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The Journal of Immunology, 2007, 178: 3837-3845.
Copyright © 2007 by The American Association of Immunologists, Inc.

Pyrin-Only Protein 2 Modulates NF-{kappa}B and Disrupts ASC:CLR Interactions

Felipe Bedoya*, Laurel L. Sandler* and Jonathan A. Harton1,*,{dagger}

* Department of Molecular Medicine, University of South Florida College of Medicine, and {dagger} Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NF-{kappa}B is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule genes and is dysregulated in many cancers, neurodegenerative disorders, and inflammatory diseases. Proteins with pyrin and/or caspase recruitment domains have roles in apoptosis, innate immunity, and inflammation. Many pyrin domain (PYD) proteins modulate NF-{kappa}B activity as well as participate in assembling both the perinuclear "apoptotic speck" and the pro-IL1beta/IL-18-converting inflammasome complex. "Pyrin-only" proteins (POP) are attractive as negative regulators of PYD-mediated functions and one such protein, POP1, has been reported. We report the identification and initial characterization of a second POP. POP2 is a 294 nt single exon gene located on human chromosome 3 encoding a 97-aa protein with sequence and predicted structural similarity to other PYDs. Highly similar to PYDs in CATERPILLER (CLR, NLR, NALP) family proteins, POP2 is less like the prototypic pyrin and ASC PYDs. POP2 is expressed principally in peripheral blood leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-{alpha}-stimulated and p65 (RelA)-induced NF-{kappa}B-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-{kappa}B nuclear import or distribution. While colocalizing with ASC in perinuclear specks, POP2 also inhibits the formation of specks by the CLR protein CIAS1/NALP3. Together, these observations demonstrate that POP2 is a negative regulator of NF-{kappa}B activity that may influence the assembly of PYD-dependent complexes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of NF-{kappa}B regulates numerous cellular processes including cytokine and adhesion molecule expression, cell cycle regulation, and cell growth. Accordingly, constitutive or excessive NF-{kappa}B activity is observed in inflammatory diseases, autoimmunity, and cancer (1, 2). Proteins containing the death domain (DD)2 fold (DDF) are associated with NF-{kappa}B signaling and apoptosis induction. Four related protein-protein interaction domains comprise the DDF superfamily: the DD; the death effector domain; the caspase recruitment domain (CARD); and the pyrin domain (PYD). These domains share a similar tertiary structure, a five to six {alpha}-helical bundle that likely function as homotypic interaction modules. DDF proteins act as adaptors, recruiting signaling proteins into complexes domains, ultimately contributing to apoptosis, innate immune responses, and cancer development (1, 2, 3, 4, 5).

Pyrin motifs are generally found at the N terminus of multidomain proteins. Pyrin (marenostrin), the founding family member, is associated with familial Mediterranean fever, an autosomal recessive disease characterized by sporadic attacks of fever and inflammation with intense abdominal, joint, and chest pain (6, 7). Pyrin also contains a B-box zinc finger and a SPRY domain functioning as adaptor and ligand binding units, respectively (8). IFN inducible (IFI) genes, coding for the HIN-200 family of hemopoietic nuclear proteins, contain one or two copies of a 200-aa protein-protein interaction domain, are preceded by a PYD, and are likely involved in cell proliferation and differentiation (9, 10). The PYD of apoptosis speck protein containing a CARD, ASC (TMS1/PyCARD), is followed by a CARD domain. ASC associates with PYD- or CARD-containing proteins through homotypic interactions via both domains (11, 12). In proapoptotic cells, ASC assembles in large multimeric perinuclear complexes or "specks" by interacting with PYD-containing proteins. The physiological significance of these interactions and the speck structure itself remains unclear. ASC also participates in forming the inflammasome complex that processes pro-IL-1beta into the mature form (13). Two thirds of the recently discovered CATERPILLER (CLR) family of intracellular pathogen receptors encode a PYD followed by a nucleotide binding domain and leucine-rich repeats domain (14, 15). CLR proteins mediate innate immune responses to certain bacterial products, are involved in autoinflammatory diseases, and may promote apoptosis (15). Some PYD-containing proteins of the CLR family induce NF-{kappa}B activation in conjunction with ASC, including PYPAF5, Monarch, and CIAS1/NALP3 (4, 16, 17). Not surprisingly, mutations in CIAS1 associated with the inflammatory syndromes familial cold urticaria, Muckle-Wells, and chronic infantile neurologic cutaneous articular syndrome, show increased capacity for ASC-dependent NF-{kappa}B activation (18, 19, 20, 21). Other CLRs, such as PAN2/PYPAF4 mediate NF-{kappa}B suppression (22). Although the nucleotide binding domain/leucine-rich repeats domain of CIAS1 inhibits NF-{kappa}B nuclear import, the PYD of PAN2/PYPAF4 inhibits NF-{kappa}B, a function likely mediated through the I{kappa}B kinase (IKK) complex (22, 23).

The NF-{kappa}B family of transcription factors is comprised of five members in mammals: p65 (Rel A), Rel B, c-Rel, p50/p105, and p52/p100, existing as homo- or heterodimers bound to the I{kappa}B inhibitory complex in the cytosol. Upon induction by proinflammatory stimuli such as TNF-{alpha} or LPS, I{kappa}B{alpha} is phosphorylated by the IKK complex, ubiquitinated, and degraded by the 26S proteasome. This process unmasks NF-{kappa}B’s nuclear localization sequence, leading to its translocation into the nucleus. Binding of NF-{kappa}B to its cognate DNA response elements induces the transcription of a host of cytokines and growth factors (e.g., IL-2, IL-8, IFN-beta, M-CSF, G-CSF, vascular endothelial growth factor), as well as various transcription factors and signaling regulators (e.g., I{kappa}B{alpha} and IFN regulatory factor-1 and -2) (2, 24). The pivotal role of NF-{kappa}B in biological processes modulating the immune response suggests that localization and subsequent activation must be rigorously controlled. Dysregulation of these events contributes to aberrant gene expression associated with numerous human diseases including cancer, neurodegenerative disorders, arthritis, and chronic inflammation (1, 2).

Solitary PYDs may disrupt PYD interactions, blocking the formation of speck and/or inflammasome complexes, thus interfering with downstream effects. Proteins of this type have recently been identified in the human genome and in the genomes of pox viruses (25, 26). Pyrin-only protein (POP)1, closely related to the PYD of ASC, is predominantly expressed in immune tissues, where it appears to inhibit NF-{kappa}B- and surprisingly enhances caspase-1-activation (27). Other genes encoding POPs have been found in the genomes of Capripoxviridae, Leporipoxviridae, Suipoxviridae, and Yatapoxviridae (28, 29, 30). Recently, Johnston et al. (31) reported that M13L-PYD, a POP from myxoma virus, inhibits both NF-{kappa}B activity and caspase-1-dependent IL-1beta production, and M13L deletion was sufficient to inhibit virus replication in vivo. Therefore, POPs can also participate as suppressors of host immunity. Modulating NF-{kappa}B signaling pathways and altering PYD-protein interactions are potential mechanisms for such suppression.

Data from the sequence of the genomes of humans and other species has resulted in the identification of novel genes and the emergence of new gene families. The recently described CLR family encompasses PYD-proteins with roles in both innate and acquired immunity. Because of our interest in this gene family, we sought to identify new PYD-encoding genes. We report the identification of a novel human POP comprised of 97 aas that we have named POP2. POP2 is encoded by a single exon gene on chromosome 3 and is expressed primarily in peripheral blood leukocytes. We demonstrate that POP2 inhibits NF-{kappa}B (RelA) activated by TNF-{alpha} or transfection of p65. NF-{kappa}B inhibition by POP2 is accompanied by a decrease in nuclear import and altered nuclear distribution of p65. In addition, POP2 associates with ASC in perinuclear speck structures, but inhibits specks composed of CIAS1/NALP3 and ASC. Collectively, these results imply an important role for POP2 as a negative regulator of the NF-{kappa}B signaling pathway and suggest a regulatory mechanism for CLR-dependent innate immune and inflammatory responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bioinformatics

TBLASTN searches of the human genome were performed at National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/genome/guide/human/) using the N-terminal-100-aa sequence of PYPAF2 (pyrin-containing Apaf-1-like protein 2) as a query. Multiple alignments of different PYDs and phylogenetic analysis were done with CLUSTALW (www.ebi.ac.uk/clustalw/) and MEGA2 (Molecular Evolutionary Genetic Analysis; www.megasoftware.net/). Tertiary structure prediction using ASC and NALP1 PYDs as a template was completed using the London Imperial College of Science, Technology and Medicine web-server, 3D-PSSM (www.sbg.bio.ic.ac.uk/~3dpssm/).

Reagents and Abs

Recombinant human TNF-{alpha} and goat anti-mouse IgG1-FITC were purchased from BD Pharmingen. Mouse-anti-FLAG M5 mAb and goat anti-mouse HRP-conjugated IgG1 Ab were purchased from Sigma-Aldrich. Mouse-anti-myc tag IgG1 mAb was purchased from Upstate Biotechnology, rabbit p65 polyclonal IgG was obtained from Santa Cruz Biotechnology, and Alexa Fluor 594 goat anti-rabbit IgG (H+L) was obtained from Molecular Probes.

Cloning and RT-PCR

A cDNA encoding POP2 was amplified from total RNA from the B cell line Ramos by RT-PCR using the reverse transcribed One-Step Kit (Qiagen) with the primers (forward 5'-aaccgcggatggcatcttctgcagag-3' and reverse 5'-aaaagcttatggcatcttctgcagag-3') designed to obtain POP2 cDNA flanked by HindIII and XhoI restrictions sites. Subsequently, POP2 was cloned into the pCDNA 3.1 vector containing an N-terminal Flag or Myc epitope. For POP2 mRNA detection, total RNA was isolated from Daudi, Ramos (B cell lymphomas), Jurkat (human T cell leukemia), THP-1 (human monocytic cell line), and K562 (human erythroid cell line) using the RNeasy RNA Isolation Kit (Qiagen) and reverse transcribed and amplified with the One-Step RT-PCR Kit (Qiagen). The same procedure was performed on total RNA from multiple human tissues (MTCC panel I) and human blood fractions (MTC panel), both obtained from BD Biosciences. Primers for GAPDH were used as a control.

Cell culture, transfection, and luciferase assays

HEK 293, HeLa, and COS-7 cells (American Type Culture Collection) were cultured in either DMEM or RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 5% L-glutamine, and 0.1% penicillin/streptomycin. Cells were passed every 3–4 days. Cell number and viability was determined by trypan blue exclusion. For transient transfection assays, cells were plated onto 6-well plates (2 x 105 cells per well) and incubated at 37°C with 5% CO2 and 95% humidity for 16 h. Transfection was conducted with FLAG-POP2 (different quantities), 100 ng of pNF-{kappa}B-Luc (a 3xNF-{kappa}B-driven firefly luciferase reporter), and either 100 ng of NF-{kappa}B-p65 using FuGene6 (3 µl:1 µg of DNA; Roche), or stimulated with varying amounts of TNF-{alpha} 24 h posttransfection. The total amount of DNA in each transfection was kept constant (1100 ng/well) by the addition of empty vector (pcDNA3). Cell lysates were prepared using 200 µl of lysis buffer (1 M Tris (pH 7.4), 1 M NaCl, 10 mM EDTA, and 1% Triton X-100), and luciferase was quantitated according to standard protocol on a VICTOR Light Luminescence Counter (Wallac).

Western blotting

Western blotting for Flag- and Myc-tagged proteins was performed essentially as described (32). Following transfection and lysis, equal amounts of protein were separated on a SDS-PAGE (15% polyacrylamide) gel, for 40 min at 200 volts, transferred to nitrocellulose (0.2 µm) for 1 h at 110 volts, and immunoblotted with either {alpha}-FLAG Ab (M5; Sigma-Aldrich; 1/5,000) or {alpha}-myc Ab (1/7,500). Bound M5 or {alpha}-myc was detected with goat anti-mouse IgG1-HRP (Southern Biotechnology Associates; 1/7,500) and visualized on Autoradiography film (Midwest Scientific) using SuperSignal West Pico HRP detection reagents (Pierce Biotechnology).

Immunofluorescence microscopy

COS-7 and HeLa cells were cultured overnight in two-chamber slides at a density of 8 x 104 cells per chamber. Cells were transiently transfected with 1 µg of pcCDNA3 as negative control and either 1 µg of GFP-POP2, 1 µg of FLAG-CIITA, or 1 µg of Myc-ASC as positive controls. Either 1 µg of FLAG-POP2, 1 µg of Myc-POP2, 1 µg of NF-{kappa}B-p65, or 1 µg of Myc-ASC was used. After 18 h, cells were fixed with a 3:2 acetone:PBS solution, washed with PBS (1% BSA), and blocked with PBS (1% BSA, 10% NGS). Cells then were incubated for 1 h at room temperature with mouse anti-FLAG or anti-Myc Ab or rabbit anti-p65 polyclonal IgG, washed with PBS (1% BSA), and incubated (1 h, room temperature) with either goat anti-mouse IgG1-FITC, goat anti-mouse IgG1-PE, or goat anti-rabbit IgG-PE. Finally, cells were washed three times with PBS, stained with 4',6'-diamidino-2-phenylindole (DAPI), and visualized using a fluorescence microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Discovery and cloning of POP2

Using the amino acid sequences of previously described PYDs (e.g., from PYPAF1/Monarch, PYPAF2/NALP2, etc.) in TBLASTN searches revealed a novel sequence: a 274-bp, single exon gene coding for 97 aas corresponding to contiguous sequence LOC152138 in the human genome on the distal teleomeric end of chromosome 3 (3q28) (Fig. 1A). The deduced amino acid sequence has 78% similarity (67% identity and 1e-25 E-value) to the PYD of PYPAF2 (data not shown). Despite the presence of an in-frame stop codon, we considered that this single exon might belong to a larger gene. Genomic analysis to determine additional predicted exons or expressed sequence tags in reasonable proximity (<±50 kbps) to this exon were uninformative. The closest identified upstream gene (68.9 kbps away) is C3orf6 (Fig. 1B), a differentially spliced 12-exon gene yielding a 306- and 482-aa protein potentially involved in spastic paraplegia (33). Downstream, the fibroblast growth factor 12 gene, Fgf12, is >630 kbps distant. The gene was isolated from DNase-treated total RNA and cloned into pcDNA3 containing either a FLAG or Myc-epitope tag. The sequence of the resulting cDNA was identical with that in the NCBI database (Fig. 1C). Determination of the 3' end of the gene from poly(A)+ mRNA reveals no evidence of splicing to a downstream exon and that the coding region terminates at the in-frame stop codon. Basic local alignment search tool (BLAST) searches of the mouse and rat genome yielded no similar murine gene, although the equivalents of C3orf6 were detected in both databases (data not shown). Given the high degree of similarity to the PYPAF PYD and the prior identification of another POP, POP1, we have coined the name POP2.


Figure 1
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  		FIGURE 1. POP2 is encoded on human chromosome 3 (3q28). A, Ideogram of human chromosome 3 indicating the location of POP2. B, Diagram of the portion of contig NT_152138-containing POP2, the relative positions of C3orf6, POP2, and Fgf12 are shown by filled boxes, distances are approximate, arrows indicate the direction of transcription. C, POP2 cDNA and protein sequences. The sequence of the 3' untranslated region (3' UTR) is indicated as is the polyadenylation signal sequence (solid line).

 
POP2 has a PYD similar to CLR pyrins

Consistent with the high degree of identity with the PYPAF2 PYD, multiple alignments using the POP2 protein sequence revealed a high homology to other PYDs (Fig. 2A). Residues L11, L15, L18, P43, A50, A68, and L84, thought to play an important role in homotypic PYD oligomerization, and the sequence motif XLXKFK generally conserved in PYDs (26, 34) are likewise conserved in POP2. Threading analysis of the POP2 protein sequence using the 3D-PSSM algorithm returns two high confidence (>90%) models based on the PYDs of ASC and NALP1 (Fig. 2B). Phylogenetic analysis indicates that the POP2 is more similar to PYDs of the CLR family than to POP1 or ASC PYDs (Fig. 2C). This result strongly suggests that this gene may have originated from a CLR-PYD gene duplication event and is likely not a paralog of POP1. These findings together demonstrate that POP2 encodes a solitary, bona fide PYD, the second such gene in the human genome.


Figure 2
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  		FIGURE 2. Multiple alignment, structural predictions, and POP2 phylogeny. A, Alignment of the POP2 protein sequence with 22 PYDs from human, mouse, or viral proteins. Sequences longer than 100 aas were truncated. Shading represents conservation (either identity (black) or functionally similar (gray)) at given positions in >50% of aligned sequences. Bold numbers (relative to POP2) indicate the location of conserved residues shown to contribute to oligomerization (residues 11, 15, 18, 43, 68, and 84) or the position of the conserved basic patch typical of PYDs (XLXKFK; residues 22–27). Abbreviations not occurring in text: MV, myxoma virus; YLDV, yaba-like disease virus; SPV, swine pox virus; SFV, Shope fibroma virus. B, Backbone structural diagram of POP2 structural predictions based on ASC (top panel) or NALP1 (bottom panel) PYDs. Structures were generated from POP2 primary sequence using the 3D-PSSM algorithm at the Imperial College of Science, Technology, and Medicine (London) (www.sbg.bio.ic.ac.uk/~3dpssm/index2.html). Both structures are high confidence models (>90%). N, amino terminus; C, C terminus. C, Phylogenetic relationships between POP2 protein and the pyrin sequences shown in A. Numbers indicate bootstrap values (percentage of 1000 replicates) reflecting the degree of relatedness between clustered sequences. Viral pyrin proteins are clustered as an outgroup.

 
POP2 is primarily expressed in peripheral blood leukocytes

To determine the expression pattern of POP2, we performed RT-PCR using mRNA isolated from various cell lines (Fig. 3A). POP2 mRNA was readily detected in the K562 (human erythroblastoid leukemia) cell line. Lower amounts of POP2 were detected in the Jurkat T cell line, Ramos B lymphoma, and the monocytoid/macrophage line THP-1. Relative to GAPDH, POP2 was more abundant in K562. The expression of POP2 mRNA in primary cells was examined using a normal human tissue cDNA panel. A product of the expected size was readily observed in peripheral blood leukocytes with some expression in testis (Fig. 3B), although expression appeared greater in peripheral blood leukocytes. With overexposure, an appropriately sized band, weaker than seen in testis was observed in both thymus and spleen (data not shown). Next, we surveyed a human blood fraction panel to determine which specific peripheral blood leukocytes cell populations express POP2 (Fig. 3C). POP2 mRNA was detected at low levels in all of the samples included in the panel. A number of important leukocyte populations are not represented in the panel, including granulocytes, macrophages, NK cells, and dendritic cells. With the exception of granulocytes, these leukocytes are not typically present in peripheral blood in significant numbers. Collectively, these data support broad expression of POP2 in hemopoietic and immune cells. An N-terminal Myc-tagged cDNA clone of POP2 was generated and HEK-293 cells were transfected. Lysates from transient transfectants yielded the expected ~12-kDa protein as demonstrated by anti-Myc immunoblot (Fig. 3D).


Figure 3
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  		FIGURE 3. Expression of POP2. A, DNase-treated total RNA was prepared from the indicated human cell lines and subjected to RT-PCR using POP2-specific primers. GAPDH-specific primer amplified controls are shown. B, POP2 and GAPDH cDNA was amplified from a panel of normal tissues. K562 cDNA is shown as a positive control. Amplification without template cDNA was used as a negative control. PBL, peripheral blood leukocytes. C, POP2 and GAPDH cDNA from a human blood fraction panel was amplified as in B. *, Activated cell populations. D, HEK293 cells were transiently transfected with 1 µg of Myc-tagged ASC (control) or POP2 (indicated lanes) and immunoblotted with anti-Myc Ab and goat anti-mouse HRP.

 
POP2 localizes to nuclear and cytoplasmic compartments

Expression of POP2 was also examined by immunofluorescence in HeLa cells to determine the subcellular distribution of POP2. POP2 was observed to be either largely cytoplasmic, present in both cytoplasm and nucleus (with little or easily observed nuclear expression), or concentrated principally in the nucleus (Fig. 4, A–D). Most POP2-expressing cells display the cytoplasmic or more evenly distributed pattern; however, a substantial minority (20%) display concentrated nuclear expression (Fig. 4E). This expression pattern is similar to that observed for POP1 (27). The presence of POP2 in both the cytoplasm and the nucleus is consistent with its size and suggests that POP2 may function in either, or both, compartments. The nuclear concentration of POP2 suggests the possibility of a nuclear localization signal sequence (NLS), although no canonical NLSs are present in the coding sequence.


Figure 4
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  		FIGURE 4. POP2 localizes to both nuclear and cytoplasmic compartments. A, C, and D, COS-7 cells were transfected with 2 µg of FLAG-tagged POP2 and visualized with anti-FLAG(M5) and goat anti-mouse FITC by fluorescence microscopy. A, C, and D show representative fields. B, DAPI-counterstained image of A. E, Cells (>100) (POP2+) from two independent transfections were counted to quantitate the proportions of cells with the indicated staining pattern. Cytoplasmic, Includes cells with either exclusive cytoplasmic staining or a combination of nuclear and cytoplasmic. Nuclear, Includes only those cells with predominating nuclear POP2.

 
POP2 inhibits NF-{kappa}B activity

Given the fact that POP1 and members of the CLR family are known to modulate NF-{kappa}B activity, and the role of NF-{kappa}B in promoting innate immune and inflammatory responses, we investigated whether POP2 could influence NF-{kappa}B activity. HEK-293 cells were transiently cotransfected with a 3x-NF-{kappa}B-Luc reporter and POP2 cDNA. Eighteen hours later, the cells were stimulated with TNF-{alpha} (10 ng/ml for 30 min) and whole cell lysates analyzed for luciferase activity (Fig. 5A). A close to 3-fold inhibition of TNF-{alpha}-stimulated NF-{kappa}B was observed following in POP2 transfectants, indicating that POP2 interferes with NF-{kappa}B signaling downstream of the TNFR. These results were confirmed in HeLa and COS-7 cells with highly concordant results (Fig. 5, B and C).


Figure 5
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  		FIGURE 5. POP2 inhibits TNF-{alpha}-induced and exogenous p65-induced NF-{kappa}B activity. HEK-293T (A), HeLa (B), and COS-7 (C) cells were transiently transfected with either 1 µg of pcDNA3 or 1 µg of POP2 and cotransfected with p65 (0.1 µg) or treated 24 h later with TNF-{alpha} (10 ng/ml; 30 min). Activation of the NF-{kappa}B-Luc reporter was measured (relative fold activation (relative to pcDNA3 control) ± SEM, representative of three experiments). D, COS-7 cells were transiently transfected with either pcDNA3 or 100 ng of NF-{kappa}B-p65 and increasing amounts of POP2 (total DNA = 1.1 µg), and activation of a NF-{kappa}B-Luc reporter was measured (fold activation relative to pcDNA3 ± SEM, representative of three identical experiments). E, Transfection of HEK-293 cells with POP2 (1 µg) does not inhibit the CMV promoter-driven pGL3-control luciferase reporter.

 
The ability of POP2 to block TNF-{alpha}-mediated NF-{kappa}B activation suggests that POP2 is involved in down-regulating the NF-{kappa}B signaling cascade. To determine the point where POP2 affects the pathway, we tested the factors involved in NF-{kappa}B activation beginning with the p65 subunit of NF-{kappa}B at the distal end of the cascade. Luciferase assays were performed using lysates from HEK-293 cells cotransfected with increasing amounts of POP2, NF-{kappa}B-Luc reporter, and active p65 (Fig. 5D). Unlike POP1, which does not inhibit p65, acting instead at the level of IKK, POP2 inhibited p65 NF-{kappa}B in a dose-dependent fashion. The highest amounts of transfected POP2 yielded inhibition comparable to experiments where TNF-{alpha} was used. These results suggest that, in contrast to POP1, which inhibits NF-{kappa}B-activation signals proximally at the level of IKK, POP2 inhibits NF-{kappa}B signals distally by affecting p65. When a CMV promoter-driven pGL3-control expression plasmid was cotransfected along with POP2 no suppression was observed, indicating that POP2 is not a general inhibitor of transcription (Fig. 5E).

POP2 colocalizes with ASC in perinuclear specks

Many members of the PYD family, including pyrin, POP1, and a number of CLRs, form a perinuclear structure (speck) by interacting with ASC, whereas others do not (4, 16, 17). CLR activation of NF-{kappa}B appears to correlate with an ability to interact with ASC and speck formation. Conversely, the PYD of PAN2/PYPAF4 that inhibits NF-{kappa}B fails to form a speck in the presence of ASC (4, 22, 35). In contrast, POP1 interacts with ASC, but inhibits NF-{kappa}B (27). To determine whether POP2 colocalizes with ASC in a speck, immunofluorescence staining was performed on HeLa cells cotransfected with GFP-POP2 and Myc-ASC (Fig. 6). Cells receiving GFP-POP2 alone displayed a nuclear and cytoplasmic staining pattern consistent with that of FLAG-POP2. No GFP-POP2 containing specks were observed. Cells receiving ASC alone evidenced concentrations of ASC in the cytoplasm, but no discrete specks. Cotransfection of GFP-POP2 together with ASC, however, resulted in the formation of perinuclear specks containing both ASC and GFP-POP2. Our results indicate that POP2 interacts with ASC and is capable of participating in speck formation.


Figure 6
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  		FIGURE 6. POP2 colocalizes with ASC in perinuclear specks. HeLa cells were transiently transfected with either GFP-POP2 or Myc-ASC, or both, fixed at 48 h and stained using mouse anti-Myc Ab. Localization was determined by indirect immunofluorescence with goat anti-mouse IgG PE-conjugated secondary Ab. Nuclei were detected by DAPI counterstaining (blue).

 
POP2 inhibits the nuclear accumulation of p65 NF-{kappa}B

Because POP2, like POP1, was able to both associate with ASC and inhibit NF-{kappa}B, we next considered how POP2 might inhibit NF-{kappa}B. Another pyrin-containing protein, CIAS1, inhibits p65 NF-{kappa}B by preventing the nuclear translocation of p65 (23). We thus considered the possibility that POP2 might function similarly. To facilitate visualization of POP2, a GFP-POP2 fusion was expressed in HeLa cells. Following TNF-{alpha} treatment, less nuclear p65-NF-{kappa}B was observed in GFP-POP2-expressing cells than in controls (Fig. 7A). POP2-expressing cells displayed one of three p65 NF-{kappa}B localization patterns: reduced nuclear, absent nuclear, or redistribution of p65 to punctuate structures within the nucleus (Fig. 7, A and B). In cells with punctuate p65, staining was not colocalized to the nucleoli. To quantitate the observed changes, >100 cells were examined for their p65 localization pattern. As shown in Fig. 7C, >90% of the TNF-treated cells without POP2 had strong nuclear expression of p65. In the presence of POP2, ~40% of POP2-expressing cells had strong nuclear p65. Nearly all of the remaining POP2-expressing cells had p65 staining in both the cytoplasm and the nucleus. These results suggest that POP2 may alter p65 accumulation (or nuclear distribution), but does not simply block p65 nuclear import.


Figure 7
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  		FIGURE 7. POP2 affects NF-{kappa}B nuclear localization. A, HeLa cells were transiently transfected with 1 µg of GFP-POP2 and treated for 30 min with TNF-{alpha} (20 ng/ml). Cells were then fixed and stained using rabbit anti-human NF-{kappa}B-p65 and goat anti-rabbit IgG-PE. Fluorescence microscopy was performed to detect GFP-POP2, NF-{kappa}B-p65, and DNA (DAPI). Two representative examples of TNF-{alpha}-treated GFP-POP2-expressing cells are shown (h–m). In h–m, arrows indicate the cells expressing GFP-POP2. B, Enlargement of i and e from A showing changes in p65 nuclear localization/distribution. C, Localization of p65 was scored as mostly nuclear (Nuc.), nuclear and cytoplasmic (Nuc./Cyto.) and mostly cytoplasmic (Cyto.). Data are representative of two similar experiments.

 
POP2 blocks the formation of CLR:ASC specks

POPs have the potential to interfere with PYD interactions by acting as a competitor. Because POP2 interacts with ASC, it seemed plausible that this interaction might prevent association of ASC with CLR PYD proteins. To determine whether POP2 is able to disrupt a functional ASC:CLR interaction, we tested its ability to disrupt the recruitment of the CIAS1 PYD to specks by ASC (Fig. 8). Cotransfection of a pyrin only CIAS1 construct (CIAS1-PYD) and ASC into HeLa cells leads to the formation of specks in nearly 50% of cells expressing ASC. Transfection of ASC alone does not yield specks. Curiously, when POP2 is coexpressed, only ~10% of the ASC-expressing cells have specks, despite POP2’s ability to form specks independently. The relative absence of speck formation in cells expressing CIAS1, POP2, and ASC suggests that CIAS1 and POP2 PYDs interact, thereby preventing the formation of an ASC-containing speck. To begin to examine the specificity of this interaction, we also tested the ability of POP2 to inhibit ASC specks formed by other CLR PYD proteins, Monarch and NALP1. Monarch and NALP1 PYD interactions with ASC were also inhibited, although to a lesser extent (Fig. 8). These findings suggest a general ability of POP2 to inhibit CLR:ASC interaction and imply that, in addition to inhibiting NF-{kappa}B, POP2 may act as an inhibitor of complexes involving ASC and CLR proteins, such as the CIAS1 and NALP1 inflammasomes.


Figure 8
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  		FIGURE 8. POP2 blocks CLR:ASC speck formation. HeLa cells were transiently cotransfected with CIAS1-PYD, Monarch-PYD, or NALP1 and Myc-ASC and GFP, or with Myc-ASC and GFP-POP2. Cells were stained with anti-Myc and goat anti-mouse IgG-PE. Three hundred to 700 ASC-positive cells were counted for each condition in two to three independent transfections. The mean percentage of ASC-expressing cells containing specks under each condition is shown ± SEM. Controls for speck formation (>200 cells counted) included GFP+ASC (8% specks) and GFP-POP2 (57% specks).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
PYD-containing proteins are becoming increasingly appreciated for their roles in signaling and inflammation. The CLR (NLR, NALP) family members NALP1 and CIAS1/NALP3 are clearly involved in the formation of the inflammasome, which depends upon ASC (TMS1, PyCARD), an adaptor protein repressed by promoter methylation in a variety of cancers, to recruit proinflammatory caspases (36, 37). These CLRs recruit ASC via the interaction of the CLR PYD with that of ASC. In a similar fashion, Caspase1 is recruited by interactions between the CARD domains of ASC and Caspase1. Proteins encoding a single competing domain disrupting PYD or CARD interactions are attractive as regulators of these interactions, and the Pyrin protein itself has been recently suggested as a regulator of ASC:Caspase-1 interactions (38). A CARD-only protein (COP) and several POPs have been described. COP inhibits the production of IL-1beta by blocking oligomerization of Caspase-1, a role similar to that of two other COPs ICEBERG and pseudo-ICE (39, 40, 41). A number of POPs are viral proteins contained in the genomes of pox viruses (42). One of these, M13L, has recently been implicated as a virulence factor capable of inhibiting the production of IL-1beta (31). The human genome contains at least two POPs: POP1, which inhibits ASC-dependent NF-{kappa}B activation (27) and POP2, the subject of this study.

We describe a PYPAF2/NALP2 PYD-related gene on chromosome 3 in the human genome, identified by BLAST search, that we have named POP2. The single exon, 274-bp gene encodes a 97-aa protein with sequence identity and predicted structure most consistent with that of a PYD. POP2 message is expressed principally in peripheral blood leukocytes and testis. Expression in purified leukocyte subpopulations is detectable but weak suggesting that POP2 is broadly expressed in immune cells. POP2 was also detected at low levels in the monocytoid cell line THP-1, B and T cell lymphoma lines, but was more highly expressed in the erythroid leukemia line K562. This expression pattern is largely consistent with a role for POP2 in regulating CLR-dependent events in immune cells. CLR proteins also play a role in embryogenesis as illustrated by MATER, a nonpyrin CLR family member required for cell division in fertilized eggs (43, 44). Likewise, mouse NALP14 is also believed to have developmental function (45). Expression of POP2 in testis may reflect a role for POP2 in regulating CLR-dependent events in development.

Not surprisingly, the POP2 protein has a high degree of sequence similarity to other PYDs and, based on structural predictions, is likely to have a similar structure. Searches with the POP1 protein sequence failed to detect POP2. Similarly, searches with the PYPAF2/NALP2 pyrin did not detect the POP1 gene (data not shown), suggesting that it may be necessary to perform extensive searches with other PYDs to enumerate the full complement of human POPs. Although POP1 is closely related to the ASC PYD and inhibits the activation of NF-{kappa}B seen upon ASC coexpression with either Pyrin or CIAS1 (27), the structurally similar POP2 is more distantly related to the ASC PYD than POP1 and more closely resembles the CLR PYDs. Given this difference, POP2 might have the potential to be a specific competitor for CLR PYDs, whereas POP1 is more specific for ASC. However, studies addressing the binding affinities of different PYD:PYD combinations have yet to be performed.

Considering a relatively small size (~12 kDa), it is not surprising that POP2 is expressed throughout the cell. Curiously, POP2 displays nuclear concentration in nearly a quarter of expressing cells. The reason for this is unclear, but suggests that POP2 may contain a NLS. Curiously, a consistent feature of PYDs is a basic patch (KKFK) near the N terminus that is similar to the SV40 NLS (46, 47). However, whereas the CLR proteins CIAS1 and Monarch as well as POP1 and ASC all have an SV40 NLS-like sequence (KKFK) in their PYDs, POP2 has the less likely NLS sequence (SKFK). Nevertheless, CIAS1 and Monarch appear to be exclusively cytoplasmic, suggesting that this sequence may not be an NLS. Similarly, the prototypic PYD protein, Pyrin (marenostrin), which is mutated in Mediterranean fever, was initially believed to function in the nucleus (48). Interestingly, Pyrin nuclear localization was leptomycin B insensitive and two putative NLS sequences were dispensable (49). Subsequently, the potential nuclear role of Pyrin has been overshadowed by its ability to interact with ASC. Despite these difficulties, some PYD proteins are clearly nuclear proteins. IFI16, an IFN-induced protein, and AIM2, which is activated in melanoma cells, both contain a HIN-200 DNA binding domain and may play roles in transcription (50, 51, 52).

Pyrin-containing proteins are reported to have diverse effects on the function of NF-{kappa}B. Consistently, pathogen recognition by CLR proteins is thought to lead to NF-{kappa}B activation via receptor-interacting protein 2 and IKK (53). This function is further supported by the ability of some CLR PYDs to cooperatively activate NF-{kappa}B when expressed as truncations (16, 23). However, other CLR PYDs, such as that of PAN2/PYPAF4 and PYPAF2/PAN1, inhibit NF-{kappa}B (22), suggesting that CLRs may perform both proinflammatory and anti-inflammatory roles. More in keeping with PAN1/2, both POP1 and POP2 appear to have NF-{kappa}B-inhibiting properties. POP2 inhibits both TNF-{alpha}-induced NF-{kappa}B activity in a variety of cells and the transcriptional activity of transiently transfected p65. The mechanisms leading to NF-{kappa}B inhibition are unclear, but both PAN2 and POP1 have been reported to interact with IKK{alpha}, which may affect the downstream phosphorylation of I{kappa}B{alpha} (22, 27). In these studies, inhibition of IKK activity correlated with POP1 or PAN2 inhibition of TNF-{alpha}-induced NF-{kappa}B activity. Although POP2 inhibits TNF-{alpha}-induced NF-{kappa}B activation, it also inhibits activation seen following transfection of the active p65 subunit. In addition, we observe that POP2 expression does not necessarily prevent nuclear import of NF-{kappa}B, but may alter nuclear distribution or accumulation as evidenced by POP2-expressing cells with either less abundant or redistributed nuclear NF-{kappa}B. These experiments suggests that inhibition occurs downstream of IKK at the nuclear level. Whether these patterns result from a relatively direct inhibition of p65 or could in some fashion involve IKK remains to be seen.

PYDs can also form complexes with the pyrin-CARD protein ASC. The interaction of Pyrin with ASC results in redistribution to a perinuclear speck that may be associated with proapoptotic cells (54). Furthermore, the PYDs of the CLR proteins NALP1, NALP2, and NALP3 recruit ASC and Caspase1 to form the pro-IL-1beta and pro-IL-18-converting inflammasome complex (55, 56). Accordingly, many CLR proteins, but not all, associate with ASC in a perinuclear speck indistinguishable from the initially described pyrin speck. Like POP1 and certain pyrin-containing CLR proteins, POP2 also forms specks when coexpressed with ASC (Fig. 6). It remains uncertain why some PYDs are able to associate with ASC and others are not. But, these PYD interactions again raise the possibility that speck and/or inflammasome formation could be regulated by single domain proteins such as COP, POP1, and POP2. In a speck formation assay using the PYD of CIAS1/NALP3, POP2 is able to nearly abolish the interaction of CIAS1 and ASC. This process strongly supports the hypothesis that ASC structures such as the speck and inflammasome are targets for regulation by pyrin or COPs.

What are the implications for POP-mediated regulation of ASC interactions? The interaction of ASC with pyrin proteins is associated with a proapoptotic state (speck formation) and with the inflammatory response to pathogen-associated molecular patterns, such as muramyl dipeptides and dsRNA through both activation of NF-{kappa}B-dependent cytokine transcription and cleavage of preformed IL-1beta and IL-18 precursors via CLR-containing inflammasomes (37, 57, 58, 59). Proteins, such as POP2, that can disrupt or otherwise impede these processes might have broad impact on cellular development, survival, or homeostasis by contributing to the control of apoptotic programs. With respect to innate immune responses, understanding of mechanisms attenuating inflammatory responses is limited. POPs may represent a mechanism for restraining proinflammatory activation events by slowing the inflammasome formation, for terminating inflammatory signals and processes by limiting the duration of inflammasomes, or both. Mutations in CIAS1/NALP3 lead to a spectrum of inflammatory diseases. POP2 disruption of ASC:CIAS1/NALP3 interactions could potentially be used to ameliorate inflammation in these individuals. Furthermore, POP2 may be able to independently inhibit NF-{kappa}B. This theory has broad implications for controlling NF-{kappa}B activity to induce or block gene expression, to prevent or favor apoptosis, etc. A better understanding of the mechanistic properties of such molecules capable of modulating NF-{kappa}B may ultimately provide alternative therapeutic strategies for the broad spectrum of diseases associated with sustained NF-{kappa}B activity including Alzheimer’s, autoimmune diseases, and cancer.


   Acknowledgments
 
We thank Drs. T. Klein and W. Kerr for their critical reading of this manuscript and helpful suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Jonathan A. Harton at the current address, Center for Immunology and Microbial Disease, Albany Medical College, 47 New Scotland Avenue, MC-151, Albany, NY 12208. E-mail address: hartonj{at}mail.amc.edu Back

2 Abbreviations used in this paper: DD, death domain; DDF, DD fold; CARD, caspase recruitment domain; PYD, pyrin domain; IKK, I{kappa}B kinase; POP, pyrin-only protein; DAPI, 4',6'-diamidino-2-phenylindole; NLS, nuclear localization signal sequence; COP, CARD-only protein. Back

Received for publication April 20, 2006. Accepted for publication December 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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