NLRP2B (pyrin-only protein 4, POP4; formerly the pseudogene NLRP2P/CLRX.1/NOD24) is a 45-amino-acid protein encoded on chromosome Xp11.21 by a processed retrocopy of an NLRP2/NLRP7-like gene that is restricted to Old World primates. Its open reading frame corresponds to the first 45 codons of NLRP2 and is predicted to fold into only the first two alpha-helices of a pyrin domain (PYD); it is about 80% identical to the N-terminal region of the pyrin-only protein POP2 (PYDC2). The ORF is under purifying selection across hominids, and the mRNA is detected in many tissues and in leukocytes and is induced by lipopolysaccharide in monocytic cells. The endogenous protein has not been detected directly. When expressed ectopically, POP4 is diffusely cytoplasmic and partly nuclear, and moves into the nucleus on TNF stimulation. It inhibits NF-kappaB RelA/p65 transactivation at or downstream of RelA/p65 by reducing RelA/p65 phosphorylation at Ser-536, and thereby lowers TNF and IL-6 release from macrophages stimulated through TLR2 or TLR4; siRNA knockdown in human THP-1 cells increases the release of these cytokines. Unlike POP2, it lacks the acidic residues and helix 4 needed to block ASC-dependent inflammasomes and does not inhibit NLRP3 inflammasome-driven IL-1beta release. In HeLa cells under cycloheximide treatment it slows S/G2 progression and, with TNF, increases apoptosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IDA PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: FLAG-tagged POP4 expressed in HeLa cells is partly nuclear at rest and becomes predominantly nuclear in most transfected cells after TNF stimulation. Reason: Direct immunofluorescence localisation, and the nuclear pool fits the proposed nuclear site of action (inhibition of the isolated RelA/p65 TAD1 in a Gal4 assay). The caveat is that this was an overexpressed tagged protein in a cell line that does not express endogenous NLRP2B; no endogenous protein localisation exists. Supporting Evidence: PMID:24871464 Further, TNFΞ± treatment also induces the nuclear translocation of POP4 in the majority (80%) of transfected cells (Fig. 4D). PMID:24871464 Although cytoplasmic localization was more predominant, some cells with nuclear concentration of POP4 were observed (Fig. 4A). |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of the UniProt subcellular location, which is itself taken from PMID:24871464. Reason: Same evidence as the IDA row above; consistent and correct. |
| GO:0005737 cytoplasm | IDA PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: Ectopically expressed FLAG-POP4 is predominantly cytoplasmic in untreated HeLa cells. Reason: Direct immunofluorescence localisation; same overexpression caveat as the nucleus annotation. Supporting Evidence: PMID:24871464 Although cytoplasmic localization was more predominant, some cells with nuclear concentration of POP4 were observed (Fig. 4A). |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of the UniProt subcellular location, derived from PMID:24871464. Reason: Same evidence as the IDA row above; consistent and correct. |
| GO:0010804 negative regulation of tumor necrosis factor-mediated signaling pathway | IMP PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: POP4 reduces TNF-induced NF-kappaB reporter activity and TNF-induced RelA/p65 Ser-536 phosphorylation. Reason: The paper (full text read) shows that POP4 suppresses NF-kappaB output in response to TNF and acts at or downstream of RelA/p65, without blocking the NF-kappaB-independent SV40 promoter. The evidence is overexpression (gain of function) rather than loss of function, so the code is generous, but the claim itself is supported. This is one of the two read-outs of the core function (dampening of NF-kappaB-dependent inflammatory signalling). Supporting Evidence: PMID:24871464 Similar to POP1 and POP2, POP4 significantly reduced NF-ΞΊB promoter/reporter activity in response to TNFΞ± (Fig. 3B). PMID:24871464 Together these results strongly suggest that POP4 limits NF-ΞΊB phosphorylation of S536 in these cells, thereby reducing transactivation by the p65 TAD1. |
| GO:0032691 negative regulation of interleukin-1 beta production | IMP NOT PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: NOT annotation. POP4 did not reduce IL-1beta release from cells with a reconstituted NLRP3 inflammasome or from ASC-overexpressing cells. Reason: The negative result is directly reported and has a structural explanation (POP4 lacks pyrin-domain helix 4 and the acidic residues at positions 6 and 16 that POP2 needs to block ASC-dependent inflammasomes). The NOT row is useful because it separates POP4 from its closest paralog POP2, which does carry this term. The scope is the reconstitution assay; inhibition of other inflammasomes was not tested. Supporting Evidence: PMID:24871464 Indeed, POP4 failed to prevent release of IL-1Ξ² from cells expressing a reconstituted NLRP3 inflammasome (Fig. 3D). PMID:24871464 Even with increasing concentrations, POP4 was unable to block ASC-dependent IL-1Ξ² release (Fig. 3E). |
| GO:0033137 negative regulation of peptidyl-serine phosphorylation | IMP PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: POP4-expressing J774A.1 clones show dose-dependent reduction of nuclear RelA/p65 phosphorylated at Ser-536 after TNF or LPS. Reason: This is the mechanistic read-out reported for POP4 and is well supported by graded clones. The kinase or phosphatase that POP4 affects is unknown, so the annotation records an effect on a phosphorylation event rather than a molecular activity; it should not be read as POP4 being a kinase inhibitor. Supporting Evidence: PMID:24871464 Nevertheless, POP4 reduces the amount of S536-phosphorylated p65 suggesting an impact of POP4 on a kinase or phosphatase targeting S536 |
| GO:0034122 negative regulation of toll-like receptor signaling pathway | IMP PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: POP4 overexpression reduces TNF and IL-6 release after TLR2 or TLR4 stimulation of macrophages, and siRNA knockdown in human THP-1 cells increases LPS-induced TNF and IL-6. Reason: The best-supported function of NLRP2B, and the only one with both gain- and loss-of-function evidence, the latter in human myeloid cells where the gene is expressed and LPS-inducible. Core function. Supporting Evidence: PMID:24871464 However, in both individual and polyclonal populations of stable-transfected POP4-expressing J774A.1 cells, POP4 reduced IL-6 and TNFΞ± release relative to vector-only controls in response to LPS (Fig. 7A) and Pam3CysK4 (Fig. 7B). PMID:24871464 In cells with reduced POP4 expression (250 ng siRNA), there was a statistically significant increase in IL-6 and TNFΞ± cytokine release as compared to control cells (Fig. 7E). |
| GO:0043065 positive regulation of apoptotic process | IMP PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | KEEP AS NON CORE | Summary: Overexpressed POP4 increased hypodiploid (apoptotic) HeLa nuclei, but only under combined cycloheximide and TNF treatment. Reason: A single, condition-dependent overexpression result in a cell line that does not express NLRP2B. The authors present it as a likely consequence of RelA/p65 inhibition, which would make it an indirect, downstream effect, and note that POP2 does not reproduce it. Retained because the curator recorded what was observed, but it is not part of the core function. Supporting Evidence: PMID:24871464 Further, apoptosis (as indicated by hypodiploid nuclei) was significantly increased by POP4, but only in the presence of both CHX and TNFΞ± (Fig. 6D). PMID:24871464 These data suggest that under appropriate conditions POP4 might restrict cell cycle progression and promote apoptotic cell death, features consistent with its ability to inhibit RelA/p65. |
| GO:0045786 negative regulation of cell cycle | IMP PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | KEEP AS NON CORE | Summary: In cycloheximide-treated HeLa cells, overexpressed POP4 reduced proliferation and disrupted progression through S/G2. Reason: Same limitations as the apoptosis row - one overexpression experiment, only under cycloheximide, in a non-expressing cell line, and probably indirect. Kept as non-core. Supporting Evidence: PMID:24871464 Cell cycle analysis revealed that in CHX-treated cells POP4 disrupts progression through the S/G2 phase (Fig. 6B&C). |
| GO:1900226 negative regulation of NLRP3 inflammasome complex assembly | IMP NOT PMID:24871464 The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional nega... | ACCEPT | Summary: NOT annotation. POP4 does not inhibit NLRP3 inflammasome function in a reconstitution assay. Reason: The paper concludes that POP4 is not an NLRP3 inflammasome inhibitor. The assay measured IL-1beta release rather than complex assembly itself, so the assembly term is slightly more specific than the read-out, but the negative claim follows (no inhibition of output implies no effective inhibition of assembly) and it distinguishes POP4 from POP2, which carries the positive annotation. Supporting Evidence: PMID:24871464 Therefore, POP4 appears not to be an inhibitor of the NLRP3 inflammasome and is unlikely to inhibit other ASC-dependent inflammasomes |
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Download this section (compressed HTML)Q: Is the 45-residue POP4 protein made from the endogenous NLRP2B locus? All functional data come from ectopic expression of the ORF, the tagged protein was not detectable by western blot, and HPA reports no protein. Detection of the endogenous peptide (for example by targeted mass spectrometry in LPS-stimulated monocytes) would confirm the product that the annotations describe.
Q: What does POP4 bind to reduce RelA/p65 Ser-536 phosphorylation - RelA/p65 itself, an IKK or other Ser-536 kinase, or a phosphatase? Only an answer to this would allow a molecular function term to be assigned.
Q: Should NLRP2B also carry a general NF-kappaB signalling regulation term (for example GO:0043124 negative regulation of canonical NF-kappaB signal transduction), or are the stimulus-specific TNF and TLR terms the intended representation?
Experiment: Ribosome profiling and targeted mass spectrometry (or an endogenous epitope knock-in) in LPS-stimulated THP-1 cells or primary monocytes, where NLRP2B mRNA is induced.
Hypothesis: The endogenous NLRP2B ORF is translated in human myeloid cells.
Type: proteomics / ribosome profiling
Experiment: Affinity purification-mass spectrometry of tagged POP4 at near-endogenous levels, followed by in vitro kinase assays with IKKbeta/TBK1 and RelA TAD1 substrate in the presence of recombinant POP4.
Hypothesis: POP4 limits RelA/p65 Ser-536 phosphorylation by binding a specific kinase or RelA/p65 itself.
Type: interaction proteomics and in vitro kinase assay
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