R118 Current Biology Vol 10 No 4

Correspondence Figure 1 (a)

Pycard HS 12: LENLPAEELKKFKLKLLSVPL.REGYGRIPRGALLSMD.ALDLTDKLVSFYLETYGAELTANVLRDMGLQEMAGQLQAAT :89 Pyrin HS 13: LEELVPYDFEKFKFKLQNTSV.QKEHSRIPRSQIQRAR.PVKMATLLVTYYGEEYAVQLTLQVLRAINQRLLAEELHRAA :90 The : a NALP1 HS 13: LEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSG..MEVASYLVAQYGEQRAWDLALHTWEQMGLRSLCAQAQEGA :90 NALP2 HS 13: LEQLSQDELSKFKYLITTFSL.AHELQKIPHKEVDKAD.GKQLVEILTTHCDSYWVEMASLQVFEKMHRMDLSERAKDEV :90 Caspy DR 13: LSNIGADNLRRFQSRLGD....RKQEPRVRKSTIEKLKDEIDLVDLLVNTFTS.DAVSVTVDILRGIKCNAVAEELLENT :87 possible member of Pycard DR 13: FEDLGADNLRKFKSKLGD....RRQEPRVTKSAIEKLKDEIDLADLMVGVFTSKDAVSVTVEILRAIKCNAVADDLLRNT :88 the -fold Consensus 1: ****-******!*-***---*-**---*****-*****--*********-*---***-**********-*-*****-*** :80 family implicated in (b) Pycard PYD CARD and Pyrin PYD B SPRY Fabio Martinon*, CASPY PYD †‡ Kay Hofmann and NALP1 PYD NACHT LRR CARD Jürg Tschopp*‡ NALP2 PYD NACHT LRR

In pro-apoptotic signaling pathways, CARD4/ CARD NACHT LRR the interaction between the Nod1 different initiator units such as the death receptor Fas, the various (c) MAGGAWGRLACYLEFLKKEELKEFQLLLANKAHSRSSSGETPAQPEKTSGMEVASYLVAQYGEQRAWDLA adaptor and is LHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAVLMPWIHELPAGCTQGSERRVLRQL primarily mediated by three PDTSGRRWREISASLLYQALPSSPDHESPSQESPNAPTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDE structurally related –protein TSGIYYTEIREREREKSEKGRPPWAAVVGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQ LLLLQRPHPRSQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA domains called death domain (DD), WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDGVDEPGWVLQ (DED), and EPSSELCLHWSQPQPADALLGSLLGKTILPEASFLITARTTALQNLIPSLEQARWVEVLGFSESSRKEYF caspase-recruitment domain YRYFTDERQAIRAFRLVKSNKELWALCLVPWVSWLACTCLMQQMKRKEKLTLTSKTTTTLCLHYLAQALQ (CARD) [1,2]. While analyzing the AQPLGPQLRDLCSLAAEGIWQKKTLFSPDDLRKHGLDGAIISTFLKMGILQEHPIPLSYSFIHLCFQEFF AAMSYVLEDEKGRGKHSNCIIDLEKTLEAYGIHGLFGASTTRFLLGLLSDEGEREMENIFHCRLSQGRNL sequence of the recently identified MQWVPSLQLLLQPHSLESLHCLYETRNKTFLTQVMAHFEEMGMCVETDMELLVCTFCIKFSRHVKKLQLI CARD-containing protein Pycard EGRQHRSTWSPTMVVLFRWVPVTDAYWQILFSVLKVTRNLKELDLSGNSLSHSAVKSLCKTLRRPRCLLE (PYD and CARD containing TLRLAGCGLTAEDCKDLAFGLRANQTLTELDLSFNVLTDAGAKHLCQRLRQPSCKLQRLQLVSCGLTSDC CQDLASVLSASPSLKELDLQQNNLDDVGVRLLCEGLRHPACKLIRLGLDQTTLSDEMRQELRALEQEKPQ protein) also known as ASC LLIFSRRKPSVMTPTEGLDTGEMSNSTSSLKRQRLGSERAASHVAQANLKLLDVSKIFPIAEIAEESSPE (apoptosis-associated speckle- like VVPVELLCVPSPASQGDLHTKPLGTDDDFWGPTGPVATEVVDKEKNLYRVHFPVAGSYRWPNTGLCFVMR protein), we realised that the second EAVTVEIEFCVWDQFLGEINPQHSWMVAGPLLDIKAEPGAVEAVHLPHFVALQGGHVDTSLFQMAHFKEE GMLLEKPARVELHHIVLENPSFSPLGVLLKMIHNALRFIPVTSVVLLYHRVHPEEVTFHLYLIPSDCSIR structural domain present at the KELELCYRSPGEDQLFSEFYVGHLGSGIRLQVKDKKDETLVWEALVKPGDLMPATTLIPPARIAVPSPLD amino terminus of Pycard — the APQLLHFVDQYREQLIARVTSVEVVLDKLHGQVLSQEQYERVLAENTRPSQMRKLFSLSQSWDRKCKDGL pyrin domain (PYD) — shows weak YQALKETHPHLIMELWEKGSKKGLLPLSS but significant sequence similarity to Current Biology a number of other proteins (a) Pyrin domain multiple alignment. For NALP1; AF310106 for NALP2; AAF66964 (Figure 1a). each block of aligned sequences, dark blue for zebrafish CASPY; AAF66956 for Pycard is a 22 kDa protein that boxes indicate > 50% sequence zebrafish Pycard. (b) Domain structure of forms aggregates when apoptosis is identity and light blue shading indicates proteins containing a Pyrin domain. induced by certain anti-tumor drugs > 50% sequence similarity through domain are named as follow: conservative amino acid substitutions. In PYD for Pyrin domain; CARD for caspase [3]. Moreover, in cells forced to the consensus line, ! indicates 100%, * recruitment domain; NACHT for NAIP, express reduced levels of Pycard, > 50% and – < 50% sequence similarity. CIITA, HET-E and TP1 domain; LRR for etoposide-mediated apoptosis is abbreviation: HS, Homo sapiens leucine-rich repeats; SPRY for domain in significantly suppressed. Using the and DR, Danio rerio. Genebank/EMBL SPla and the ryanodine receptor. B for accession numbers are: AF310103 for B-box. (c) Amino acid sequence of NALP1. PYD of Pycard, a simple BLAST human Pycard; AF310104 for murine The shadings of the boxes correspond to search of protein databases Pycard; O15553 for Pyrin; AF310105 for the domains as presented in Figure 1b. identified Pyrin as an additional PYD- containing protein as previously reported [3]. Pyrin was Mediterranean fever [4,5], a Based on the PYD sequence initially identified as the product of hereditary periodic fever syndrome alignment between Pyrin and the MEFV , which is mutated characterized by episodic fever and Pycard — 32.5% identity with an in patients with familial serosal or synovial inflammation. E-value of <1 × 10–5 —a Magazine R119

Figure 2

helix 1 helix 2helix 3 helix 4 helix 5 (a) ααααααααααααα αααααα αααααα αααααααααααα αααααααααααααααααααααααααααhelix 6 (b) PycardHS 12: L EN LPA EE LKKFKLKL LS. VPLREGY...... GRIPRDLGALLSMDALL ...TDK.. D L VSFYLETY..GAELT ANVLRDMGLQEMAGQLQAAT PYD Pyrin HS 13: LEELVPYDFEKFKFKL QN.TSVQKEH...... SRIPRKMATLSQIQRARPVV..... L VTYYGEEY..AVQLT LQVLRAINQRLLAEELHRAA αααααααααααααααα αααααααααα ααααααααα ααααααααααααααα αααααααααααααα ααααααα FLAG-

APAF-1HS 5: A RN CLLQHREALEKDIKTSYIMDHM MISDGF.. LTISEEEKVVR NEPTQQ.. .Q RAAML IKMILKKD..NDSYV.SFYNAV L LHEGYK.DLAALL NALP1-PYD Mock RAIDD NALP1-CARD Pycard -PYD Apaf1-CARD CARD CED-3CE 5: R RS LLERNIMMFSSHL KVDEIL EVLIAKQV.. LNSDNGDMINSCGTVRI .. .E KRREIVKAVQRRG... DVA FDAFYDAL RSTGHE.GLAEVL IgGH CED-4CE 6: E CR ALSTAHTRLIHDFEPRDALTYL LEGKNI.. FTEDHSELISKMSTRLI .. .E RIANFLRIL YRRQA....SELGL PLIDFFNYNNQS.HLADFL IP α-FLAG αααααααααα ααααααααααα αααααααααα ααααααααααααα ααααααααααααα ααααααα WB α-VSV FASHS 204: S DV DLSKYITTIAGVMT.QVKGFVRKNG LS V .. VNEAKI DEIKNDNVQDTA.EQKVQI E L LRNL WHQLHGKK. EAYDTLIKDL KKANLC.TLAEKI VSV-Pycard DD FADDHS 93: G EE DLCAAFNVICDNVG. . KDWRRLARQLK.. VSDTKDSI IEDRYPRNLT.ERVRESI E LRIL WKNTEKEN. ATVAHV LVGAL RSCQMN.LVADLV RIPHS 579: T TS LTD KH LDPIRENL GKH.. WKNCARKLG.. FTQSQIDEIDHDYERDGLKEKVYQI E MLQKL WVMREGIKGATVGKV LAQAL HQCSRIDLLSSLI ααααααααααααα ααααααααα αααααααα ααααααααααα ααααααααααα ααααααα Cell extracts Caspase8HS 1: M D.. FS RN LYDIGEQL DS.EDL ASLKFLL SLDYIPQRKQEPIIKDKL...... ALMLFQRLQEKRMLEESNLL S FLKEL LFRINRI .... LDL LITY VSV-Pycard WB α-VSV DED FADDHS 1: M DD PFL VL LHSVSSSL SS.SEL TELKFLCLGL RVGKRKLERVQS...... GLDLFSMLLEQNDLEPGHTML E LLLREL LASLRRL .... HDL LRRV PEA-15HS 1: M VE. YG TL FQD LTNNITL.EDL EQLKSACKEDL IPSEKSEEITTI ...... GSAWFSFLESHNKLDKDNLL S IIIEHIFEISRR.... PDL LTMV WB α-FLAG FLAG-tagged proteins Current Biology

(a) Alignment of representative pyrin domains DD, CARD and DED solution structures NALP1 (NALP1-CARD), and an empty vector (PYD), death effector domains (DED), respectively [11]. (b) Pycard homodimerizes (Mock) were cotransfected in 293T cells with caspase recruitment domains (CARD) and via its PYD and interacts with the PYD of a VSV-tagged Pycard construct. Anti-FLAG death domains (DD) respectively, showing NALP1. FLAG-tagged construct containing immunoprecipitates were analyzed for the the similarity of these interaction domains. the PYD of Pycard (Pycard-PYD), RAIDD, presence of VSV-Pycard. Expression of the α lines indicate the predicted α-helices for the CARD of Apaf-1 (Apaf1-CARD), the PYD different constructs was analyzed in the cell the PYD [14], and the indicated α-helices in of NALP1 (NALP1-PYD), the CARD of lysates (lower panel).

generalized PYD profile was (Figure 1b,c). The NACHT and to form both homodimers and generated [6] and used in a LRR domain architecture is found heterodimers, and each domain is subsequent search of protein in proteins known to be involved highly conserved [10]. NMR databases. In addition to zebrafish in inflammation or apoptosis, in analysis has confirmed the Pycard, two other PYD-containing particular in CARD4/Nod1 structure prediction for these proteins, NALP1 and NALP2 (Figure 1b), an NF-κB-inducing domains [11], and their overall (NACHT-, LRR- and PYD- molecule, the neuronal apoptosis topology is very similar, in containing proteins) were found, inhibitor protein NAIP, and the particular with respect to the with an E-value of <1 × 10–2. A MHC class II transcription structural core formed by helices further combination profile activator CIITA [8]. Interestingly, α2 to α5 (Figure 2a). idendified the zebrafish caspase, the PYD of NALP2 is replaced by a Including the PYD-containing CASPY, with an E-value of CARD in CARD4/Nod1, whereas sequences in a general alignment <1 × 10–10. The PYD family could the overall structural organization is with the DED, CARD and DD, we also be assembled when the amino identical (Figure 1b). CASPY is a identified PYD as a potential acid sequence of Pyrin between PYD- and caspase-domain- fourth member of the death- position 11 and 89 was used as the containing protein that was initially domain-fold superfamily query for searching protein identified in a database search for (Figure 2a). To demonstrate that databases; the BLAST program zebrafish homologues to PYDs are indeed functional retrieved Pycard with an E-value of mammalian apoptosis regulators protein–protein interaction <1 × 10–5. A single iteration [9]. This caspase is most similar to domains, we generated expression BLAST search using the PSI- CASPASE-13, which in human vectors for several PYD-containing BLAST program [7] — with the contains a CARD instead of the proteins and tested their cut-off for inclusion of sequences PYD. interactions with other proteins in in he profile set at E = 3 × 10–3 — Based on sequence alignments, co-immunoprecipitation retrieved NALP2 with E = 2 × 10–3, we previously proposed that DD, experiments. Despite their and the zebrafish homologue of DED, and CARD are structurally similarity in folding, DD, DED Pycard with E = 3 × 10–3. A second related protein–protein interaction and CARD were found to interact iteration retrieved NALP1 with modules [10]. All three domain exclusively with members of their E=6× 10–2 and CASPY with types have a similar size, and own subfamily. As shown in E=1× 10–11. secondary structure prediction Figure 2b, when a VSV-tagged Sequence analysis revealed that analysis proposed a similar Pycard was co-expressed with a NALPs contain a PYD, NACHT arrangement of six α-helices. The FLAG-tagged construct and LRR modular organization three domains also share the ability containing the PYD of Pycard, R120 Current Biology Vol 10 No 4

homodimerization of the PYDs was latter fold is an almost exact mirror 7. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: observed. Moreover, Pycard also image of the six-helix fold of DD, Gapped BLAST and PSI-BLAST: a new interacted with the PYD of DED and CARD. While none of generation of protein database search NALP1, indicating that PYDs can these results prove unequivocally programs. Nucleic Acids Res 1997, 25:3389-3402. form specific heteromers. No that PYD is related to the death 8. Koonin EV, Aravind L: The NACHT family - interactions of Pycard’s PYD with domains by evolution, the a new group of predicted NTPases DDs, CARDs or DEDs (Figure 2b cumulative evidence presented implicated in apoptosis and MHC transcription activation. TIBS 2000, and data not shown) were found. here makes this relationship very 25:223-224. This experiment identifies PYD as likely. 9. Inohara N, Nunez G: with homology to mammalian apoptosis a protein–protein interaction In conclusion, we have regulators identified in zebrafish. Cell domain capable of binding to other identified PYD as a novel Death Differ 2000, 7:509-510. PYDs. protein–protein interaction 10. Hofmann K, Bucher P, Tschopp J: The CARD domain: a new apoptotic While there is no strong module that is presumably a signalling motif. Trends Biochem Sci sequence similarity between the member of the death-domain-fold 1997, 22:155-156. 11. Fesik SW: Insights into domain subfamilies, they family. PYDs are found in death through structural biology. Cell share an interaction domain association with proteins 2000, 103:273-282. containing six α-helices, a similar implicated in apoptosis and 12. Kelley LA, MacCallum RM, Sternberg MJ: Enhanced genome annotation using three-dimensional fold, a common inflammation as best illustrated by structural profiles in the program 3D- mode of interaction, and occur the zebrafish caspase CASPY. The PSSM. J Mol Biol 2000, 299:499-520. 13. Itoh N, Nagata S: A novel protein domain together in apoptotic signaling identification of novel PYD- required for apoptosis. Mutational proteins. PYD also tends to occur in containing proteins is therefore analysis of human Fas antigen. J Biol the same proteins as CARD, or likely to facilitate the Chem 1993, 268:10932-10937. 14. Rost B, Sander C: Secondary structure replaces CARD in the case of the characterization of novel pro- prediction of all-helical proteins in two zebrafish caspase. Moreover, PYD apoptotic or pro-inflammatory states. Protein Eng 1993, 6:831-836. is of the same size as DD, DED signaling pathways, as was the and CARD, and secondary case after the identification of Acknowledgements structure prediction programs the DD of Fas a few years ago [13]. We thank Kim Burns for critical reading of the manuscript and Marco Pagni and Thomas Junier indicate that PYD has a similar for help with the HIT database. helical structure. Finally, different Supplementary material PYDs interact with each other, Supplementary material including detailed Addresses: *Institute of Biochemistry, strongly suggesting that PYDs are methods for sequence and structure analysis, University of Lausanne, BIL Biomedical and for cloning, expression and interaction not only structurally similar to the Research Center, Chemin des Boveresses analysis of the PYD-containing protein is 155, CH-1066 Epalinges, Switzerland. other six-helix interaction domains available at http://current-biology.com/ † supmat/supmatin.htm. MEMOREC Stoffel GmbH, Stoeckheimer but also serve a similar adaptor Weg 1, D-50829 Köln, Germany. function in a different set of proteins. References The 1. Aravind L, Dixit VM, Koonin EV: ‡These authors contributed equally to this To investigate whether PYDs domains of death: evolution of the have both the same arrangement of apoptosis machinery. TIBS 1999, work. helices, and a topology that is 24:47-53. 2. Hofmann K: The modular nature of similar to the established apoptotic apoptotic signaling proteins. Cell Mol Life Correspondence: Jürg Tschopp signaling domain, we performed a Sci 1999, 55:1113-1128. 3. Masumoto J, Taniguchi S, Ayukawa K, E-mail: [email protected] threading analysis with the 3D- Sarvotham H, Kishino T, Niikawa N, Hidaka PSSM program (Enhanced Genome E, Katsuyama T, Higuchi T, Sagara J: ASC, Annotation using Structural Profiles a novel 22 kDa protein, aggregates during apoptosis of human in the Program 3D-PSSM [12]). promyelocytic leukemia HL-60 cells. J The editors of Current Biology welcome Most of the isolated PYDs failed to Biol Chem 1999, 274:33835-33838. correspondence on any article in the give significant scores but 4. InternationalFMFConsortium: Ancient journal, but reserve the right to reduce missense in a new member of the length of any letter to be published. consistently had death-domain-like the RoRet gene family are likely to cause All Correspondence containing data or familial Mediterranean fever. The folds — PDB:1NGR, 1FAD, 1A1W, scientific argument will be refereed. International FMF Consortium. Cell 1997, 1CY5A — among the five top- 90:797-807. Items for publication should either be scoring hits. Using the PYD of 5. FrenchFMFConsortium: A candidate gene submitted typed, double-spaced to: The zebrafish CASPY as query, we for familial Mediterranean fever. The Editor, Current Biology, Elsevier French FMF Consortium. Nat Genet 1997, found three hits with E-values <1: Science London, 84 Theobald’s Road, 17:25-31. London, WC1X 8RR, UK, or sent by the death domains of FADD and 6. Bucher P, Karplus K, Moeri N, Hofmann K: A flexible search technique based on electronic mail to NGF-receptor, and the saposin fold generalized profiles. Computer Chem [email protected] of NK-Lysin. Interestingly, the 1996, 20:3-24.