cif (PA2934) encodes Cif, "CFTR inhibitory factor", a secreted alpha/beta-hydrolase-fold epoxide hydrolase of Pseudomonas aeruginosa and a virulence factor of the airway. The 319-residue precursor carries a signal peptide and the mature protein is released from the cell, largely in outer-membrane vesicles, through which it is delivered into human airway epithelial cells. Cif is a homodimer with the canonical epoxide hydrolase catalytic triad Asp129-His297-Glu153, in which Asp129 is the nucleophile that opens the epoxide ring; purified Cif hydrolyses epibromohydrin and cis-stilbene oxide, with a narrow substrate range, and lacks the haloacetate and haloalkane dehalogenase activities of the two neighbouring enzyme classes that share the same Asp-nucleophile fold. Its physiological epoxide substrate is unknown. In host cells Cif reduces the amount of CFTR chloride channel in the apical membrane and so reduces CFTR-mediated chloride secretion: it stabilises a G3BP1-mediated inhibition of the deubiquitinase USP10, which leaves endocytosed CFTR ubiquitinated and diverts it from recycling endosomes to lysosomal degradation. The catalytic histidine is required for this host effect, implying that turnover of an endogenous epoxide substrate, rather than mere binding, drives it. Reduced chloride secretion impairs mucociliary clearance, and cif is expressed by P. aeruginosa in the cystic fibrosis lung.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004301 epoxide hydrolase activity | IDA PMID:20118260 Crystal structure of the cystic fibrosis transmembrane condu... | ACCEPT | Summary: PseudoCAP row from the Cif crystal structure and enzymology paper, and the sole seed of the PTN002458043 epoxide hydrolase IBD (38 leaf IBAs, 15 taxa). It also contributes one of 13 seeds behind the broad PTN000863140 hydrolase activity IBD (791 leaf IBAs). Reason: A sound seed, and the core molecular function. The evidence is direct enzymology on purified recombinant protein rather than a phenotype: a 1.8 A structure resolving the complete epoxide hydrolase triad Asp129-His297-Glu153, measured hydrolysis of epibromohydrin (0.25 U/mg) and of cis-stilbene oxide, and tested absence of the haloacetate and haloalkane dehalogenase activities of the two other Asp-nucleophile alpha/beta hydrolase classes, which is what makes the epoxide hydrolase assignment a result rather than a family default. The activity is independently reproduced with purified protein in PMID:17502391. Supporting Evidence: PMID:20118260 detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis PMID:20118260 We measured a specific activity of EBH hydrolysis of 0.25 U/mg (1 U = 1 ΞΌmol substrate hydrolyzed per minute) for Cif-WT. PMID:20118260 Cif has a catalytic triad consisting of residues Asp129, His297, and Glu153 PMID:20118260 Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase |
| GO:0016787 hydrolase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA from PTN000863140, a broad alpha/beta hydrolase node seeded by 13 genes including characterised mammalian epoxide hydrolases (e.g. human EPHX2 P34913), and propagating to 791 leaves across 88 taxa. This gene is among the seeds, which is why it appears in its own WITH/FROM; that marks experimental grounding on the target, not circularity. Reason: The node annotation is not disputed: for a clade of this breadth the generic term is the right choice, because the Asp-nucleophile alpha/beta hydrolases contain epoxide hydrolases, haloacetate dehalogenases and haloalkane dehalogenases, and Cif itself shows that membership does not predict which (it has epoxide hydrolase activity and demonstrably lacks both dehalogenase activities). For this gene, however, the propagated term is less specific than the evidence supports: epoxide hydrolase activity (GO:0004301) is an is_a descendant that the gene already carries by IDA from direct enzymology on the purified protein, so the row is refined to it. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000863140 Β· PTN000863140 SUPPORTS TRANSFER Broad alpha/beta hydrolase node with 13 seeds, among them well characterised epoxide hydrolases from mouse, rat, human (EPHX2, P34913), zebrafish, C. elegans and M. tuberculosis (P9WMR9), propagating to 791 leaves in 88 taxa. The generic term suits that breadth and substrate diversity; a specific epoxide hydrolase IBD at this depth would over-annotate. No defect. UniProtKB:Q9HZR3 Β· PA2934 cif SUPPORTS TRANSFER The target's own PseudoCAP IDA row (PMID:20118260, purified protein, structure plus turnover of two epoxides) is one of the seeds, so it is expected in the WITH/FROM list. It is also what allows the row to be refined on this gene. Proposed replacements: epoxide hydrolase activity Supporting Evidence: PMID:20118260 detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis PMID:20118260 Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase |
| GO:2001226 negative regulation of chloride transport | IDA PMID:16236828 Pseudomonas aeruginosa inhibits endocytic recycling of CFTR ... | ACCEPT | Summary: PseudoCAP row for Cif's host effect, citing Swiatecka-Urban et al. 2006. That paper's abstract reports the activity of an unfractionated cell-free filtrate and predates the identification of the responsible protein (full text not read); purified Cif was shown to be necessary and sufficient for the same effect in PMID:17502391. Reason: The claim is correct for this gene and is the reason it is named Cif: purified recombinant Cif reduces apical-membrane CFTR and CFTR-mediated chloride secretion, and the mechanism has since been resolved to diversion of endocytosed CFTR from recycling endosomes to lysosomes. Cif itself performs the step that reduces the channel's abundance, so involved_in a negative regulation of chloride transport is a claim about this protein's own activity, not a downstream consequence. Retained despite the reference problem, which is recorded as a source defect: on its abstract the 2006 paper used a filtrate, so it looks like the wrong citation for an IDA on PA2934, which is not a reason to doubt the annotation. Supporting Evidence: PMID:17502391 sufficient to mediate the reduction in apical membrane expression of CFTR and a PMID:17502391 concomitant reduction in CFTR-mediated Cl(-) ion secretion. Cif demonstrates PMID:21455491 Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by |
| GO:1990215 symbiont-mediated perturbation of host vesicle-mediated transport | IDA PMID:21455491 A Pseudomonas aeruginosa toxin that hijacks the host ubiquit... | NEW | Summary: Proposed annotation. Cif delivered in outer-membrane vesicles redirects endocytosed host CFTR away from recycling endosomes into the lysosomal pathway, by stabilising G3BP1-mediated inhibition of the deubiquitinase USP10. Reason: This is the mechanism by which Cif acts, and the step it performs is a perturbation of host vesicle-mediated transport: the protein is delivered into the host cell and acts there on the trafficking machinery, with the trafficking change (recycling to lysosome) demonstrated directly for Cif-containing vesicles and absent for vesicles from a cif mutant. It is not an indirect or pleiotropic effect, and it is not entailed by the chloride-transport row, which names the outcome rather than the subverted host process. Both rows describe the same single activity from different sides, which is why only this one is proposed rather than a set of trafficking terms. Supporting Evidence: PMID:21455491 Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by PMID:21455491 stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), PMID:21455491 Cif redirects CFTR from recycling endosomes to the lysosomal, degradative pathway |
| GO:0005576 extracellular region | IDA PMID:17502391 The Pseudomonas aeruginosa secreted protein PA2934 decreases... | NEW | Summary: Proposed annotation. Cif is a secreted protein, recovered from culture supernatant and from outer-membrane vesicles, consistent with the SignalP signal peptide 1-24 in the UniProt entry. Reason: Where Cif acts is essential to understanding it, and the GOA record carries no cellular component row at all. Secretion was shown directly in the paper that identified the protein, and the mature chain begins after a predicted signal peptide. The root extracellular term is used rather than a vesicle term because the association is with bacterial outer-membrane vesicles, which the available component terms do not capture well. Supporting Evidence: PMID:17502391 is a secreted protein and is found associated with outer membrane-derived file:PSEAE/cif/cif-uniprot.txt SIGNAL 1..24 |
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Download this section (compressed HTML)Q: Which endogenous epoxide does Cif hydrolyse in the host cell, and is its diol product (or the loss of the parent epoxide) what inhibits USP10 through G3BP1?
Q: Does Cif serve any function in P. aeruginosa's own physiology, such as detoxification of environmental or host-derived epoxides, in hosts and niches where no CFTR is present?
Q: Is GO:1990215 (symbiont-mediated perturbation of host vesicle-mediated transport) plus GO:2001226 the right pair for a secreted effector of this kind, or should the chloride-transport effect be expressed only through a host-perturbation term?
Experiment: Lipidomic comparison of airway epithelial cells exposed to Cif-containing versus cif-mutant outer-membrane vesicles, and of purified Cif incubated with host lipid extracts, looking for epoxide substrates consumed and diols formed; then test whether the candidate diol reproduces the USP10/G3BP1 effect on its own.
Experiment: Screen a panel of epoxides (including epoxyeicosatrienoic acids and other arachidonate-derived epoxides) against purified Cif and the catalytically dead D129N and H297A variants to define the substrate range quantitatively, in parallel with the apical CFTR assay, to establish whether host effect and catalytic preference track together.
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The endogenous epoxide substrate of Cif is unidentified, so the chemistry that links its catalytic activity to the inhibition of host USP10 is unknown.
BIOLOGY MF_DARK
What is known: Firmly established: Cif is an epoxide hydrolase with a complete, structurally resolved triad, turns over model epoxides, and needs its catalytic histidine for the host CFTR effect, which proceeds through G3BP1-dependent inhibition of USP10 and lysosomal routing of CFTR. What is missing is the natural substrate and whether the active species is the consumed epoxide or the diol product.
Significance: Without the substrate, neither the enzyme's role in P. aeruginosa's own metabolism nor the step at which it intersects host signalling can be modelled, and inhibitor design has no target reaction.
What would resolve it: Identify epoxides consumed and diols produced in host cells exposed to Cif, and test whether a candidate product reconstitutes the USP10 effect without the enzyme.
Provenance (the field's own admissions):
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