IpaH4.5 is a plasmid-encoded type III secretion system effector of Shigella flexneri and a member of the IpaH/SspH family of bacterial E3 ubiquitin ligases. Its 574-residue sequence has two modules: an N-terminal leucine-rich-repeat solenoid (ten LRRs, residues 63-270) that selects host substrates, and a C-terminal NEL (Novel E3 Ligase) catalytic domain (residues 295-574) whose active-site cysteine, Cys379, forms a thioester with the C-terminal glycine of ubiquitin before transferring it to a substrate lysine. This is a HECT-like, cysteine-dependent mechanism rather than a RING scaffold, and a C379A substitution abolishes activity. The protein is exported by the Mxi-Spa injectisome and acts in the cytoplasm of the infected host cell, where it hijacks the host ubiquitin-proteasome system to remodel innate immune signalling. Characterized substrates are all host cytosolic signalling proteins, and the N-terminal LRR region, which differs between IpaH paralogs, is what makes the choice of substrate paralog-specific. IpaH4.5 polyubiquitinates the NF-kappaB subunit p65 and suppresses NF-kappaB-dependent transcription, so cells and mice infected with an ipaH4.5 deletion mutant mount a stronger pro-inflammatory cytokine response; it attaches K48 chains to the kinase TBK1, driving its proteasomal degradation and preventing IRF3 phosphorylation, nuclear import and activation; and it degrades the 19S proteasome subunit RPN13, which disorganizes the 26S proteasome and reduces MHC class I antigen cross-presentation to CD8+ T cells. Not all of its effects are suppressive: IpaH4.5 also attaches K63 chains to NLRP3, stabilizing the protein without changing its mRNA level and thereby increasing caspase-1 activation, interleukin-1 beta release and pyroptosis in infected macrophages. Together these activities make IpaH4.5 a virulence factor that both blunts the interferon and NF-kappaB arms of innate immunity and reshapes inflammasome output in favour of bacterial colonization.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000209 protein polyubiquitination | IDA PMID:23083102 Shigella flexneri T3SS effector IpaH4.5 modulates the host i... | ACCEPT | Summary: IpaH4.5 is a NEL-domain E3 ligase that builds polyubiquitin chains. The cited paper reports in vitro ligase activity towards both free ubiquitin (i.e. chain elongation) and the NF-kappaB subunit p65. Two independent later papers map the chain linkage directly: K48 chains on TBK1 and K63 chains on NLRP3, the latter with a C379A catalytic-dead control. Reason: CACAO row (ASSIGNED BY = CACAO, IDA, PMID:23083102). The cited paper's in vitro ubiquitination assay with ubiquitin and p65 as acceptors is a direct assay of this gene product and matches the term at the right granularity; nothing here is a downstream phenotype read as a process. The claim is corroborated beyond the single paper by explicit linkage-specific mapping in PMID:26700764 (K48 on TBK1) and PMID:33117724 (K63 on NLRP3, Figure 2B), so it does not depend on panels we cannot see. This is a core molecular activity of the effector. Supporting Evidence: PMID:23083102 In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. PMID:26700764 IpaH4.5 interacts with TBK1 and promotes its K48-linked polyubiquitylation. PMID:33117724 the ubiquitination of NLRP3 was catalyzed by IpaH4.5 mainly at position K63 (Figure 2B) |
| GO:0004842 ubiquitin-protein transferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the NEL domain (IPR029487), which UniProt places at residues 295-574 of P18009 with an active site at Cys379. The activity is real, but the gene also carries the more specific GO:0061630 ubiquitin protein ligase activity, which is what the experimental work demonstrates. Reason: Correct but less specific than the evidence supports. GO:0061630 ubiquitin protein ligase activity is an is_a child of GO:0004842 and is already annotated to this gene from the EC mapping; direct assays show IpaH4.5 transferring ubiquitin onto substrate lysines (p65, TBK1, NLRP3), which is the ligase reaction, not merely transferase activity. Per the refinement rule the general term is modified to the specific descendant the gene already carries rather than kept. Proposed replacements: ubiquitin protein ligase activity Supporting Evidence: file:SHIFL/ipaH4.5/ipaH4.5-uniprot.txt DOMAIN 295..574 PMID:33117724 mutation of the IpaH4.5 E3 ligase motif (C379A mutant) did not upregulate NLRP3 ubiquitination to the same intensity as wildtype IpaH4.5 (Figure 2A) |
| GO:0005576 extracellular region | IDA PMID:11418613 Shigella protein IpaH(9.8) is secreted from bacteria within ... | KEEP AS NON CORE | Summary: Congo-red-induced secretion of IpaH into the culture medium, detected by immunoblot. The paper's title and its nuclear-import experiments concern the paralog IpaH9.8, but the secretion experiments are explicitly framed as a survey of the IpaH proteins, and the UniProt curator cites this paper on the P18009 entry under SUBCELLULAR LOCATION AND SECRETION VIA TYPE III SECRETION SYSTEM. Reason: The claim is sound: IpaH4.5 is a confirmed member of the pWR100 T3SS secretome and the cited paper assays secretion of IpaH proteins as a group, so this is not a wrong-gene annotation and is not removed. It is non-core because the extracellular medium is where a T3SS effector is caught in transit under artificial secretion induction, not where it acts. Every characterized IpaH4.5 substrate (p65, TBK1, RPN13, NLRP3) is a host cytosolic protein, so GO:0030430 host cell cytoplasm is the functionally relevant compartment. Note also that the nuclear localization reported in this paper is specific to IpaH9.8 and must not be propagated to IpaH4.5. Supporting Evidence: PMID:11418613 we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells PMID:11418613 the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location keyword mapping from the "Secreted" annotation on P18009, which rests on the same T3SS secretion data as the IDA row for this term. Reason: Same claim as the IDA row for GO:0005576 and given the same action for consistency. The secreted state is a genuine part of the effector's itinerary, but it is a transit compartment rather than the site of function, which is the host cytosol. Supporting Evidence: PMID:11418613 the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD |
| GO:0006511 ubiquitin-dependent protein catabolic process | IMP PMID:26700764 Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri T... | MODIFY | Summary: IpaH4.5 attaches K48 polyubiquitin chains to TBK1 and the modified kinase is then destroyed by the proteasome, with loss of downstream IRF3 phosphorylation and nuclear translocation. A second, independent paper reports the same route for the proteasome subunit RPN13, degraded via the ubiquitin-proteasome system. Reason: CACAO row (ASSIGNED BY = CACAO, IMP, PMID:26700764). What the paper shows for this gene is right for the term but more specific than the term states: the degradation is explicitly proteasome-dependent, and K48 chains are the canonical proteasomal targeting signal. The claim is about the ligase doing the tagging step of the pathway, so IpaH4.5 does participate in the process rather than merely being required for it. Modified to the is_a descendant GO:0043161, supported twice over by different substrates (TBK1, RPN13). Not a phenotype-as-process error and not a wrong-paper error; only the granularity is off. Proposed replacements: proteasome-mediated ubiquitin-dependent protein catabolic process Supporting Evidence: PMID:26700764 polyubiquitylated TBK1 undergoes proteasome-dependent degradation, which perturbs the phosphorylation, nuclear translocation, and activation of IRF3 PMID:30414351 targets the proteasome regulatory particle non-ATPase 13 (RPN13) and induces its degradation via the ubiquitin-proteasome system (UPS) |
| GO:0016567 protein ubiquitination | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the NEL domain. True of the gene, but the chains IpaH4.5 builds have been characterized as polyubiquitin on specific substrates, and GO:0000209 protein polyubiquitination is already annotated from direct assay. Reason: Correct but less specific than the evidence supports. GO:0000209 is an is_a descendant of GO:0016567 and the gene already carries it with experimental evidence, including explicit K48 and K63 linkage mapping. Per the refinement rule the general term is modified to that descendant. Proposed replacements: protein polyubiquitination Supporting Evidence: PMID:26700764 IpaH4.5 interacts with TBK1 and promotes its K48-linked polyubiquitylation. PMID:33117724 the ubiquitination of NLRP3 was catalyzed by IpaH4.5 mainly at position K63 (Figure 2B) |
| GO:0030430 host cell cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping for a T3SS effector delivered into the host cytoplasm. This is the compartment where all the characterized biochemistry happens: p65, TBK1, RPN13 and NLRP3 are host cytosolic proteins, and the ubiquitination, stabilization and degradation events are all measured in host cell lysates during infection or transfection. Reason: Although the row is an electronic keyword mapping, the underlying claim is experimentally grounded for this gene: ipaH4.5 deletion and complementation (including a catalytically dead C379A complement) change NLRP3 stability, caspase-1 activation and pyroptosis in infected macrophages, which requires the effector to be inside the host cell. No sub-compartment is warranted; in particular the nuclear import reported for the paralog IpaH9.8 is not evidence for IpaH4.5. Accepted as the core location. Supporting Evidence: PMID:33117724 We demonstrate that IpaH4.5 mediates NLRP3 protein stability, caspase-1 activation, IL-1Ξ² production, and cell death in Shigella-infected macrophages. PMID:26700764 TBK1 is a novel target of the IpaH4.5 protein, a Shigella type III effector possessing E3 ubiquitin ligase activity |
| GO:0035821 modulation of process of another organism | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning association rule placing IpaH4.5 in the generic inter-organism modulation branch, on the strength of its being a secreted bacterial effector. True, but two levels above what has been demonstrated. Reason: The term is an effector-generic placeholder. The literature establishes specific descendants: suppression of the host innate immune response through TBK1 degradation and loss of IRF3 activation (GO:0052170, already annotated to this gene), and suppression of the host NF-kappaB cascade through p65 polyubiquitination and loss of NF-kappaB-driven transcription (GO:0085034). Modified to both rather than one, since the gene acts on two distinct host signalling arms. Proposed replacements: symbiont-mediated suppression of host innate immune response symbiont-mediated suppression of host NF-kappaB cascade Supporting Evidence: PMID:26700764 polyubiquitylated TBK1 undergoes proteasome-dependent degradation, which perturbs the phosphorylation, nuclear translocation, and activation of IRF3 PMID:23083102 the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF-ΞΊB signalling pathway |
| GO:0039604 symbiont-mediated suppression of host translation | IDA PMID:23083102 Shigella flexneri T3SS effector IpaH4.5 modulates the host i... | MODIFY | Summary: GO:0039604 is defined as a symbiont inhibiting or disrupting the translation of host mRNA into protein, for example by cleavage of the host mRNAs. The cited paper contains no assay of host protein synthesis. What it reports is that IpaH4.5 binds the NF-kappaB subunit p65, ubiquitinates it in vitro, and suppresses NF-kappaB-driven transcription measured with an NF-kappaB-response-element dual-luciferase reporter, with reduced MIP-2 and TNF-alpha in vivo as the downstream consequence. Reason: CACAO row (ASSIGNED BY = CACAO, IDA, PMID:23083102). The assay behind the row is the NF-kappaB promoter-response-element luciferase reporter plus the in vitro p65 ubiquitination; the gene product does the work of that step (it is the ligase that modifies p65), so the aspect is right and the evidence code fits. The term, however, names the wrong molecular step: suppression of host translation, not of host transcription or signalling. This is a wrong-step substitution within the correct region of the ontology, not a false claim and not a downstream phenotype, so it is modified rather than removed, in keeping with the curator-deference rule (the full text and the GONUTS evidence notes could not be read: gowiki.tamu.edu is offline and no archived snapshot of the PMID page exists). GO:0085034 symbiont-mediated suppression of host NF-kappaB cascade is exactly the mechanism the paper demonstrates and is the proposed replacement. Proposed replacements: symbiont-mediated suppression of host NF-kappaB cascade Supporting Evidence: PMID:23083102 Yeast two-hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF-ΞΊB. PMID:23083102 the transcriptional activity of NF-ΞΊB was shown to be inhibited by IpaH4.5 utilizing a dual-luciferase reporter gene detection system containing NF-ΞΊB promoter response elements PMID:23083102 the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF-ΞΊB signalling pathway |
| GO:0051865 protein autoubiquitination | IDA PMID:23083102 Shigella flexneri T3SS effector IpaH4.5 modulates the host i... | KEEP AS NON CORE | Summary: Self-ubiquitination is a general property of the IpaH/SspH NEL ligases and UniProt records it for P18009 as a PTM. The cited abstract reports in vitro ligase activity towards ubiquitin and p65 but does not mention the enzyme itself as an acceptor, and the figure that would settle it is not accessible. Reason: CACAO row (ASSIGNED BY = CACAO, IDA, PMID:23083102). The underlying claim is credible: an in vitro E1/E2/E3/ubiquitin reaction of the kind the abstract describes is where IpaH autoubiquitination is normally observed, and UniProt independently annotates P18009 as being ubiquitinated in the presence of host E1, E2 and ubiquitin. Because only the abstract is readable and the GONUTS record of the student's evidence could not be retrieved, the row is not removed or second-guessed, per the curator-deference rule. It is marked non-core rather than accepted because autoubiquitination is a self-directed side reaction of the catalytic cycle, not one of the gene's evolved functions; the biologically meaningful activity is the modification of host substrates. Supporting Evidence: PMID:23083102 In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. file:SHIFL/ipaH4.5/ipaH4.5-uniprot.txt Ubiquitinated in the presence of host E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and ubiquitin. |
| GO:0052170 symbiont-mediated suppression of host innate immune response | IMP PMID:26700764 Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri T... | ACCEPT | Summary: IpaH4.5 degrades TBK1, the kinase that activates IRF3, so IRF3 phosphorylation, nuclear translocation and activation are all lost and the host antibacterial response is blunted. The suppression is corroborated on a second arm by the p65/NF-kappaB work (reduced MIP-2 and TNF-alpha in wild-type versus ipaH4.5-deletion infections) and on a third by RPN13 degradation reducing MHC class I cross-presentation. Reason: CACAO row (ASSIGNED BY = CACAO, IMP, PMID:26700764). The paper's mutant-based evidence is directly about this gene, and the term matches what it shows at the right granularity: a symbiont effector suppressing the host innate immune response. The gene product performs the step itself (it is the ligase that tags TBK1), so this is participation and not merely a downstream phenotype. Three independent substrate arms support the same term, which is why no narrower child is substituted. Core biological process. Supporting Evidence: PMID:26700764 Because IRF3 and TBK1 are required for restricting Shigella growth, we propose that the polyubiquitylation and degradation of TBK1 during Shigella infection are new bacterial strategies to modulate the host antibacterial responses. PMID:23083102 the Shigella ΞipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro-inflammatory cytokine levels (MIP-2 and TNF-Ξ±) |
| GO:0061630 ubiquitin protein ligase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.3.2.27 mapping, matching the NEL catalytic domain at residues 295-574 and the Cys379 active site that forms a glycyl thioester with ubiquitin. The activity is experimentally demonstrated for IpaH4.5 itself: a C379A substitution abolishes NLRP3 ubiquitination, NLRP3 stabilization, IL-1 beta induction and pyroptosis. Reason: This is the core molecular function of the protein and it is directly supported, not merely inferred from the EC number: the catalytic cysteine predicted by UniProt is the residue whose mutation (C379A) abolishes ligase output in cells, and substrate-specific polyubiquitination has been shown for p65, TBK1, RPN13 and NLRP3. Accepted at this granularity. Supporting Evidence: PMID:33117724 IL-1Ξ² production was specifically upregulated by WT IpaH4.5 but not the C379A mutant (Figure 4C), demonstrating the importance of IpaH4.5 E3 ligase activity in inflammasome activation. file:SHIFL/ipaH4.5/ipaH4.5-uniprot.txt /note="Glycyl thioester intermediate" |
| GO:0141087 positive regulation of inflammasome-mediated signaling pathway | IDA PMID:33117724 The Shigella Type III Secretion Effector IpaH4.5 Targets NLR... | NEW | Summary: IpaH4.5 binds NLRP3 and attaches K63-linked polyubiquitin chains to it. NLRP3 protein rises without any change in NLRP3 mRNA, so the effect is post-transcriptional stabilization, and caspase-1 activation, IL-1 beta secretion and LDH release all increase. A C379A catalytic-dead IpaH4.5 fails to do any of this, and macrophages infected with an ipaH4.5 deletion strain release less LDH than those infected with wild type or with the complemented strain, while the C379A complement does not restore it. Reason: No row in the existing GOA set covers this arm of IpaH4.5 biology, and it is opposite in sign to the suppression terms: the same ligase blunts NF-kappaB and IRF3 signalling but increases inflammasome output. Participation test: IpaH4.5 itself catalyses the K63 modification that stabilizes NLRP3, so it performs the regulatory step rather than merely being required for the outcome, and the catalytic-dead C379A control separates the two. Comparator test: host E3 ligases that ubiquitinate NLRP3 (for example TRIM31, ARIH2, PELI2) carry inflammasome regulation terms, so an E3 acting on NLRP3 taking such a term follows the established convention rather than inventing a use for it. GO:0141087 is preferred over GO:1900227 positive regulation of NLRP3 inflammasome complex assembly because complex assembly as such was not assayed; what was measured was signalling output (caspase-1, IL-1 beta, pyroptosis). This paper is the only one on this gene with full text available, and all the key panels are visible. Supporting Evidence: PMID:33117724 the ubiquitination of NLRP3 was catalyzed by IpaH4.5 mainly at position K63 (Figure 2B) PMID:33117724 Quantitative RT-PCR showed that NLRP3 mRNA levels were not affected by IpaH4.5 overexpression (Figure 3D), suggesting that IpaH4.5 stabilized NLRP3 protein by posttranscriptional modification. PMID:33117724 Mutation of the IpaH4.5 E3 ligase motif not only abolishes NLRP3 inflammasome signaling but also dramatically reduces the ability of Shigella to induce cell pyroptosis. PMID:33117724 We observed significantly lower LDH release in BMDMs infected with IpaH4.5-deficient Shigella when compared to that of WT and ΞIpaH4.5/IpaH4.5 Shigella (Figure 5B). |
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Download this section (compressed HTML)Q: The CACAO annotation GO:0039604 symbiont-mediated suppression of host translation (IDA, PMID:23083102) is keyed to a paper whose only gene-expression readout is an NF-kappaB-response-element luciferase reporter. Can the CACAO or GOA curators confirm from the full text, or from the GONUTS evidence record, that no host protein-synthesis assay was performed, so that the row can be re-pointed to GO:0085034 symbiont-mediated suppression of host NF-kappaB cascade?
Q: UniProt P18009 carries the alternative name "Probable RING-type E3 ubiquitin transferase ipaH4.5" while the same record annotates a NEL domain and a Cys379 glycyl-thioester active site, and PMID:33117724 shows C379A is catalytically dead. Should the alternative name be corrected to the NEL/HECT-like class, and should the same check be run across the other IpaH and SspH entries?
Q: Does the reported Rab31 GAP activity of IpaH4.5 use the NEL catalytic cysteine, a distinct TBC-like surface, or neither? A C379A variant retaining full GAP activity would establish bifunctionality; one losing it would suggest the GAP readout is downstream of ubiquitination.
Q: What determines the ubiquitin chain linkage IpaH4.5 builds, given that it makes K48 chains on TBK1 but K63 chains on NLRP3 with the same catalytic cysteine? Is linkage set by the host E2 recruited, by the substrate's presentation on the LRR surface, or by the local lysine environment?
Q: Each IpaH paralog has a distinct N-terminal LRR region and a distinct substrate set, while the molecular function term is identical across the family. Would these effector annotations be better served by recording the substrate explicitly, for example as has_input in a GO-CAM, so that the paralog-specific information is not lost?
Experiment: Settle the GO:0039604 question directly by measuring host translation during Shigella infection: polysome profiling or ribosome profiling, plus puromycin or O-propargyl-puromycin incorporation, in cells infected with wild type, an ipaH4.5 deletion strain, and the deletion strain complemented with wild-type or C379A ipaH4.5. A negative result would confirm that IpaH4.5 suppresses NF-kappaB-driven transcription rather than translation; a positive one would rehabilitate the existing annotation.
Type: translational profiling in infection
Experiment: Resolve the opposite-signed arms by single-cell time-course analysis of infected macrophages and epithelial cells: track p65, TBK1, RPN13 and NLRP3 levels, IRF3 and NF-kappaB reporter output, and caspase-1 activation in the same cells over the course of invasion, comparing wild type with the ipaH4.5 deletion and C379A complement strains, and titrating effector dose with an inducible promoter.
Type: single-cell infection time course
Experiment: Test the bifunctionality claim with purified protein: assay recombinant IpaH4.5 and IpaH4.5 C379A side by side for ubiquitin ligase activity (E1/E2/ubiquitin with NLRP3 or TBK1 as acceptor) and for GTP hydrolysis stimulation on Rab31 using a single-turnover GAP assay, and attempt to localize any GAP activity to a defined fragment by limited proteolysis or deletion mapping. Solve a structure of the putative GAP region if activity is confirmed.
Type: enzymology and structural biology
Experiment: Map the determinants of paralog-specific substrate choice by swapping the N-terminal LRR regions between IpaH4.5 and IpaH7.8 or IpaH9.8 (the boundary is well defined: residues 1-284 of IpaH4.5) and assaying each chimera for ubiquitination of p65, TBK1, RPN13, NLRP3, glomulin and NEMO. This would test whether the LRR solenoid alone encodes the substrate set and would provide the evidence needed to annotate substrates per paralog rather than per family.
Type: chimeric protein substrate mapping
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: It is not known how IpaH4.5 produces opposite-signed outcomes on host innate immunity in the same infection: it suppresses NF-kappaB and IRF3 signalling by destroying p65 and TBK1, yet stabilizes NLRP3 with K63 chains and thereby increases caspase-1 activation and pyroptosis. Whether these arms are separated by cell type, by time after invasion, by effector dose, or by competition between substrates for the LRR surface is undetermined, and so is what decides the chain linkage (K48 versus K63) on a given substrate.
BIOLOGY BP_DARK
What is known: Firmly established: the substrates (p65, TBK1, RPN13, NLRP3), the linkage on TBK1 (K48) and on NLRP3 (K63), the requirement for Cys379 in every case, and that each arm is demonstrable on its own with deletion and complementation strains.
Significance: Which way IpaH4.5 shifts the balance decides whether it is a net anti-inflammatory or pro-inflammatory effector, and bears on why Shigella infection is simultaneously immunosuppressive and intensely inflammatory.
Gap: Whether IpaH4.5 has a second catalytic activity as a Rab31 GTPase-activating protein is unresolved. One report describes TBC-like dual-finger motifs and potent RabGAP activity towards Rab31 with consequences for CD-MPR trafficking and lysosomal function, but no TBC-like module is detectable in P18009 by UniProt, Pfam or PROSITE, and the finding has not been reproduced independently.
BIOLOGY MF_DARK
What is known: Firmly established: the only recognized catalytic module in IpaH4.5 is the NEL domain at residues 295-574 with its Cys379 active site, and all reproduced activities of the protein are ubiquitin ligase activities.
Significance: A genuine second activity would make IpaH4.5 a bifunctional effector and would need a separate molecular function annotation; a spurious one would otherwise propagate across the IpaH family by similarity.
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