype across oxidative, DNA-damage and oleate (peroxisome-inducing) conditions, with epistasis to NUC1.
Hypothesis: Single-deletion silence of FSH3 is due to redundancy with FSH1/FSH2.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate of FSH3 and the specific reaction it catalyzes are undetermined. Only an uncommitted esterase activity (EC 3.1.-.-) is assigned, and UniProt describes FSH3 as a "serine hydrolase of unknown specificity".
OPEN BIOLOGY MF_DARK
What is known: FSH3 has a complete, canonical Ser-Asp-His serine-hydrolase catalytic triad (Ser117 in a GXSXG motif, Asp180, His209) on an alpha/beta-hydrolase fold and was detected as a catalytically active serine hydrolase by activity-based proteomics in yeast; the class of chemistry (serine-nucleophile ester/amide hydrolysis) is therefore known, but no natural substrate has been identified.
Significance: FSH3 is the yeast counterpart of the human candidate tumor-suppressor esterase OVCA2; identifying its substrate would define the molecular function of a conserved but functionally dark eukaryotic serine-hydrolase family.
What would resolve it: In-vitro substrate profiling (e.g. fluorogenic ester/lipid panels, competitive activity-based protein profiling with FP-probes) of recombinant FSH3 and a catalytic-serine (S117A) mutant, paired with untargeted metabolomics/lipidomics of fsh3-delta vs wild type to identify accumulating candidate substrates.
Provenance (the field's own admissions):
Gap: The precise biological role/pathway of FSH3 is not fully resolved. Its single deletion causes no growth defect under standard conditions, and its overexpression phenotypes (PC reduction, NUC1-dependent apoptosis) cannot be read as its native role. A loss-of-function clue does exist: a double mutant lacking both FSH3 and the peroxisomal lipase Lpx1 (but neither single mutant) has reduced peroxisomal fatty-acid beta-oxidation activity, implicating FSH3 in peroxisomal fatty-acid beta-oxidation (GO:0006635; peroxisomal in yeast) redundantly with Lpx1 -- but FSH3's specific molecular step in that pathway is undetermined.
NARROWING BIOLOGY BP_DARK
What is known: FSH3 localizes to the peroxisome, is induced by the DNA-damage-checkpoint effector Crt1/RFX1 and by H2O2, its overexpression reduces phosphatidylcholine and triggers NUC1-dependent apoptosis, and a double deletion of FSH3 with LPX1 shows a significant reduction in beta-oxidation activity relative to control and both single mutants (an overlapping role for Fsh3 with Lpx1). Single-deletion growth is silent, so the specific pathway step and whether the beta-oxidation contribution is direct (an acyl-ester hydrolase step) or indirect remain open.
Significance: The Fsh3/Lpx1 double-mutant beta-oxidation defect provides a candidate pathway (peroxisomal fatty-acid beta-oxidation / lipid mobilization) and explains why single deletions look silent (redundancy with Lpx1); resolving FSH3's specific role there would move it from BP-dark to an annotatable biological process (candidate GO:0006635 fatty acid beta-oxidation), and let the overexpression apoptosis phenotype be interpreted.
What would resolve it: Condition-specific phenotyping of fsh3-delta and fsh3/lpx1 combinations on fatty-acid (oleate) growth and direct beta-oxidation-flux assays, epistasis with FSH1/FSH2 and NUC1, to define FSH3's step in peroxisomal fatty-acid metabolism and justify a GO:0006635 (fatty acid beta-oxidation) annotation.
Provenance (the field's own admissions):
Gap: The degree and direction of functional redundancy among the three paralogous S. cerevisiae serine hydrolases FSH1, FSH2 and FSH3 is unresolved; whether they share substrates or act in overlapping/opposing pathways is unknown.
OPEN BIOLOGY MF_DARK
What is known: All three are members of the Fsh serine-hydrolase family sharing conservation with human OVCA2, and fsh double deletions (fsh1 fsh2, fsh1 fsh3, fsh2 fsh3) grow faster than wild type, indicating some shared or overlapping influence on growth; but no shared molecular substrate or pathway has been established.
Significance: Redundancy among paralogs is the most likely reason single deletions are silent; resolving it is a prerequisite for detecting loss-of-function phenotypes and for assigning family-level function.
What would resolve it: Comparative biochemistry of purified FSH1/FSH2/FSH3 on shared substrate panels and phenotyping of single, double, and triple fsh deletions across stress conditions.
Provenance (the field's own admissions):
Gap: It is unknown whether peroxisomal FSH3 acts directly on a glycerophospholipid substrate (e.g. phosphatidylcholine) or whether the phosphatidylcholine reduction seen on FSH3 overexpression is an indirect consequence of a different activity.
OPEN BIOLOGY MF_DARK
What is known: FSH3 overexpression produces the strongest lipidome change (PC reduction) among a panel of dark peroxisomal proteins, and FSH3 has the catalytic machinery of a serine ester hydrolase; but no in-vitro assay has shown FSH3 hydrolyzing PC or any defined lipid.
Significance: A direct link between FSH3 and glycerophospholipid turnover would give the family a concrete molecular function and connect it to peroxisomal lipid metabolism.
What would resolve it: Direct in-vitro lipase/phospholipase assays on recombinant FSH3 (and S117A) against PC and related glycerophospholipids, with lipidomics of fsh3-delta to test for the reciprocal (accumulation) effect.
Provenance (the field's own admissions):
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