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 pyrin domain: 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 death domain-fold Consensus 1: ****-******!*-***---*-**---*****-*****--*********-*---***-**********-*-*****-*** :80 family implicated in (b) Pycard PYD CARD apoptosis and inflammation Pyrin PYD B SPRY Fabio Martinon*, CASPY PYD CASPASE †‡ 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 proteins and caspases is LHTWEQMGLRSLCAQAQEGAGHSPSFPYSPSEPHLGSPSQPTSTAVLMPWIHELPAGCTQGSERRVLRQL primarily mediated by three PDTSGRRWREISASLLYQALPSSPDHESPSQESPNAPTSTAVLGSWGSPPQPSLAPREQEAPGTQWPLDE structurally related protein–protein TSGIYYTEIREREREKSEKGRPPWAAVVGTPPQAHTSLQPHHHPWEPSVRESLCSTWPWKNEDFNQKFTQ LLLLQRPHPRSQDPLVKRSWPDYVEENRGHLIEIRDLFGPGLDTQEPRIVILQGAAGIGKSTLARQVKEA domains called death domain (DD), WGRGQLYGDRFQHVFYFSCRELAQSKVVSLAELIGKDGTATPAPIRQILSRPERLLFILDGVDEPGWVLQ death effector domain (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% amino acid 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 Homology 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 Species 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 gene, 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