Pyrin-Only 2 Modulates NF-κB and Disrupts ASC:CLR Interactions Felipe Bedoya, Laurel L. Sandler and Jonathan A. Harton This information is current as J Immunol 2007; 178:3837-3845; ; of September 23, 2021. doi: 10.4049/jimmunol.178.6.3837 http://www.jimmunol.org/content/178/6/3837 Downloaded from References This article cites 59 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/178/6/3837.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Pyrin-Only Protein 2 Modulates NF-␬B and Disrupts ASC:CLR Interactions

Felipe Bedoya,* Laurel L. Sandler,* and Jonathan A. Harton1*†

NF-␬B is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule and is dysregulated in many , neurodegenerative disorders, and inflammatory . with pyrin and/or recruitment domains have roles in apo- ptosis, innate immunity, and inflammation. Many (PYD) proteins modulate NF-␬B activity as well as participate in assembling both the perinuclear “apoptotic speck” and the pro-IL1␤/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 located on 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 Downloaded from leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-␣-stimulated and p65 (RelA)- induced NF-␬B-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-␬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-␬B activity that may influence the assembly of PYD-dependent complexes. The Journal of Immunology, 2007, 178: 3837–3845. http://www.jimmunol.org/ ctivation of NF-␬B regulates numerous cellular processes inducible (IFI) genes, coding for the HIN-200 family of hemopoi- including cytokine and adhesion molecule expression, cell etic nuclear proteins, contain one or two copies of a 200-aa pro- A cycle regulation, and cell growth. Accordingly, constitutive tein-protein interaction domain, are preceded by a PYD, and are or excessive NF-␬B activity is observed in inflammatory diseases, likely involved in cell proliferation and differentiation (9, 10). The autoimmunity, and (1, 2). Proteins containing the PYD of speck protein containing a CARD, ASC (TMS1/ (DD)2 fold (DDF) are associated with NF-␬B signaling and apoptosis PyCARD), is followed by a CARD domain. ASC associates with induction. Four related protein-protein interaction domains comprise PYD- or CARD-containing proteins through homotypic interac- the DDF superfamily: the DD; the ; the tions via both domains (11, 12). In proapoptotic cells, ASC as- caspase recruitment domain (CARD); and the pyrin domain sembles in large multimeric perinuclear complexes or “specks” by by guest on September 23, 2021 (PYD). These domains share a similar tertiary structure, a five to interacting with PYD-containing proteins. The physiological sig- six ␣-helical bundle that likely function as homotypic interaction nificance of these interactions and the speck structure itself re- modules. DDF proteins act as adaptors, recruiting signaling pro- mains unclear. ASC also participates in forming the inflammasome teins into complexes domains, ultimately contributing to apoptosis, complex that processes pro-IL-1␤ into the mature form (13). Two innate immune responses, and cancer development (1–5). thirds of the recently discovered CATERPILLER (CLR) family of Pyrin motifs are generally found at the N terminus of multido- intracellular pathogen receptors encode a PYD followed by a nu- main proteins. Pyrin (marenostrin), the founding family member, cleotide binding domain and leucine-rich repeats domain (14, 15). is associated with familial Mediterranean fever, an autosomal re- CLR proteins mediate innate immune responses to certain bacterial cessive characterized by sporadic attacks of fever and in- products, are involved in autoinflammatory diseases, and may pro- flammation with intense abdominal, joint, and chest pain (6, 7). mote apoptosis (15). Some PYD-containing proteins of the CLR Pyrin also contains a B-box zinc finger and a SPRY domain func- family induce NF-␬B activation in conjunction with ASC, includ- tioning as adaptor and ligand binding units, respectively (8). IFN ing PYPAF5, Monarch, and CIAS1/NALP3 (4, 16, 17). Not sur- prisingly, in CIAS1 associated with the inflammatory syndromes familial cold urticaria, Muckle-Wells, and chronic in- *Department of Molecular Medicine, University of South Florida College of Med- fantile neurologic cutaneous articular syndrome, show increased icine, and †Immunology Program, H. Lee Moffitt Cancer Center and Research ␬ Institute, Tampa, FL 33612 capacity for ASC-dependent NF- B activation (18–21). Other CLRs, such as PAN2/PYPAF4 mediate NF-␬B suppression (22). Received for publication April 20, 2006. Accepted for publication December 28, 2006. Although the nucleotide binding domain/leucine-rich repeats do- 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 main of CIAS1 inhibits NF- B nuclear import, the PYD of PAN2/ with 18 U.S.C. Section 1734 solely to indicate this fact. PYPAF4 inhibits NF-␬B, a function likely mediated through the 1 Address correspondence and reprint requests to Dr. Jonathan A. Harton at the cur- I␬B kinase (IKK) complex (22, 23). rent address, Center for Immunology and Microbial Disease, Albany Medical Col- The NF-␬B family of transcription factors is comprised of five lege, 47 New Scotland Avenue, MC-151, Albany, NY 12208. E-mail address: [email protected] members in : p65 (Rel A), Rel B, c-Rel, p50/p105, and ␬ 2 Abbreviations used in this paper: DD, death domain; DDF, DD fold; CARD, p52/p100, existing as homo- or heterodimers bound to the I B caspase recruitment domain; PYD, pyrin domain; IKK, I␬B kinase; POP, pyrin-only inhibitory complex in the cytosol. Upon induction by proinflam- protein; DAPI, 4Ј,6Ј-diamidino-2-phenylindole; NLS, nuclear localization signal se- matory stimuli such as TNF-␣ or LPS, I␬B␣ is phosphorylated by quence; COP, CARD-only protein. the IKK complex, ubiquitinated, and degraded by the 26S pro- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 teasome. This process unmasks NF-␬B’s nuclear localization

www.jimmunol.org 3838 POP2 BLOCKS NF-kB ACTIVITY AND ASC:CLR INTERACTION sequence, leading to its translocation into the nucleus. Binding of clonal IgG was obtained from Santa Cruz Biotechnology, and Alexa Fluor 594 NF-␬B to its cognate DNA response elements induces the tran- goat anti-rabbit IgG (HϩL) was obtained from Molecular Probes. scription of a host of cytokines and growth factors (e.g., IL-2, IL-8, IFN-␤, M-CSF, G-CSF, vascular endothelial growth factor), as Cloning and RT-PCR well as various transcription factors and signaling regulators (e.g., A cDNA encoding POP2 was amplified from total RNA from the B cell I␬B␣ and IFN regulatory factor-1 and -2) (2, 24). The pivotal role line Ramos by RT-PCR using the reverse transcribed One-Step Kit (Qia- of NF-␬B in biological processes modulating the immune response gen) with the primers (forward 5Ј-aaccgcggatggcatcttctgcagag-3Ј and re- suggests that localization and subsequent activation must be rig- verse 5Ј-aaaagcttatggcatcttctgcagag-3Ј) designed to obtain POP2 cDNA orously controlled. Dysregulation of these events contributes to flanked by HindIII and XhoI restrictions sites. Subsequently, POP2 was cloned into the pCDNA 3.1 vector containing an N-terminal Flag or Myc aberrant associated with numerous human dis- epitope. For POP2 mRNA detection, total RNA was isolated from Daudi, eases including cancer, neurodegenerative disorders, arthritis, and Ramos (B cell lymphomas), Jurkat (human leukemia), THP-1 (hu- chronic inflammation (1, 2). man monocytic cell line), and K562 (human erythroid cell line) using the Solitary PYDs may disrupt PYD interactions, blocking the for- RNeasy RNA Isolation Kit (Qiagen) and reverse transcribed and amplified with the One-Step RT-PCR Kit (Qiagen). The same procedure was per- mation of speck and/or inflammasome complexes, thus interfering formed on total RNA from multiple human tissues (MTCC panel I) and with downstream effects. Proteins of this type have recently been human blood fractions (MTC panel), both obtained from BD Biosciences. identified in the and in the genomes of pox Primers for GAPDH were used as a control. (25, 26). Pyrin-only protein (POP)1, closely related to the PYD of ASC, is predominantly expressed in immune tissues, where it ap- Cell culture, transfection, and luciferase assays pears to inhibit NF-␬B- and surprisingly enhances caspase-1-acti- Downloaded from HEK 293, HeLa, and COS-7 cells (American Type Culture Collection) vation (27). Other genes encoding POPs have been found in the were cultured in either DMEM or RPMI 1640 medium supplemented with genomes of Capripoxviridae, Leporipoxviridae, Suipoxviridae, 10% heat-inactivated FBS, 5% L-glutamine, and 0.1% penicillin/strepto- and Yatapoxviridae (28–30). Recently, Johnston et al. (31) re- mycin. Cells were passed every 3–4 days. Cell number and viability was ported that M13L-PYD, a POP from myxoma , inhibits both determined by trypan blue exclusion. For transient transfection assays, ϫ 5 NF-␬B activity and caspase-1-dependent IL-1␤ production, and cells were plated onto 6-well plates (2 10 cells per well) and incubated at 37°C with 5% CO2 and 95% humidity for 16 h. Transfection was con- M13L deletion was sufficient to inhibit virus replication in vivo. ducted with FLAG-POP2 (different quantities), 100 ng of pNF-␬B-Luc (a http://www.jimmunol.org/ Therefore, POPs can also participate as suppressors of host immu- 3xNF-␬B-driven firefly luciferase reporter), and either 100 ng of NF-␬B- nity. Modulating NF-␬B signaling pathways and altering PYD- p65 using FuGene6 (3 ␮l:1 ␮g of DNA; Roche), or stimulated with varying protein interactions are potential mechanisms for such suppression. amounts of TNF-␣ 24 h posttransfection. The total amount of DNA in each transfection was kept constant (1100 ng/well) by the addition of empty Data from the sequence of the genomes of and other vector (pcDNA3). Cell lysates were prepared using 200 ␮l of lysis buffer has resulted in the identification of novel genes and the (1 M Tris (pH 7.4), 1 M NaCl, 10 mM EDTA, and 1% Triton X-100), and emergence of new gene families. The recently described CLR fam- luciferase was quantitated according to standard protocol on a VICTOR ily encompasses PYD-proteins with roles in both innate and ac- Light Luminescence Counter (Wallac). quired immunity. Because of our interest in this gene family, we sought to identify new PYD-encoding genes. We report the iden- Western blotting by guest on September 23, 2021 tification of a novel human POP comprised of 97 aas that we have Western blotting for Flag- and Myc-tagged proteins was performed essen- named POP2. POP2 is encoded by a single exon gene on chromo- tially as described (32). Following transfection and lysis, equal amounts of some 3 and is expressed primarily in peripheral blood leukocytes. protein were separated on a SDS-PAGE (15% polyacrylamide) gel, for 40 We demonstrate that POP2 inhibits NF-␬B (RelA) activated by min at 200 volts, transferred to nitrocellulose (0.2 ␮m)for1hat110volts, ␣ TNF-␣ or transfection of p65. NF-␬B inhibition by POP2 is ac- and immunoblotted with either -FLAG Ab (M5; Sigma-Aldrich; 1/5,000) or ␣-myc Ab (1/7,500). Bound M5 or ␣-myc was detected with goat anti- companied by a decrease in nuclear import and altered nuclear mouse IgG1-HRP (Southern Biotechnology Associates; 1/7,500) and vi- distribution of p65. In addition, POP2 associates with ASC in pe- sualized on Autoradiography film (Midwest Scientific) using SuperSignal rinuclear speck structures, but inhibits specks composed of CIAS1/ West Pico HRP detection reagents (Pierce Biotechnology). NALP3 and ASC. Collectively, these results imply an important role for POP2 as a negative regulator of the NF-␬B signaling path- Immunofluorescence microscopy way and suggest a regulatory mechanism for CLR-dependent in- COS-7 and HeLa cells were cultured overnight in two-chamber slides at a nate immune and inflammatory responses. density of 8 ϫ 104 cells per chamber. Cells were transiently transfected with 1 ␮g of pcCDNA3 as negative control and either 1 ␮g of GFP-POP2, 1 ␮g of FLAG-CIITA, or 1 ␮g of Myc-ASC as positive controls. Either 1 Materials and Methods ␮g of FLAG-POP2, 1 ␮g of Myc-POP2, 1 ␮gofNF-␬B-p65, or 1 ␮gof Bioinformatics 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, TBLASTN searches of the human genome were performed at National 10% NGS). Cells then were incubated for1hatroom temperature with Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/ mouse anti-FLAG or anti-Myc Ab or rabbit anti-p65 polyclonal IgG, genome/guide/human/) using the N-terminal-100-aa sequence of PYPAF2 washed with PBS (1% BSA), and incubated (1 h, room temperature) with (pyrin-containing Apaf-1-like protein 2) as a query. Multiple alignments of either goat anti-mouse IgG1-FITC, goat anti-mouse IgG1-PE, or goat anti- different PYDs and phylogenetic analysis were done with CLUSTALW rabbit IgG-PE. Finally, cells were washed three times with PBS, stained (www.ebi.ac.uk/clustalw/) and MEGA2 (Molecular Evolutionary Genetic with 4Ј,6Ј-diamidino-2-phenylindole (DAPI), and visualized using a fluo- Analysis; www.megasoftware.net/). Tertiary structure prediction using rescence microscope. 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/ϳ3dpssm/). Results Discovery and cloning of POP2 Reagents and Abs Using the sequences of previously described PYDs (e.g., Recombinant human TNF-␣ and goat anti-mouse IgG1-FITC were purchased from BD Pharmingen. Mouse-anti-FLAG M5 mAb and goat anti-mouse HRP- from PYPAF1/Monarch, PYPAF2/NALP2, etc.) in TBLASTN conjugated IgG1 Ab were purchased from Sigma-Aldrich. Mouse-anti-myc searches revealed a novel sequence: a 274-bp, single exon gene cod- tag IgG1 mAb was purchased from Upstate Biotechnology, rabbit p65 poly- ing for 97 aas corresponding to contiguous sequence LOC152138 in The Journal of Immunology 3839

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, Downloaded from arrows indicate the direction of transcription. C, POP2 cDNA and protein sequences. The sequence of the 3Ј untranslated region (3Ј UTR) is indicated as is the polyadenylation signal sequence (solid line).

the human genome on the distal teleomeric end of chromosome 3 codes a solitary, bona fide PYD, the second such gene in the hu- http://www.jimmunol.org/ (3q28) (Fig. 1A). The deduced amino acid sequence has 78% sim- man genome. ilarity (67% identity and 1e-25 E-value) to the PYD of PYPAF2 (data not shown). Despite the presence of an in-frame stop codon, POP2 is primarily expressed in peripheral blood leukocytes we considered that this single exon might belong to a larger gene. To determine the expression pattern of POP2, we performed RT- Genomic analysis to determine additional predicted exons or ex- PCR using mRNA isolated from various cell lines (Fig. 3A). POP2 pressed sequence tags in reasonable proximity (ϽϮ50 kbps) to mRNA was readily detected in the K562 (human erythroblastoid this exon were uninformative. The closest identified upstream gene leukemia) cell line. Lower amounts of POP2 were detected in the (68.9 kbps away) is C3orf6 (Fig. 1B), a differentially spliced 12- Jurkat T cell line, Ramos B lymphoma, and the monocytoid/mac- exon gene yielding a 306- and 482-aa protein potentially involved rophage line THP-1. Relative to GAPDH, POP2 was more abun- by guest on September 23, 2021 in spastic paraplegia (33). Downstream, the fibroblast growth fac- dant in K562. The expression of POP2 mRNA in primary cells tor 12 gene, Fgf12,isϾ630 kbps distant. The gene was isolated was examined using a normal human tissue cDNA panel. A prod- from DNase-treated total RNA and cloned into pcDNA3 contain- uct of the expected size was readily observed in peripheral blood ing either a FLAG or Myc-epitope tag. The sequence of the re- leukocytes with some expression in testis (Fig. 3B), although ex- sulting cDNA was identical with that in the NCBI database (Fig. pression appeared greater in peripheral blood leukocytes. With over- 1C). Determination of the 3Ј end of the gene from poly(A)ϩ exposure, an appropriately sized band, weaker than seen in testis was mRNA reveals no evidence of splicing to a downstream exon and observed in both thymus and spleen (data not shown). Next, we sur- that the coding region terminates at the in-frame stop codon. Basic veyed a human blood fraction panel to determine which specific pe- local alignment search tool (BLAST) searches of the mouse and rat ripheral blood leukocytes cell populations express POP2 (Fig. 3C). genome yielded no similar murine gene, although the equivalents POP2 mRNA was detected at low levels in all of the samples included of C3orf6 were detected in both databases (data not shown). Given in the panel. A number of important leukocyte populations are not the high degree of similarity to the PYPAF PYD and the prior represented in the panel, including granulocytes, macrophages, NK identification of another POP, POP1, we have coined the cells, and dendritic cells. With the exception of granulocytes, these name POP2. leukocytes are not typically present in peripheral blood in significant numbers. Collectively, these data support broad expression of POP2 POP2 has a PYD similar to CLR pyrins in hemopoietic and immune cells. An N-terminal Myc-tagged cDNA Consistent with the high degree of identity with the PYPAF2 PYD, clone of POP2 was generated and HEK-293 cells were transfected. multiple alignments using the POP2 protein sequence revealed a Lysates from transient transfectants yielded the expected ϳ12-kDa high homology to other PYDs (Fig. 2A). Residues L11, L15, L18, protein as demonstrated by anti-Myc immunoblot (Fig. 3D). P43, A50, A68, and L84, thought to play an important role in homotypic PYD oligomerization, and the sequence motif POP2 localizes to nuclear and cytoplasmic compartments XLXKFK generally conserved in PYDs (26, 34) are likewise con- Expression of POP2 was also examined by immunofluorescence in served in POP2. Threading analysis of the POP2 protein sequence HeLa cells to determine the subcellular distribution of POP2. using the 3D-PSSM algorithm returns two high confidence POP2 was observed to be either largely cytoplasmic, present in (Ͼ90%) models based on the PYDs of ASC and NALP1 (Fig. 2B). both cytoplasm and nucleus (with little or easily observed nuclear Phylogenetic analysis indicates that the POP2 is more similar to expression), or concentrated principally in the nucleus (Fig. 4, PYDs of the CLR family than to POP1 or ASC PYDs (Fig. 2C). A–D). Most POP2-expressing cells display the cytoplasmic or This result strongly suggests that this gene may have originated more evenly distributed pattern; however, a substantial minority from a CLR-PYD gene duplication event and is likely not a para- (20%) display concentrated nuclear expression (Fig. 4E). This ex- log of POP1. These findings together demonstrate that POP2 en- pression pattern is similar to that observed for POP1 (27). The 3840 POP2 BLOCKS NF-kB ACTIVITY AND ASC:CLR INTERACTION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 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 Ͼ50% 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/ϳ3dpssm/ index2.html). Both structures are high confidence models (Ͼ90%). 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. presence of POP2 in both the cytoplasm and the nucleus is suggests the possibility of a nuclear localization signal sequence consistent with its size and suggests that POP2 may function in (NLS), although no canonical NLSs are present in the coding either, or both, compartments. The nuclear concentration of POP2 sequence.

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 ,ء transiently transfected with 1 ␮g of Myc-tagged ASC (control) or POP2 (indicated lanes) and immunoblotted with anti-Myc Ab and goat anti-mouse HRP. The Journal of Immunology 3841

FIGURE 4. POP2 localizes to both nuclear and cy- toplasmic compartments. A, C, and D, COS-7 cells were transfected with 2 ␮g of FLAG-tagged POP2 and visu- alized with anti-FLAG(M5) and goat anti-mouse FITC by fluorescence microscopy. A, C, and D show repre- sentative fields. B, DAPI-counterstained image of A. E, Cells (Ͼ100) (POP2ϩ) from two independent trans- fections 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 predominat- ing nuclear POP2.

POP2 inhibits NF-␬B activity with the p65 subunit of NF-␬B at the distal end of the cascade. Lu- Given the fact that POP1 and members of the CLR family are ciferase assays were performed using lysates from HEK-293 cells ␬ known to modulate NF-␬B activity, and the role of NF-␬B in pro- cotransfected with increasing amounts of POP2, NF- B-Luc reporter, and active p65 (Fig. 5D). Unlike POP1, which does not inhibit p65, moting innate immune and inflammatory responses, we investi- Downloaded from ␬ gated whether POP2 could influence NF-␬B activity. HEK-293 acting instead at the level of IKK, POP2 inhibited p65 NF- Bina cells were transiently cotransfected with a 3x-NF-␬B-Luc reporter dose-dependent fashion. The highest amounts of transfected POP2 ␣ and POP2 cDNA. Eighteen hours later, the cells were stimulated yielded inhibition comparable to experiments where TNF- was used. with TNF-␣ (10 ng/ml for 30 min) and whole cell lysates analyzed These results suggest that, in contrast to POP1, which inhibits NF- ␬ for luciferase activity (Fig. 5A). A close to 3-fold inhibition of B-activation signals proximally at the level of IKK, POP2 inhibits ␣ ␬ NF-␬B signals distally by affecting p65. When a CMV promoter-

TNF- -stimulated NF- B was observed following in POP2 trans- http://www.jimmunol.org/ fectants, indicating that POP2 interferes with NF-␬B signaling driven pGL3-control expression plasmid was cotransfected along with downstream of the TNFR. These results were confirmed in HeLa POP2 no suppression was observed, indicating that POP2 is not a and COS-7 cells with highly concordant results (Fig. 5, B and C). general inhibitor of transcription (Fig. 5E). The ability of POP2 to block TNF-␣-mediated NF-␬B activation suggests that POP2 is involved in down-regulating the NF-␬B sig- POP2 colocalizes with ASC in perinuclear specks naling cascade. To determine the point where POP2 affects the path- Many members of the PYD family, including pyrin, POP1, and a way, we tested the factors involved in NF-␬B activation beginning number of CLRs, form a perinuclear structure (speck) by interacting by guest on September 23, 2021

FIGURE 5. POP2 inhibits TNF-␣-induced and exogenous p65-induced NF-␬B activity. HEK-293T (A), HeLa (B), and COS-7 (C) cells were transiently transfected with either 1 ␮g of pcDNA3 or 1 ␮g of POP2 and cotransfected with p65 (0.1 ␮g) or treated 24 h later with TNF-␣ (10 ng/ml; 30 min). Activation of the NF-␬B-Luc reporter was measured (relative fold activation (relative to pcDNA3 control) Ϯ SEM, representative of three experiments). D, COS-7 cells were transiently transfected with either pcDNA3 or 100 ng of NF-␬B-p65 and increasing amounts of POP2 (total DNA ϭ 1.1 ␮g), and activation of a NF-␬B-Luc reporter was measured (fold activation relative to pcDNA3 Ϯ SEM, representative of three identical experiments). E, Trans- fection of HEK-293 cells with POP2 (1 ␮g) does not inhibit the CMV promoter-driven pGL3-control luciferase reporter. 3842 POP2 BLOCKS NF-kB ACTIVITY AND ASC:CLR INTERACTION

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 immuno- fluorescence with goat anti-mouse IgG PE-conjugated secondary Ab. Nuclei were detected by DAPI coun- terstaining (blue). Downloaded from

with ASC, whereas others do not (4, 16, 17). CLR activation of ceiving GFP-POP2 alone displayed a nuclear and cytoplasmic NF-␬B appears to correlate with an ability to interact with ASC staining pattern consistent with that of FLAG-POP2. No GFP- and speck formation. Conversely, the PYD of PAN2/PYPAF4 that POP2 containing specks were observed. Cells receiving ASC alone inhibits NF-␬B fails to form a speck in the presence of ASC (4, 22, evidenced concentrations of ASC in the cytoplasm, but no discrete http://www.jimmunol.org/ 35). In contrast, POP1 interacts with ASC, but inhibits NF-␬B specks. Cotransfection of GFP-POP2 together with ASC, however, (27). To determine whether POP2 colocalizes with ASC in a resulted in the formation of perinuclear specks containing both speck, immunofluorescence staining was performed on HeLa cells ASC and GFP-POP2. Our results indicate that POP2 interacts with cotransfected with GFP-POP2 and Myc-ASC (Fig. 6). Cells re- ASC and is capable of participating in speck formation. by guest on September 23, 2021

FIGURE 7. POP2 affects NF-␬B nuclear localization. A, HeLa cells were transiently transfected with 1 ␮g of GFP-POP2 and treated for 30 min with TNF-␣ (20 ng/ml). Cells were then fixed and stained using rabbit anti-human NF-␬B-p65 and goat anti-rabbit IgG-PE. Fluorescence microscopy was performed to detect GFP-POP2, NF-␬B-p65, and DNA (DAPI). Two representative examples of TNF-␣-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. The Journal of Immunology 3843

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 Ϯ SEM. Controls for speck formation (Ͼ200 cells counted) included GFPϩASC (8% specks) and GFP-POP2 (57% specks).

POP2 inhibits the nuclear accumulation of p65 NF-␬B Discussion Downloaded from Because POP2, like POP1, was able to both associate with ASC PYD-containing proteins are becoming increasingly appreciated and inhibit NF-␬B, we next considered how POP2 might inhibit for their roles in signaling and inflammation. The CLR (NLR, NF-␬B. Another pyrin-containing protein, CIAS1, inhibits p65 NALP) family members NALP1 and CIAS1/NALP3 are clearly NF-␬B by preventing the nuclear translocation of p65 (23). We involved in the formation of the inflammasome, which depends thus considered the possibility that POP2 might function similarly. upon ASC (TMS1, PyCARD), an adaptor protein repressed by

To facilitate visualization of POP2, a GFP-POP2 fusion was ex- promoter methylation in a variety of cancers, to recruit proinflam- http://www.jimmunol.org/ pressed in HeLa cells. Following TNF-␣ treatment, less nuclear matory (36, 37). These CLRs recruit ASC via the inter- p65-NF-␬B was observed in GFP-POP2-expressing cells than in action of the CLR PYD with that of ASC. In a similar fashion, controls (Fig. 7A). POP2-expressing cells displayed one of three Caspase1 is recruited by interactions between the CARD domains p65 NF-␬B localization patterns: reduced nuclear, absent nuclear, of ASC and Caspase1. Proteins encoding a single competing do- or redistribution of p65 to punctuate structures within the nucleus main disrupting PYD or CARD interactions are attractive as reg- (Fig. 7, A and B). In cells with punctuate p65, staining was not ulators of these interactions, and the Pyrin protein itself has been colocalized to the nucleoli. To quantitate the observed changes, recently suggested as a regulator of ASC:Caspase-1 interactions Ͼ100 cells were examined for their p65 localization pattern. As (38). A CARD-only protein (COP) and several POPs have been de- shown in Fig. 7C, Ͼ90% of the TNF-treated cells without POP2 scribed. COP inhibits the production of IL-1␤ by blocking oligomer- by guest on September 23, 2021 had strong nuclear expression of p65. In the presence of POP2, ization of Caspase-1, a role similar to that of two other COPs ϳ40% of POP2-expressing cells had strong nuclear p65. Nearly all ICEBERG and pseudo-ICE (39–41). A number of POPs are viral of the remaining POP2-expressing cells had p65 staining in both proteins contained in the genomes of pox viruses (42). One of these, the cytoplasm and the nucleus. These results suggest that POP2 M13L, has recently been implicated as a virulence factor capable of may alter p65 accumulation (or nuclear distribution), but does not inhibiting the production of IL-1␤ (31). The human genome contains simply block p65 nuclear import. at least two POPs: POP1, which inhibits ASC-dependent NF-␬B ac- tivation (27) and POP2, the subject of this study. POP2 blocks the formation of CLR:ASC specks We describe a PYPAF2/NALP2 PYD-related gene on chromo- POPs have the potential to interfere with PYD interactions by acting some 3 in the human genome, identified by BLAST search, that we as a competitor. Because POP2 interacts with ASC, it seemed plau- have named POP2. The single exon, 274-bp gene encodes a 97-aa sible that this interaction might prevent association of ASC with CLR protein with sequence identity and predicted structure most con- PYD proteins. To determine whether POP2 is able to disrupt a func- sistent with that of a PYD. POP2 message is expressed principally tional ASC:CLR interaction, we tested its ability to disrupt the re- in peripheral blood leukocytes and testis. Expression in purified cruitment of the CIAS1 PYD to specks by ASC (Fig. 8). Cotransfec- leukocyte subpopulations is detectable but weak suggesting that tion of a pyrin only CIAS1 construct (CIAS1-PYD) and ASC into POP2 is broadly expressed in immune cells. POP2 was also de- HeLa cells leads to the formation of specks in nearly 50% of cells tected at low levels in the monocytoid cell line THP-1, B and T cell expressing ASC. Transfection of ASC alone does not yield specks. lymphoma lines, but was more highly expressed in the erythroid leu- Curiously, when POP2 is coexpressed, only ϳ10% of the ASC-ex- kemia line K562. This expression pattern is largely consistent with a pressing cells have specks, despite POP2’s ability to form specks in- role for POP2 in regulating CLR-dependent events in immune cells. dependently. The relative absence of speck formation in cells express- CLR proteins also play a role in embryogenesis as illustrated by ing CIAS1, POP2, and ASC suggests that CIAS1 and POP2 PYDs MATER, a nonpyrin CLR family member required for cell di- interact, thereby preventing the formation of an ASC-containing vision in fertilized eggs (43, 44). Likewise, mouse NALP14 is speck. To begin to examine the specificity of this interaction, we also also believed to have developmental function (45). Expression tested the ability of POP2 to inhibit ASC specks formed by other CLR of POP2 in testis may reflect a role for POP2 in regulating PYD proteins, Monarch and NALP1. Monarch and NALP1 PYD CLR-dependent events in development. interactions with ASC were also inhibited, although to a lesser extent Not surprisingly, the POP2 protein has a high degree of se- (Fig. 8). These findings suggest a general ability of POP2 to inhibit quence similarity to other PYDs and, based on structural predic- CLR:ASC interaction and imply that, in addition to inhibiting NF-␬B, tions, is likely to have a similar structure. Searches with the POP1 POP2 may act as an inhibitor of complexes involving ASC and CLR protein sequence failed to detect POP2. Similarly, searches with proteins, such as the CIAS1 and NALP1 inflammasomes. the PYPAF2/NALP2 pyrin did not detect the POP1 gene (data not 3844 POP2 BLOCKS NF-kB ACTIVITY AND ASC:CLR INTERACTION shown), suggesting that it may be necessary to perform extensive IL-1␤ and pro-IL-18-converting inflammasome complex (55, 56). searches with other PYDs to enumerate the full complement of Accordingly, many CLR proteins, but not all, associate with ASC human POPs. Although POP1 is closely related to the ASC PYD in a perinuclear speck indistinguishable from the initially de- and inhibits the activation of NF-␬B seen upon ASC coexpression scribed pyrin speck. Like POP1 and certain pyrin-containing CLR with either Pyrin or CIAS1 (27), the structurally similar POP2 is proteins, POP2 also forms specks when coexpressed with ASC more distantly related to the ASC PYD than POP1 and more (Fig. 6). It remains uncertain why some PYDs are able to associate closely resembles the CLR PYDs. Given this difference, POP2 with ASC and others are not. But, these PYD interactions again might have the potential to be a specific competitor for CLR PYDs, raise the possibility that speck and/or inflammasome formation whereas POP1 is more specific for ASC. However, studies ad- could be regulated by single domain proteins such as COP, POP1, dressing the binding affinities of different PYD:PYD combinations and POP2. In a speck formation assay using the PYD of CIAS1/ have yet to be performed. NALP3, POP2 is able to nearly abolish the interaction of CIAS1 Considering a relatively small size (ϳ12 kDa), it is not surpris- and ASC. This process strongly supports the hypothesis that ASC ing that POP2 is expressed throughout the cell. Curiously, POP2 structures such as the speck and inflammasome are targets for reg- displays nuclear concentration in nearly a quarter of expressing ulation by pyrin or COPs. cells. The reason for this is unclear, but suggests that POP2 may What are the implications for POP-mediated regulation of ASC contain a NLS. Curiously, a consistent feature of PYDs is a basic interactions? The interaction of ASC with pyrin proteins is asso- patch (KKFK) near the N terminus that is similar to the SV40 NLS ciated with a proapoptotic state (speck formation) and with the (46, 47). However, whereas the CLR proteins CIAS1 and Monarch inflammatory response to pathogen-associated molecular patterns, as well as POP1 and ASC all have an SV40 NLS-like sequence such as muramyl dipeptides and dsRNA through both activation of Downloaded from (KKFK) in their PYDs, POP2 has the less likely NLS sequence NF-␬B-dependent cytokine transcription and cleavage of pre- (SKFK). Nevertheless, CIAS1 and Monarch appear to be exclu- formed IL-1␤ and IL-18 precursors via CLR-containing inflam- sively cytoplasmic, suggesting that this sequence may not be an masomes (37, 57–59). Proteins, such as POP2, that can disrupt or NLS. Similarly, the prototypic PYD protein, Pyrin (marenostrin), otherwise impede these processes might have broad impact on cel- which is mutated in Mediterranean fever, was initially believed to lular development, survival, or homeostasis by contributing to the

function in the nucleus (48). Interestingly, Pyrin nuclear localiza- control of apoptotic programs. With respect to innate immune re- http://www.jimmunol.org/ tion was leptomycin B insensitive and two putative NLS sequences sponses, understanding of mechanisms attenuating inflammatory were dispensable (49). Subsequently, the potential nuclear role of responses is limited. POPs may represent a mechanism for re- Pyrin has been overshadowed by its ability to interact with ASC. straining proinflammatory activation events by slowing the inflam- Despite these difficulties, some PYD proteins are clearly nuclear masome formation, for terminating inflammatory signals and proteins. IFI16, an IFN-induced protein, and AIM2, which is ac- processes by limiting the duration of inflammasomes, or both. tivated in melanoma cells, both contain a HIN-200 DNA binding Mutations in CIAS1/NALP3 lead to a spectrum of inflammatory domain and may play roles in transcription (50–52). diseases. POP2 disruption of ASC:CIAS1/NALP3 interactions Pyrin-containing proteins are reported to have diverse effects on could potentially be used to ameliorate inflammation in these in- the function of NF-␬B. Consistently, pathogen recognition by CLR dividuals. Furthermore, POP2 may be able to independently inhibit by guest on September 23, 2021 proteins is thought to lead to NF-␬B activation via receptor-inter- NF-␬B. This theory has broad implications for controlling NF-␬B acting protein 2 and IKK (53). This function is further supported activity to induce or block gene expression, to prevent or favor by the ability of some CLR PYDs to cooperatively activate NF-␬B apoptosis, etc. A better understanding of the mechanistic proper- when expressed as truncations (16, 23). However, other CLR ties of such molecules capable of modulating NF-␬B may ulti- PYDs, such as that of PAN2/PYPAF4 and PYPAF2/PAN1, inhibit mately provide alternative therapeutic strategies for the broad NF-␬B (22), suggesting that CLRs may perform both proinflam- spectrum of diseases associated with sustained NF-␬B activity in- matory and anti-inflammatory roles. More in keeping with cluding Alzheimer’s, autoimmune diseases, and cancer. PAN1/2, both POP1 and POP2 appear to have NF-␬B-inhibiting properties. POP2 inhibits both TNF-␣-induced NF-␬B activity in a Acknowledgments variety of cells and the transcriptional activity of transiently trans- We thank Drs. T. Klein and W. Kerr for their critical reading of this manu- fected p65. The mechanisms leading to NF-␬B inhibition are un- script and helpful suggestions. clear, but both PAN2 and POP1 have been reported to interact with IKK␣, which may affect the downstream phosphorylation of I␬B␣ Disclosures (22, 27). In these studies, inhibition of IKK activity correlated with The authors have no financial conflict of interest. POP1 or PAN2 inhibition of TNF-␣-induced NF-␬B activity. Al- though POP2 inhibits TNF-␣-induced NF-␬B activation, it also References inhibits activation seen following transfection of the active p65 1. Dolcet, X., D. Llobet, J. Pallares, and X. Matias-Guiu. 2005. NF-␬B in devel- subunit. In addition, we observe that POP2 expression does not opment and progression of human cancer. Virchows Arch. 446: 475–482. 2. Hayden, M. S., and S. Ghosh. 2004. Signaling to NF-␬B. Genes Dev. 18: necessarily prevent nuclear import of NF-␬B, but may alter nuclear 2195–2224. distribution or accumulation as evidenced by POP2-expressing 3. Bruey, J. M., N. Bruey-Sedano, R. Newman, S. Chandler, C. Stehlik, and ␬ J. C. Reed. 2004. 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