Molecular and genetic characterisation of sea bass caspases

Molecular and genetic characterisation of sea bass caspases. Marta Inês Rocha Reis Tese de doutoramento em Ciências Biomédicas 2012 Marta Inês Rocha Reis Molecular and genetic characterisation of sea bass caspases. Tese de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutor Nuno Miguel Simões dos Santos Categoria – Investigador auxiliar Afiliação – Instituto de Biologia Molecular e Celular Co-orientador - Doutora Ana Maria Silva do Vale Categoria – Investigadora auxiliar Afiliação – Instituto de Biologia Molecular e Celular iii De acordo com o disposto no Decreto-Lei nº 74/2006 de 24 de Março, esclarece-se serem da nossa responsabilidade a execução das experiências que estiveram na origem dos resultados apresentados, assim como a sua interpretação, discussão e redação. Nesta tese foram apresentados os resultados contidos nos artigos já publicados e seguidamente discriminados: Marta I.R. Reis, Carolina Costa-Ramos, Ana do Vale and Nuno M.S. dos Santos. (2010) Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis. Fish & Shellfish Immunology 29(1): 58-65. Nuno M.S. dos Santos, Ana do Vale, Marta I.R. Reis and Manuel T. Silva (2008) Fish and Apoptosis: Molecules and Pathways. Current Pharmaceutical Design 14(2): 148-169. Marta I.R. Reis, Ana do Vale, Cristina Pinto, Diana S. Nascimento, Carolina CostaRamos, Daniela S.P. Silva, Manuel T. Silva and Nuno M.S. dos Santos (2007) First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia. Molecular Immunology 44(7): 1754-1764. Marta I.R. Reis, Diana S. Nascimento, Ana do Vale, Manuel T. Silva and Nuno M.S. dos Santos (2007) Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene. Molecular Immunology 44(5): 774-783. Durante esta longa jornada contei com a ajuda de muitas pessoas que directa ou indirectamente tornaram esta tese possível. A todas elas deixo aqui o meu profundo agradecimento. vii TABLE OF CONTENTS Abbreviation list ................................................................................................................ ix Abstract ........................................................................................................................... xi Resumo .......................................................................................................................... xv Chapter I - Introduction ..................................................................................................... 1 1. Caspases ........................................................................................................... 3 1.1. Classification ........................................................................................ 3 1.2. Functions ............................................................................................. 6 1.3. Activation and regulation ..................................................................... 8 2. Fish caspases .................................................................................................. 12 2.1. Fish apoptotic initiator caspases ........................................................ 14 2.2. Fish apoptotic executioner caspases ................................................. 18 2.3. Fish putative apoptotic caspases ....................................................... 20 2.4. Fish inflammatory caspases .............................................................. 21 References .......................................................................................................... 25 Chapter II - Project Aims ................................................................................................ 37 Chapter III - Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis. ............ 41 Chapter IV - First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia. .................................................................. 51 Chapter V - Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene. ............................................................................................................. 65 Chapter VI - Caspase-1 and IL-1β processing in sea bass. ............................................ 77 Chapter VII - Concluding remarks and future perspectives. .......................................... 121 Annex I - Fish and Apoptosis: Molecules and Pathways. ............................................. 129 xv Resumo As caspases são proteases cisteínicas com especificidade para aspartatos extremamente conservadas ao longo da filogenia. Tornaram-se conhecidas principalmente devido ao seu envolvimento na apoptose e inflamação embora, hoje em dia, a sua participação noutros processos biológicos esteja bem documentada. Ainda assim, as caspases continuam a ser classificadas como apoptóticas ou pró-inflamatórias. As caspases apoptóticas podem ser subdivididas em (i) iniciadoras (caspase-2, -8, -9 e -10) e (ii) executoras ou efetoras (caspase-3, -6 and -7). As caspases iniciadoras são mais especializadas em clivar os seus próprios precursores, as caspases efetoras e um pequeno número de outros substratos, enquanto que as caspases efetoras estão sobretudo envolvidas na clivagem de vários substratos celulares, levando às alterações morfológicas típicas da apoptose. As caspases pro-inflamatórias (caspase-1, -4, -5, -11 e -12) têm uma função crucial no processamento de citoquinas inflamatórias. As caspases são sintetizadas como pro-enzimas compostas por um pro-domínio amino-terminal extremamente variável em sequência e tamanho e envolvido na regulação da ativação da pro-enzima, uma subunidade grande (p20) contendo a cisteína reativa no contexto de um motivo QAC(G/R/Q/)G conservado, e uma subunidade pequena (p10). Algumas pro-caspases também têm um pequeno fragmento de ligação entre as subunidades grande e pequena. Em resposta a diversos estímulos externos ou internos, as pro-caspases são ativadas por processamento proteolítico específico no lado carboxilo de um resíduo de ácido aspártico (Asp), resultando na remoção do pro-domínio e na formação de um hetero-tetrâmero com dois locais ativos, composto por duas subunidades p20 e duas subunidades p10. As caspases clivam os seus substratos num resíduo Asp específico que tem de estar no contexto de um motivo tetrapeptídico. Diferentes caspases reconhecem motivos tetrapeptídicos distintos, estando esta diferença na base da diversidade das funções biológicas das caspases. A ativação das caspases apoptóticas iniciadoras e das caspases pró-inflamatórias ocorre em complexos multiproteicos, conhecidos como DISC para a caspase-8, apoptosoma para a caspase-9, e inflamasoma para a caspase-1. As referidas plataformas multiproteicas conduzem a um aumento da concentração local das procaspases, permitindo a auto ativação das mesmas por um mecanismo de dimerização induzido por proximidade. As caspases efetoras não necessitam de um mecanismo tão complexo de ativação, uma vez que são normalmente ativadas por processamento proteolítico efetuado por caspases previamente ativadas ou por outras proteases, como por exemplo a Granzima B. Desta forma, as caspases formam uma cascata de eventos xvi proteolíticos, onde as caspases iniciadoras despoletam a maquinaria efetora da morte, enquanto que as caspases efetoras clivam principalmente substratos celulares, levando à ocorrência das alterações bioquímicas e morfológicas características da apoptose. A atividade das caspases encontra-se altamente regulada a vários níveis, desde a sua expressão genética até aos vários passos da sua produção, processamento e atividade. A ativação do programa apoptótico, em resposta a um sinal de morte, está regulada por um balanço preciso entre os mecanismos pro-apoptóticos e antiapoptóticos, no qual os membros da família Bcl-2 têm um papel fundamental através da regulação da permeabilização da membrana externa mitocondrial (MOMP), a qual permite a saída de diversas proteínas reguladoras do espaço intermembranar da mitocôndria. Para além da regulação efetuada através de diferentes fatores proteicos, as atividades das caspases também são reguladas por isoformas. Aquando do início do presente trabalho, o envolvimento das caspases dos mamíferos em diversos processos biológicos estava bem documentado. No entanto, a informação relativa às caspases dos peixes era bastante limitada, e apenas algumas caspases de peixes tinham sido identificadas e caracterizadas. Dada a importância da investigação em peixes, não só para apoiar o desenvolvimento da aquacultura mas também como contribuição para o estudo de diversos aspetos fisiológicos e patológicos em mamíferos, e dada a participação da apoptose e inflamação em vários processos biológicos e patologias de vertebrados, a investigação das caspases em peixes é, obviamente, relevante. O presente trabalho foi desenvolvido no grupo de Imunologia e Vacinologia de Peixes do IBMC que, durante os últimos anos, tem vindo a usar o robalo para estudar o mecanismo de patogenicidade da Photobacterium damselae ssp. piscicida como um exemplo de uma infeção bacteriana onde o agente patogénico subverte as maquinarias apoptótica e pro-inflamatória do hospedeiro. O plano de trabalho desta tese foi desenvolvido nesse contexto, tendo como objetivo a clonagem, sequenciação e caracterização genética de representantes de todas as classes de caspases no robalo. As caspases iniciadoras da via extrínseca e intrínseca, caspase-8 e -9, respetivamente, assim como a principal caspase executora da apoptose, a caspase-3, foram identificadas no genoma do robalo. A nível molecular, as sequências da caspase-8, -9 e -3 obtidas retêm os motivos que são funcionalmente importantes, tais como o motivo catalítico pentapeptídico (QACXG) e os locais de processamento (em ácidos aspárticos) que resultam na organização característica das caspases em pro-domínio, grande e pequena subunidades. Na caspase-8 de robalo, dois domínios efetores de morte (DED) foram previstos por análise bioinformática. A mesma abordagem permitiu identificar um domínio de recrutamento de caspases (CARD) no pro-domínio da caspase-9 de robalo. Tanto o DED como o CARD são domínios importantes envolvidos em interações xvii proteína-proteína necessários para mediar a dimerização e consequente ativação das caspases-8 e -9, respetivamente. Dados obtidos durante este trabalho revelaram baixos níveis de expressão das caspases-8, -9 e -3 em diversos órgãos de robalo não estimulados e um aumento de expressão das mesmas no baço em resposta a um estímulo apoptótico. Este trabalho originou informação que contribuiu para uma melhor compreensão da biologia das caspases em peixes e que permitiu o desenvolvimento de ferramentas para estudar os mecanismos da apoptose em robalo. Neste trabalho, o gene da caspase-1 de robalo foi também clonado, sequenciado e caraterizado. Foi mostrado que a caspase-1 de robalo é auto-processada de forma semelhante à da sua homóloga de mamíferos, resultando em heterodímeros ativos de p24/p10 e p20/p10. A existência de variantes da caspase-1 originadas por processamento alternativo em robalo foi também reportada. A existência de isoformas da caspase-1 em peixes e em mamíferos sugere que foram mantidas durante a evolução filogenética e portanto muito provavelmente desempenham uma função reguladora na resposta inflamatória. O papel da caspase-1 no processamento de citoquinas inflamatórias em vertebrados que não mamíferos tem vindo a ser objeto de intenso debate. Os resultados aqui apresentados contribuíram para clarificar essa questão, uma vez que mostraram que as IL-1β de robalo e de aves são clivadas especificamente pela caspase-1 em aspartatos conservados filogeneticamente, distintos do local de clivagem presente na IL-1β de mamíferos. Em conclusão, as caspases apoptóticas-8, -9 e -3 assim como a caspase-1 do robalo, foram sequenciadas e caracterizadas. A identificação destas caspases em robalo permitiu a produção de novas ferramentas que foram usadas para estudar o envolvimento das caspases de robalo em processos apoptóticos e para caracterizar bioquimicamente a caspase-1. No futuro, estas ferramentas poderão ser aplicadas na clarificação do envolvimento das caspases do robalo em mais situações envolvendo processos apoptóticos e/ou inflamatórios assim como no estudo de outras funções desempenhadas pelas caspases. Adicionalmente, a demonstração de que a caspase-1 de um peixe teleósteo processa a proIL-1β na sua forma matura abre portas ao estudo do papel da caspase-1 em processos imunológicos em vertebrados que não mamíferos. Nos últimos anos, tornou-se evidente a importância dos inflamasomas em diversas patologias, tais como doenças auto-imunes e infeciosas. Em diversas infeções de mamíferos, foi mostrado que a ativação dos inflamasomas é acompanhada pela indução de piroptose, como tentativa do hospedeiro controlar a replicação do agente infecioso. No entanto, em peixes, não existem quaisquer dados no que respeita a inflamasomas ou piroptose. A clarificação dos mecanismos moleculares envolvidos na ativação da caspase-1 ao nível dos inflamasomas nos peixes, assim como a subversão os inflamasomas por diferentes xviii agentes patogénicos, apresentam-se como novas linhas de investigação que podem vir a ser exploradas no futuro. Por último, é de salientar que há dados recentes sobre novos mecanismos de regulação das caspases de mamíferos pelas suas isoformas e, assim sendo, o estudo do papel das isoformas da caspase-1 do robalo na regulação da atividade da caspase-1 é de grande importância. CHAPTER I Introduction* * Parts of this chapter were adapted from dos Santos NM, do Vale A, Reis MI, Silva MT. Fish and apoptosis: molecules and pathways. Current Pharmaceutical Design (2008) 14(2):148-69 (Annex I). Chapter I - Introduction 3 Introduction 1. Caspases The family of intracellular Cysteine Aspartyl-Specific Proteases, known as caspases, is highly conserved in multicellular organisms and their members share common features such as having high specificity to cleave their substrates after aspartic acid (Asp) residues [1]. Caspases are usually described as being the key players responsible for the morphological and biochemical changes occurring during apoptosis (Box 1). Nevertheless, when the first caspase was identified, there was no indication of its involvement in apoptosis. In 1992, the mammalian interleukin-1 beta (IL-1β) converting enzyme (ICE or caspase-1) was cloned and described as being responsible for the processing of pro-inflammatory cytokines, in particular interleukin-1 beta, into their active forms [2, 3]. The association of a caspase to cell death was reported for the first time one year later, when the ced-3 gene, the mammalian caspase-9 homolog, was cloned in the nematode Caenorhabditis elegans and was shown to encode a protein that was implicated in cell death [4]. The crucial role of caspases in apoptosis and inflammation is well recognised almost since their discovery and recent and very elegant studies have, associated apoptotic caspases with the regulation of multiple cellular processes beyond cell death, such as cell migration, proliferation and differentiation, thus amplifying the functions of the apoptotic caspases to non-apoptotic purposes [5-9]. Nowadays, the idea that caspases are important proteins involved in a wide range of biological reactions besides cell death is well established [6, 8, 9]. For this reason, the interest to understand what drives the activation and regulation of caspases increased and currently caspases are among the most studied proteins in science biology, with thousands of scientific papers on the subject being publish every year. 1.1. Classification All caspases share similarities in amino acid sequence, structure and substrate specificity [10]. These proteases are constitutively expressed as zymogens (pro-enzymes) of about 30 to 55 kDa and are composed of a NH2-terminal pro-domain, which is highly variable in sequence and length and is involved in the regulation of the pro-enzyme activation, a large subunit of approximately 20 kDa (p20) containing the cysteine active site within a conserved QAC(G/R/Q/)G motif, and a C-terminal small subunit of approximately 10 kDa (p10) (Figure 1). Some pro-caspases have also a short linker of about 10 amino acids between the large and small subunits [10-13]. Functionally, caspases can be classified as apoptotic or pro-inflammatory. Chapter I - Introduction 4 Box 1. Apoptosis Apoptosis [14] is a genetically controlled and evolutionarily conserved form of active cell death, with an increase in complexity during phylogeny. Apoptosis is critical in the elimination of redundant, ectopic, damaged, mutated or infected cells and, therefore, plays an essential role in normal development, tissue homeostasis and immunity [7, 15-17]. Cells dying by apoptosis shrink and loose the connections with the surrounding cells. In general, organelles remain intact in the cytoplasm, despite some observations that describe an increase in endoplasmic reticulum size, the release and aggregation of ribosomes, the enlargement of the cisternae to form vesicles and vacuoles and the occurrence of changes in the mitochondria network [18, 19]. The main changes in the nucleus involve the chromatin condensation (pyknosis), DNA fragmentation by endonucleases into small fragments and nuclear fragmentation (karyorrhexis) [19]. Finally, the blebbing of the plasma membrane of the cell that has already lost the contact with their neighbours becomes perceptive. In some cases, the cell breaks into small fragments of various sizes containing the cellular contents and surrounded by membrane (apoptotic bodies) [19]. Apoptotic cells and apoptotic bodies are, in vivo, engulfed and digested mainly by macrophages, resulting in the elimination of the dying cells before they lyse and, therefore, without induction of an inflammatory response [14, 19-22]. In mammals, apoptosis may follow two principal pathways (Box 2): (i) the extrinsic/death-receptor pathway, initiated at the tumour necrosis factor (TNF) family receptors or at the dependence receptors [23] and (ii) the intrinsic/Bcl-2regulated/mitochondrial pathway, regulated by the Bcl-2 family proteins and mainly characterised by mitochondrial membrane permeabilization (MMP) [23]. Apoptotic caspases can be further subdivided into: i) upstream initiator caspases (caspase-2, -8, -9 and -10) and (ii) downstream executioner or effector caspases (caspase-3, -6 and -7). The pro-forms of the initiator caspases possess large NH2terminal pro-domains, containing either death effector domains (DED) (caspases-8 and - 10) or a caspase-associated recruitment domain (CARD) (caspases-2 and -9); these domains are responsible for the interaction with upstream adaptor molecules and thereby Adapted from [24] Cells undergoing apoptosis in response to the apoptogenic toxin AIP56 [25]. Chapter I - Introduction 5 have a relevant role in the activation of these caspases. The initiator caspases are more specialised to cleave their own precursors, downstream effector caspases and a small number of other substrates [13, 26]. The executioner or effector caspases have pro-forms containing only short NH2-terminal pro-domains thought to be involved in subcellular targeting. Effector caspases cleave and inactivate cellular proteins, which will lead to cellular destruction, although they can also activate other caspases. Pro-inflammatory caspases (caspase-1, -4, -5, -11, -12, -13 and -14) are synthesised as pro-forms with large NH2-terminal pro-domains containing CARD, and their main role lies in cytokine maturation rather than apoptotic activity [10, 13, 27]. Figure 1. Structure and classification of the caspase family. (A) Diagrammatic representation of the three major groups of caspases: pro-inflammatory (group I), apoptotic initiator (group II), apoptotic executioner (group III). (B) Schematic representation of pro-caspase activation. (C) Three-dimensional structure of the caspase-3 heterotetramer composed of two heterodimers p17/p12. NH2and CH2termini of the small and large subunits are indicated. Adapted from [28]. Chapter I - Introduction 12 preventing the formation of a functional apoptosome [86, 87]. A caspase-9 isoform, Casp9-Carboxyl-terminal divergent (CTD), was also identified in rat and CTD was shown to have an alternative peptide sequence replacing the C-terminal of the full-length caspase9. Like the previous discussed caspase isoforms, CTD is expressed in several tissues and was implicated in the regulation of cell death, since it interferes with the activation of caspase-9 [88]. Another important mechanism for regulating the caspase-8 is the multiple splicing of caspase-8 gene. Caspase-8 co-expressed with caspase-8 isoforms possessing incomplete caspase regions resulted in decreased cell death [89, 90]. For example, caspase-8L, modulates the caspase-8 activity by the homotypic binding to FADD. This isoform exerts its dominant effect by preventing the coupling of pro-caspases-8 and subsequent apoptosis by the receptor pathway [91]. In human, four additional isoforms of caspase-1 were identified: the isoform β and γ, lacking part of the pro-domain, and the isoforms δ and ε, where most of the pro-domain and the region containing the active site have been deleted [92]. Like the caspase isoforms described above, these isoforms may have a regulatory role in the activation process of caspase-1. For instance, in the isoform ε most of the pro-domain and all the p20 subunit were deleted, meaning that this isoform has only the p10 subunit with 19 different amino acids in the NH2-terminus. This incomplete p10 prevents the formation of an active tetramer by competing with the p10 subunit of caspase-1 [92]. 2. Fish caspases A high conservation at the molecular and functional levels has been described between the players of the apoptotic machinery in invertebrates (Caenorhabditis elegans and Drosophila) and mammals [93, 94]. However, fish represent an excellent and advantageous model for the study of vertebrate development and disease [95-98], bridging the gap between the C. elegans/Drosophila and mouse/human models [99]. Moreover, contrary to C. elegans and Drosophila, fish can be used for studying the development and function of vertebrate-specific organs and have a fully developed immune system similar to that of mammals [100]. Despite the increasing interest in caspases and their evolutionarily conserved function, until the beginning of the 21st century, the information on caspases was limited to mammals (human and mouse), Drosophila, C. elegans and to a few reports on chicken [101], frog [102], and a few fish species [103-105]. Since then, the number of sequenced caspases from different species has been increasing. At the moment, 11 and 10 caspase genes were identified in the human and mouse genomes, respectively. In humans, the 11 genes encode caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 and -14. The mouse caspases are functional orthologs of Chapter I - Introduction 13 Box 5. Bcl-2 family Bcl-2 family members have been extensively studied in mammalian systems, mainly due to their importance as key regulators of apoptosis, playing a central role in some pathological conditions, including cancer, neurodegenerative disorders and autoimmune diseases [45, 73, 106]. Briefly, Bcl-2 family members include proteins with sequence and structural similarity in the so-called Bcl-2 homology (BH1 to BH4) domains. Bcl-2 proteins regulate the response to diverse cytotoxic conditions such as UVand γ-irradiation, anti-neoplastic drugs, virus and bacteria, but normally have limited impact on the signals induced by the death receptors of the TNF family [107]. Bcl-2 members can be anti-apoptotic (like Bcl-2 and Bcl-xL) or pro-apoptotic (such as Bad, Bik, Bid, Bim). The anti-apoptotic members are mainly characterised by containing three to four BH domains and for inducing cell survival by sequestering/inhibiting pro-apoptotic scaffold or adaptor proteins and pro-apoptotic members needed for activation of caspases, and/or by maintaining organelle integrity [45, 73, 108]. Pro-apoptotic members can be subdivided into BH3-only proteins (“activators” and “derepressors/facilitators”), which, as the name stands for, only contain the third BH domain (Bid, Bim, Bad) [109111], or multidomain pro-apoptotic proteins (Bax and Bak). These share a high degree of structural similarity with pro-survival Bcl-2 family members, containing two to three BH domains, and all having the BH3 domain responsible for the pro-apoptotic activity [76, 108]. At least in some instances, cell death can be prevented at the mitochondrial level by anti-apoptotic Bcl-2 members which, depending on their relative abundance, can inhibit the release of apoptogenic molecules into the cytosol [108]. In addition, Bcl-2 family members are also involved in the regulation of several types of pro-apoptotic signals that are generated in organelles other than mitochondria, such as nucleus, endoplasmic reticulum and lysosomes [45]. That fish have functionally conserved Bcl-2 family members, similar to the ones found in mammals, has been shown by several lines of evidence. Fish have, in their genomes, homologs of virtually all mammalian Bcl-2 family members regulated by similar transcriptional and posttranslational mechanisms. Most of the data related with the Bcl-2 family members of fish refer to zebrafish although their presence in other fish species was also reported such as Bax and Bcl-XL in trout [112], Bcl-2 in gudgeon [113], and Bcl-2 and mlc-1 in carp [114]. Bcl-2 family members in other fish species can also be found in non-redundant and EST (Expressed Sequence Tags) public databases as well as genomic sequence databases (Table 2 in Annex 1). Several fish Bcl-2 family genes have two copies (Table 2 in Annex 1 and [115]) resulting from the early whole-genome duplication in the teleost lineage after the divergence from tetrapods about 450 million years ago [116-118]. Chapter I - Introduction 14 human caspases and maintain the same names with the exception of caspase-11 and -12 that are functional orthologs of caspase-4 and -5, respectively. The other exception is caspase-10 which is absent in the mouse genome [6, 11]. 2.1. Fish apoptotic initiator caspases Caspase-8 (also known as FLICE/MACH1/Mch5) is an essential component of the extrinsic/death-receptor pathway (Box 2 and [23]). The identification and characterisation of caspase-8 from medaka, stickleback, zebrafish and sea bass (Chapter III and [119121]) have recently been reported. At the genomic level, whereas zebrafish caspase-8 genes show a similar structure as their mammalian equivalents, being organised in 10 exons and 9 introns with coincident splice junctions sites [119, 122], the sea bass, medaka and stickleback caspase-8 genes present 12 exons and 11 introns, with the additional exons located at the first death effector and protease domains (Chapter III and [120]). Although results from Southern blot analysis suggested that the sea bass caspase8 gene is a single copy gene (Chapter III), the caspase-8 gene is apparently duplicated in the genome of several species of fish (Table 2 in Annex 1). Chromosome mapping showed that the chromosomal segment around the zebrafish, medaka, fugu and stickleback caspase-8 genes has an organisation syntenic with that of other vertebrates [119, 120]. Fish caspase-8 molecules revealed about 35% identity and 55% similarity to the human counterpart, which together with the above data supports that fish caspase-8 genes are orthologs of mammalian caspase-8. As their amphibian and mammalian homologs, fish caspase-8 possess two DEDs at their NH2-terminal pro-domains, and a carboxyl-terminal catalytic domain (CASc) including conserved amino acids essential for catalytic activity and the characteristic pentapeptide active site QACQG (QACRG in zebrafish caspase-8b) (Chapter III and [119, 120]). That zebrafish caspase-8 associates with the adaptor molecule FADD through homophilic DED interactions was shown by detecting zebrafish caspase-8 and human FADD in immunoprecipitates of HEK293T cells co-expressing HA-tagged human FADD with Flag-tagged zebrafish caspase-8 [119]. Additionally, exogenous expression of zebrafish caspase-8 in caspase-8-deficient mouse embryonic fibroblast (MEF) cells triggered cell death with or without ligation of Fas, the former showing an increased sensitivity, with 80% of the transfectants dying following Fas ligation. This indicates that zebrafish caspase-8 directly transmits apoptotic signals via Fas to downstream molecules, including, as shown in zebrafish caspase-8-expressing HeLa cells, caspase-3; the same holds true for TNFα induced-cell death [119]. Similarly, exogenous expression of medaka caspase-8 induced apoptotic death in transfected HeLa cells, but apoptotic death was inhibited by either zVAD-fmk, the anti-apoptotic cowpox virus serpin cytokine response modifier A (CrmA) [123], or the pan-caspase inhibitor p35 Chapter I - Introduction 15 [120]. Furthermore, co-injection of zebrafish FLIP, or CrmA, reduced zebrafish caspase8a-mediated embryonic mortality, confirming that caspase-8 functions similarly in zebrafish and mammals [124]. The long splice form of the extrinsic pathway inhibitor cellular FLIP (cFLIP) is a key regulator of the extrinsic pathway of caspase-8 activation, inhibiting the recruitment and activation of caspase-8 by death receptors [78, 82]. Like mammalian FLIP, which contains two DEDs that interact with the DED in FADD, zebrafish FLIP is structurally similar to caspase-8 and -10 and lacks an active-site cysteine [124]. Inhibition of caspase-8-mediated apoptosis in early zebrafish embryos has been also shown to occur through interactions of Yaf2 with the DED of caspase-8, with implications on cell survival during zebrafish embryogenesis [121]. Yaf2 is a member of the Rybp/Yaf2 protein family, with Rybp (also known as DEDAF) being described in mammals as interacting with DED-containing proteins, including FADD, caspase-8, caspase-10, and DEDD, for promoting Rybp pro-apoptotic activity [125]. Finally, induction of extensive apoptosis in zebrafish embryos injected with zebrafish caspase-8a mRNAs [124] as well as detection of caspase-8 transcription by RT-PCR in both maternal and zygotic transcripts during zebrafish embryonic development, with ubiquitous expression in whole embryos [119], suggested that caspase-8 plays an important role during embryonic development of fish embryos similarly to mammalian/amphibian embryos. Moreover, in sea bass treated with Photobacterium damselae ssp. piscicida (Phdp) culture supernatants (known to induce apoptosis of sea bass macrophages and neutrophils), the expression levels of caspase-8 in the spleens were increased (Chapter III). The involvement of sea bass caspase-8 in the Phdp infection model is further supported by the detection of caspase-8 activity in sea bass peritoneal phagocytes treated with recombinant AIP56 [126]. The above data confirm that, like its mammalian homolog [90, 127], fish caspase-8 is an initiator caspase recruited to the DISC via binding to the adaptor protein FADD, transmitting apoptotic signals from death receptors to downstream effector caspases; it can also mediate cross-talk between the extrinsic and intrinsic apoptotic pathways, since fish Bid sequences reveal conservation of the caspase-8 cleavage site. Caspase-10, similarly to caspase-8, contains two DEDs and a CASc, and is involved in the Fas ligand-mediated cell death pathway [10, 47]. Both caspases are not clearly distinguished since, for instance, the difference in the activation process is not well studied in detail [128]. Recent studies demonstrated that caspase-10 inhibition prevents apoptosis mediated by the TRAIL-R2 receptor in human immune cells, proving that caspase-10 has an important role in apoptosis mediated by this receptor [129]. Despite caspase-10-like genes with high degree of similarity to human caspase-10 have been reported for fish and can be found in the databases (Table 2 in Annex 1), the only Chapter I - Introduction 16 characterisation of a fish caspase-10 belongs to the Japanese flounder [130]. The gene has 6.6 kb and is organised in 11 exons and 10 introns similar to the human caspase-10. Additionally, the sequence conserves characteristic features such as the DEDs, the large and small subunits and the pentapeptide active site (QACQG), suggesting that it may have a similar function as the human caspase-10. Although the sequence of Japanese flounder caspase-10 revealed 35.3% and 32.6% similarity to human caspase-8 and -10, respectively, the phylogenetic tree clusters the fish caspase-10 with other caspase-10 sequences. The Japanese flounder caspase-10 mRNA was found to be expressed in several tissues and an over-expression in the cell line HINAE resulted in the induction of apoptosis [130]. Since, in addition to caspase-8 and caspase-10, other components essential for forming the DISC, as well as death ligands and the BH3-only member Bid, have been identified in fish (Annex 1), these data strongly point to the existence of a structural and functional conserved extrinsic/death receptor apoptotic pathway in vertebrates. Although caspase-2 was one of the first caspases to be discovered and has been implicated in the regulation of cell cycle progression [131], its role in apoptosis remains a matter of considerable debate [131]. Caspase-2 genes have been identified in fish (Table 2 in Annex 1 and [99, 124]), but no detailed structural and functional characterisation of a fish caspase-2 has been reported so far. The fact that fish caspase-2 gene presents a conserved synteny with the human ortholog and that fish caspase-2 contains a CARD in its pro-domain (Figure 2), suggests that this protease plays a similar role in the apoptotic process of fish and mammals. Caspase-9 (also known as ICE-LAP6/MCH-6/Apaf-3) is a member of the CED-3 subfamily, bearing high similarity to caspase-3, and is a key component of the intrinsic/mitochondrial death-pathway (Box 2 and [23, 132, 133]). Caspase-9 genes have been identified in some species of fish (Table 2 in Annex 1), and the primary structures of sea bass (Chapter IV), and large yellow croaker [134] caspase-9 were characterised. BLAST analysis revealed homology between fish caspase-9 and their vertebrate equivalents, and, as expected, fish caspase-9 presented higher similarity (>90%) and identity (>84%) with other fish counterparts than with that of non-fish vertebrates (46-48% identity and 59-64% similarity). Sea bass and large yellow croacker caspase-9 genes are organised in 10 exons and 9 introns, which, when compared with the human homolog, represents an extra exon located in the predicted pro-domain (Chapter IV and [134]). Southern blot analysis of sea bass genomic DNA suggested that, in fish, caspase-9 gene exists as a single copy gene, an hypothesis that is supported by the fact that only a single caspase-9 gene has been identified in other fish species (Table 2 in Annex 1 and [124]). Additionally, fish caspase-9 genes likely preserve a conserved synteny with the human Chapter I - Introduction 17 ortholog, as indicated by the reported analysis of the zebrafish caspase-9 [124]. Moreover, fish caspase-9 conserves characteristic caspase-9 features (Figure 2) [10] such as a CARD in the NH2-terminal pro-domain, a large (p20) and small (p10) subunits, a caspase family signature, and all important catalytic residues, including the active site pentapeptide QACGG (Table 2 in Annex 1). The presence of CARD in fish caspase-9 is relevant, taking into account that fish apoptotic protease-activating factor 1 (Apaf-1) was identified and contains CARD, a nucleotide-binding oligomerization domain (NO) and WD40 repeats (Table 2 in Annex 1 and [99]). This suggests that, like mammals [135], fish might regulate caspase-9 activity in an Apaf-1/cytochrome c-dependent manner within the apoptosome context [75, 136]. In addition, a homolog of Nod1, an Apaf-1-like molecule that regulates apoptosis and NF-κB activation [137, 138], was found in zebrafish [99]. As with the mammalian equivalents [47, 78, 132, 139], further regulation of fish caspase-9 may be also achieved through inhibition by IAPs, as both IAP and XIAP homologs have been identified in fish (Table 2 in Annex 1 and [99, 124]). Structural studies suggest that the NH2-terminal BIR2 linker region of XIAP occupies the catalytic site in active caspase-3 and -7, whereas the XIAP BIR3 domain is required for inhibiting caspase-9 [47, 78, 132, 139]. Figure 2. Fish apoptotic and inflammatory caspases. Diagrammatic representation of the domain architecture of the executioner and initiator apoptotic caspases, pro-inflammatory caspase-1 and putative apoptotic/proinflammatory caspase pro-enzymes of fish. Chapter I - Introduction 18 The identification of Smac/DIABLO homologs and several serine proteases highly similar to HtrA2/Omi in fish (Table 2 in Annex 1) also indicates that the inhibitory effect of IAPs may be neutralised in a similar way as in mammals, i.e., during apoptotic signalling, Smac/DIABLO and HtrA2 are released from the mitochondria into the cytoplasm where they bind to XIAP and prevent its inhibitory activity [47, 78, 132, 139]. Indications of caspase-9 function and involvement in apoptosis in fish were obtained by detection of caspase-9 transcription by RT-PCR in head kidney of sea bass infected with the apoptotic-inducing bacteria Phdp. The increased expression of caspase-9 correlated with the presence of apoptotic cells and putative caspase-9 activity in the head kidney, the latest detected by a luminescence assay using a proluminescent substrate containing the caspase-9 specific cleavage tetrapeptide LEHD (Chapter IV). Recent studies in large yellow croaker suggested that the intrinsic apoptotic pathway might be implicated in the immune response in spleens and kidneys stimulated with poly(I:C), since the levels of caspase-9 expression and the enzyme activity increase in these conditions [134]. Taken together, the above data represent strong evidence that apoptotic processes involving caspase-9 as well as the mechanisms of caspase-9 regulation are evolutionarily conserved among vertebrates, pointing to the existence of a conserved functional intrinsic apoptotic pathway in vertebrates. 2.2. Fish apoptotic executioner caspases Caspase-3 is the main downstream effector caspase that, once activated by initiator caspases, except caspase-2 [10, 132, 140], cleaves the majority of the cellular substrates in apoptosing cells [10, 132, 140]. Caspase-3 is the most well characterised caspase in fish, including zebrafish [103, 141], salmon [142], sea bass (Chapter V) and large yellow croaker [143]. BLAST search revealed a close relation between fish caspase3 and the caspase-3 molecules of other vertebrates, which is supported by the relatively high percentage of amino acid identities (50-80%) (Chapter V and [103]). Fish caspase-3 presents smaller introns than the human equivalent, and presents a 6 exon/5 intron (7 exon/6 intron in zebrafish) structure, while the human gene has an 8 exon/7 intron structure. However, they all conserve an identical gene organization within the coding region. Although Southern blotting has suggested that sea bass caspase-3 is a single copy gene (Chapter V), it is likely that it is present in all fish genomes as a duplicate gene, as two caspase-3 genes have been identified in other fish species (Table 2 in Annex 1). Whether the duplicates have acquired distinct spatiotemporal functions requires further investigation. As their mammalian homologs, fish caspase-3 present a short NH2-terminal pro-domain and a catalytic domain containing both caspase p20 and p10 subunits, a Chapter I - Introduction 19 caspase family signature and all important catalytic residues, including the characteristic pentapeptide active-site QACRG (Figure 2) (Chapter V and [103, 142, 143]). The apoptotic function of fish caspase-3 has been widely demonstrated. Thus, high proteolytic activity towards the mammalian caspase-3 and -7 substrate Ac-DEVD-MCA was reported for the recombinant zebrafish caspase-3, whereas it had low activity against the caspase1, caspase-6/ -8, and caspase-9 substrates (Ac-YVAD-MCA, Ac-IETD-MCA and AcLEHD-MCA, respectively), indicating a similar substrate specificity to that of mammalian caspase-3 [103]. Moreover, overexpression of the zebrafish caspase-3 in cultured fish cells and zebrafish embryos induced apoptosis that could be repressed by the inhibitor of mammalian caspase-3 Z-DEVD-FMK [103]. Zebrafish embryos microinjected with a zebrafish caspase-3 expression vector also had high Ac-DEVD-MCA-hydrolysing activity. In the same study, Yabu et al also showed that the expression of the zebrafish caspase-3 gene was regulated in both a tissueand stage-specific manner, with caspase-3 mRNA from maternal origin being present in early embryogenesis and after gastrulation by zygotic genome activation, especially in the pectoral fin bud, optic vesicle and hindbrain [103]. Finally, an increase of caspase-3 expression was observed in the head kidney of sea bass infected with the apoptotic-inducing bacteria Phdp, which was accompanied by the occurrence, in that organ, of cells with apoptotic morphology and activated caspase-3 (Chapter V). In the large yellow croaker it was shown that caspase-3 expression and activity are increased in spleen and kidney stimulated with poly(I:C) [134, 143]. Caspase-7 is highly similar to caspase-3 (Figure 2) and has similar substrate specificity [10, 132]. However, at least for apoptosis induced via the intrinsic pathway, a combined function of caspase-3 and -7 is apparently required for cell death execution [10, 132, 144]. Caspase-7 has been identified in several fish species as a duplicated gene (Table 2 in Annex 1) and its primary structure has been recently characterised in salmon [142]. The caspase-7 gene from salmon is organised in a 5 exon/4 intron structure, differing from the mouse and human counterparts, which have a 6 exon/5 intron structure [142]. Caspase-7 mRNA was exclusively detected by whole-mount in situ hybridization in the lens of salmon embryos, while caspase-3b, caspase-6a and -6b were expressed in multiple organs of heat-stressed and control salmon embryos. Moreover, cell death induced in heat-stressed salmon embryos could be correlated with the expression of caspase-7, as well as caspase-6 and -3, in a similar way as in mouse embryos [142]. Although structurally similar to caspase-3 and -7, caspase-6 (Figure 2) has a somewhat different substrate specificity, its optimal substrate being VEHD rather than DEVD, the preferred caspase-3 and -7 substrate [145]. Caspase-6 was demonstrated to have an important role in neurodegenerative diseases, such as Huntington and Alzheimer diseases, through the involvement in neuronal degeneration and death [146]. In fish, it has Chapter I - Introduction 20 been characterised in trout [104] and salmon [142], being identified as a duplicate [142] or even triplicate [124] gene (Table 2 in Annex 1). As caspase-3 and -7, fish caspase-6a and -6b have a relatively short NH2-terminal pro-domain followed by a catalytic C-terminal domain comprising the large and small subunits, the caspase family signature as well as the amino acids involved in catalysis, including the QACRG catalytic site, and those forming the P1 carboxylate binding pocket. Caspase-6a and -6b share high identity (98% amino acid identity between salmon caspase-6a and -6b) as well as high identity with other vertebrate counterparts (>60% amino acid identity). At genomic level, salmon caspase-6a presents a 7 exon/6 intron organization, as its human homolog, whereas the insertion of an intron within the first exon generated an additional exon in the caspase-6b gene, similar to the chicken caspase-6 gene [142]. In human caspase-6, intron 3 is involved in activation by binding of the pro-apoptotic transcription factor p53 [147]. However, as hypothesised by Takle et al [142] for salmon, the different genomic structure of caspase-6a and -6b may cause different regulation of the two genes that may be worth investigating. RT-PCR analysis revealed that the trout caspase-6 gene is expressed in brain, blood, gill, liver, head kidney and spleen [104]. In salmon, caspase-6a and -6b were also expressed in multiple tissues of heat-exposed and control embryos, although cardiac expression of caspase-6a and -6b was only observed in heart of the heat-stressed embryos, with an apparent correlation with cell death [142]. Trout caspase-6 expression in head kidney leukocytes could be specifically increased by addition of LPS, although only in the presence of and following a pre-incubation with cortisol [104]. This effect was mediated by glucocorticoid receptors, as incubation with the glucocorticoid receptor blocker RU486 abrogated the expression of caspase-6 in LPS-stimulated trout leukocytes. Moreover, a marked up-regulation of caspase-6 expression in the trout head kidney was observed upon in vivo induction of confinement stress. Although both glucocorticoids and stress are regarded as inducers of apoptosis in fish [148, 149], no correlation of the observed induction of caspase-6 with cell death was reported by Laing et al [104]. The above data clearly indicate that fish possess a caspase cascade, similar to that characterised in mammals. 2.3. Fish putative apoptotic caspases A caspase-8-like gene, showing high similarity to human and other vertebrate caspase-8 and caspase-10 sequences, but possessing a CARD at their NH2-terminal prodomains instead of DEDs (and, therefore, tentatively named card-caspase-8), has been reported in fish (Figure 2) [105, 119, 120]. The card-caspase-8 is not a third caspase-8/- 10 gene as the one identified in the frog and chicken genomes; instead, it apparently results from replacement of its DED by a CARD upon a tandem duplication of the Chapter I - Introduction 21 ancestral caspase-8 gene over the course of evolution [119, 120]. In addition to the CARD in its pro-domain, card-caspase-8 contains the caspase family signature, the active catalytic site, and the large (p20) and small (p10) subunits at the CASc. Moreover, cardcaspase-8 exhibits pro-apoptotic activity [105, 120]. However, it would be interesting to find out whether fish card-caspase-8 is activated by interacting with death receptors via a CARD-containing adapter molecule such as RAIDD or by recruitment to large multiprotein complexes (as caspase-2) [10, 131, 132], or by heterotypic interaction (DED-CARD) with FADD. Other caspases mostly homologous to mammalian caspase-8 (zebrafish caspaseXa and -Xb), but containing a QACRG active site and lacking obvious NH2-terminal DEDs (Figure 2), have also been reported (Table 2 in Annex 1 and [142]). 2.4. Fish inflammatory caspases Caspase-1, also known as ICE (interleukin-1β converting enzyme), is an inflammatory caspase that normally exists in the cytoplasm and is involved in inflammatory processes and in pyroptosis (Box 3) [68, 150]. As previously mentioned, caspase-1 was the first cysteine protease of the caspase family to be identified and it was also the first in which the three-dimensional structure has been solved, revealing that active caspases are heterotetramers originated from the inactive zymogens [151, 152]. The first caspase-1 homologue identified in fish was the sea bass caspase-1 (Chapter VI) followed by the caspase-1 of sea bream [153]. Both fish caspase-1 retain the CARD in the NH2-terminal pro-domain and the caspase catalytic domain as their mammalian counterpart (Chapter VI and [153]), indicating that a strikingly conserved structure has been kept throughout evolution. In addition, the sea bass recombinant caspase-1 (Chapter VI) is auto-catalytically converted in vitro into two active heterodimeric complexes consisting of p10 paired either with p24 or p20 as described for human caspase-1 [3, 154-156]. Similarly to what has been described for human caspase-1, alternative splicing isoforms were also found in sea bass, all containing the proand p20 domains, including the fully conserved active site QACRG sequence. The differences introduced by the alternative splicing events in the sea bass caspase-1 isoforms are all located downstream from the active site (Chapter VI), contrary to some of the human isoforms [92]. 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Lopez-Castejon, G., et al., Molecular and functional characterization of gilthead seabream Sparus aurata caspase-1: the first identification of an inflammatory caspase in fish. Mol Immunol, 2008. 45(1): p. 49-57. 154. Ramage, P., et al., Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor. J Biol Chem, 1995. 270(16): p. 9378-83. 155. Yamin, T.T., J.M. Ayala, and D.K. Miller, Activation of the native 45-kDa precursor form of interleukin-1-converting enzyme. J Biol Chem, 1996. 271(22): p. 13273-82. Chapter I - Introduction 35 156. Miller, D.K., et al., Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells. J Biol Chem, 1993. 268(24): p. 18062-9. 157. Lopez-Castejon, G., et al., Characterization of ATP-gated P2X7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1 beta. Mol Immunol, 2007. 44(6): p. 1286-99. 158. Masumoto, J., et al., Caspy, a zebrafish caspase, activated by ASC oligomerization is required for pharyngeal arch development. J Biol Chem, 2003. 278(6): p. 4268-76. 159. Liepinsh, E., et al., The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition. J Mol Biol, 2003. 332(5): p. 1155-63. CHAPTER II Project Aims Chapter III - Sea bass Caspase-8 44 Chapter III - Sea bass Caspase-8 45 Chapter III - Sea bass Caspase-8 46 Chapter III - Sea bass Caspase-8 47 Chapter III - Sea bass Caspase-8 48 Chapter III - Sea bass Caspase-8 49 Chapter III - Sea bass Caspase-8 50 CHAPTER IV First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia. Marta I.R. Reis, Ana do Vale, Cristina Pinto, Diana S. Nascimento, Carolina CostaRamos, Daniela S.P. Silva, Manuel T. Silva and Nuno M.S. dos Santos Molecular Immunology (2007) 44(7): 1754-1764 Chapter IV - Sea bass Caspase-9 53 Chapter IV - Sea bass Caspase-9 60 Chapter IV - Sea bass Caspase-9 61 Chapter IV - Sea bass Caspase-9 62 Chapter IV - Sea bass Caspase-9 63 CHAPTER V Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene. Marta I.R. Reis, Diana S. Nascimento, Ana do Vale, Manuel T. Silva and Nuno M.S. dos Santos Molecular Immunology (2007) 44(5): 774-783. Chapter V - Sea bass Caspase-3 67 Chapter V - Sea bass Caspase-3 68 Chapter V - Sea bass Caspase-3 69 Chapter V - Sea bass Caspase-3 76 CHAPTER VI Caspase-1 and IL-1β processing in sea bass. Marta I. R. Reis,* Ana do Vale,* Pedro J. B. Pereira,† Jorge E. Azevedo,‡§ and Nuno M. S. dos Santos*1 *Fish Immunology and Vaccinology, †Biomolecular Structure and ‡Organelle Biogenesis and Function; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. §Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal. Unpublished manuscript Chapter VI - Sea bass Caspase-1 79 Abstract IL-1β is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in those reported so far in non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding its processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, which in turn are distinct from the cleavage site of mammalian IL1β. Chapter VI - Sea bass Caspase-1 80 Introduction IL-1β is the most studied pro-inflammatory cytokine, much due to its role in mediating auto-inflammatory diseases (see e.g., [1-4]). It is mainly produced by activated macrophages, monocytes, and dendritic cells and affects almost every cell type, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges, although it can also have detrimental effects (see e.g., [1-6]). IL-1β exerts its activity by binding to IL-1 type I receptor (IL-1RI), which then recruits IL-1 receptor accessory protein (IL-1RAP) forming a complex that triggers a series of phosphorylation events leading to the activation of IκB kinase (IKK) and MAPK pathways (see e.g., [4, 7, 8]). These signalling pathways activate the transcription factors NF-κB and AP-1, resulting in the induction of genes encoding chemokines, cytokines, acute-phase proteins, cell adhesion molecules, and enzymes involved in the production of small proinflammatory substances [5]. In mammals, IL-1β is synthesized as an inactive 31 kDa signal peptide-less precursor molecule (proIL-1β) [9, 10]. In humans and mice, caspase-1, also known as ICE (interleukin-1β converting enzyme) [11-13], specifically cleaves proIL1β after aspartate 116 and 117, respectively, yielding a C-terminal 17.5 kDa secreted active form [11, 12, 14-18]. Non-caspase-1-mediated cleavage mechanisms generating different active forms of IL-1β have also been described (reviewed in [2, 4, 5, 19]). IL-1β has been identified in several fish species [20-30]. However, although it was reported that processing of IL-1β by a gilthead sea bream fibroblast cell line is abrogated by a specific caspase-1 inhibitor [31], neither direct evidence for the participation of caspase-1 in this processing event was provided nor the cleavage site in proIL-1β was defined. This last issue is particularly important because the caspase-1 cleavage site reported for mammalian proIL-1β is absent in the proIL-1β sequences of non-mammalian vertebrates. Putative trout [32], sea bass [33], and chicken [34-36] recombinant mature IL1β starting respectively at Ala95, Ala86, and Ala106, equivalent sequence-wise to the Nterminal residue of mature mammalian IL-1β, were shown to be biologically active. However, in none of these is the putative mature N-terminal preceded by an aspartate residue, making those sites rather improbable caspase-1 cleavage residues. In trout, a 29 kDa IL-1β precursor expressed in RTS-11 cells was processed to a 24 kDa peptide [37] and a 15 kDa band has been detected by Western blotting in PHA activated carp leucocyte culture supernatant [38]. However, the mechanism and exact cleavage site of processing were not clarified [39-41]. Caspase-1 is the prototype of a family of inflammatory caspases that contain a caspase recruitment domain (CARD) within their amino-terminal pro-domains (see e.g., [5, 42]). Normally, it exists in the cytoplasm as a 45 kDa inactive precursor [43], being Chapter VI - Sea bass Caspase-1 81 activated by CARD oligomerization in a multiprotein complex known as the inflammasome (see e.g., [44-51]). Once activated, caspase-1 processes the pro-inflammatory cytokines, starting an inflammatory response. Caspase-1 was recently found to be also involved in pyroptosis, a rapid active cell death programme characterised by early plasma-membrane rupture and release of pro-inflammatory intracellular contents ([52]; reviewed in [53, 54]). In humans, caspase-1 is activated by cleavage after Asp103, D119, D297 and D316, releasing the 11.5 kDa pro-domain and a 2 kDa linker peptide and originating the active heterodimer p20/p10 [17, 18, 55-57]. Fish inflammatory caspases have been primarily sequenced in zebrafish [58] and sea bass (GenBank accession no.: DQ198377), the later being a caspase-1 homologue that has recently also been reported for sea bream [59] but there is still no functional data from these enzymes. Here, the characterisation of the sea bass caspase-1 homologue, its processing and activity are reported. It is shown that sea bass caspase-1 auto-processing follows the mechanism described for the mammalian enzyme, yielding p24/p10 and p20/10 active heterodimers. In addition, three other caspase-1 isoforms have been identified. Finally, analysis of sea bass IL-1β processing by active sea bass caspase-1 revealed that the cytokine is cleaved at a phylogenetically conserved aspartate residue, distinct from the cleavage site in birds. Chapter VI - Sea bass Caspase-1 82 Materials and Methods Fish Sea bass (Dicentrarchus labrax) were kept in a recirculating, ozone-treated salt-water (25-30‰) system at 20 ± 1 ºC, and fed at a ratio of 2 % body weight per day. For organ collection fish were euthanized with 2-phenoxyethanol (Panreac; >5 ml/10 L). This study was carried out in accordance with European and Portuguese legislation for the use of animals for scientific purposes (Directive 86/609/EEC; Decreto-Lei 129/92; Portaria 1005/92). The work was approved by Direcção Geral de Veterinária, the Portuguese authority for animal protection. Complementary and genomic DNA cloning and sequencing, southern blotting, sequence analysis of caspase-1 and expression analysis of caspase-1 isoforms Standard techniques for molecular biology have been used, as described in supporting information. Production of recombinant sea bass caspase-1 isoforms The coding region of sea bass caspase-1 isoform 1 was amplified from the pGEMT Easy plasmid DNA carrying the full length cDNA using specific primers DLCASP1FWNdeI and DLCASP1RVXhoI (Table SII). The PCR product was cloned into pGEM-T Easy, the plasmid DNA was digested with NdeI and XhoI (Fermentas), and the insert cloned into pET-28a (Novagen) in frame with Nand C-terminal His-tags. Recombinant sea bass caspase-1 was expressed in E. coli BL21 Rosetta (DE3) overnight at 37 ºC with 1 mM IPTG. The recombinant protein was extracted from bacterial cells as inclusion bodies and solubilized with 8 M Urea, 50 mM Tris-HCl, 0.2 M NaCl, 2 mM EDTA pH 8.0. The protein was refolded by dilution in 50 volumes of refolding buffer (50 mM Taps pH 8.5, 1.5 M Sorbitol, 1 mM TCEP, 24 mM NaCl and 1 mM KCl) overnight at 22 ºC. As a control, the refolding process was also performed in the presence of 100 µM of a specific caspase-1 inhibitor (Ac-YVAD-CHO, Caspase-1 inhibitor I, Calbiochem). The refolded protein was bound to an IMAC column (HisTrap HP, GE Healthcare) in refolding buffer. The column was washed with the same buffer supplemented with 10 mM imidazole and the protein was then eluted in 4 steps with increasing concentrations of imidazole (50, 100, 250 and 500 mM). The purified protein was analysed by SDS-PAGE and, after blotted onto polyvinylidine difluoride membrane (PVDF), fragments of 24, 20 and 10 kDa were subjected to N-terminal Edman sequencing (Proteome Factory AG, Germany), in Chapter VI - Sea bass Caspase-1 83 order to determine the cleavage sites between the large and small subunits of sea bass caspase-1. The cDNAs encoding caspase-1 isoforms 2, 3 and 4 were obtained with the forward primer DLCASP1FWNdeI together with DLISO2/3RVXhoI for isoform 2 and 3 and with DLISO4RVXhoI for isoform 4 (Table SII). The cloning strategy, expression, purification and refolding protocols were those described for isoform 1, except that isoform 2 was cloned in pET-30a (Novagen). In vitro processing of caspase-1 isoforms Two polyclonal antibodies directed against sea bass caspase-1 were produced (Davids Biotechnologie GmbH, Germany). An anti-p10 polyclonal antibody was raised against the peptide VHKEKDFISLLSST, and detected all caspase-1 forms containing the p10 domain. The antibody produced against the peptide QACRGNAGGAVLVSD corresponding to the carboxyl-terminal residues adjacent to the proteolytic cleavage site, detected processed forms that were cleaved at the p20 cleavage site (therefore, lacking the linker and the small subunit), but did not recognise unprocessed caspase-1. Auto-processing of the caspase-1 isoforms was analysed by SDS-PAGE and Western blotting. Samples of the isoforms collected after urea solubilisation or after an overnight refolding step in the presence or absence of 50-100 µM caspase-1 inhibitor were subjected to SDS-PAGE. Proteins in the gels were stained with Coomassie brilliant blue or were transferred to nitrocellulose membranes and probed with the rabbit anti-p10 or anti-p20 antibodies (both at a 1/10000 dilution) for 1 h at room temperature. Goat antirabbit Ig conjugated with alkaline phosphatase (Sigma) was used as the secondary antibody and the detection performed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT). The ability of active sea bass caspase-1 isoform 1 to process isoforms 2, 3 and 4 was tested by incubating 2 µg of the purified isoforms with recombinant active caspase-1 (p24/p10) at a ratio of 2:1 (w/w) in a caspase-1 substrate buffer (0.1% v/w CHAPS, 0.1 M HEPES, 10 % v/w sucrose, 10 mM TCEP, pH 7.5) overnight at 22 ºC. As control, similar incubations were performed in the presence of 100 µM caspase-1 inhibitor. The proteins in the samples were precipitated with TCA and analysed by SDS-PAGE and Western blotting using the anti-p10 and anti-p20 antibodies, as described above, or transferred to a PVDF membrane for N-terminal Edman sequencing. Chapter VI - Sea bass Caspase-1 84 Activity of recombinant sea bass caspase-1 isoforms The activities of the refolded sea bass caspase-1 isoforms were tested by a fluorimetric assay using 0.1-2 µg of enzyme and a specific substrate for caspase-1 (ZYVAD-AFC, Caspase-1 substrate VI, Calbiochem). To control cleavage specificity, 100 μM caspase-1 inhibitor was used. All assays were performed in duplicate. In each reaction, 71.5 μl of reaction buffer (0.2% CHAPS, 0.2 M HEPES, 20% sucrose, 29 mM DTT, pH 7.5) and 16 μM of caspase-1 substrate were used. The mixtures were incubated at 25 ºC and the fluorescence recorded for up to 3 h with 20 min intervals on a Spectra Max Gemini XS fluorimeter (Molecular Devices) at an excitation wavelength of 405 nm and an emission wavelength of 492 nm. The results were expressed as relative fluorescence units (RFU). In vitro production of sea bass, chicken and human IL-1β by rabbit reticulocytes lysates Sea bass Plasmids harboring different IL-1β forms have been produced as detailed below. pET23proIL1β[His]: the coding region of sea bass proIL-1β was first amplified by PCR with primers DLIL1βFWNde3 and DLIL1βRVXho4 (Table SII) using a pGEX-4T-3 plasmid (GE Healthcare) carrying the proIL-1β cDNA (previously amplified from sea bass head kidney) as template [23]. The PCR product was purified and cloned into the pGEM-T Easy vector. After digestion with NdeI and XhoI, the sea bass proIL-1β was cloned into the expression vector pET-23a (Novagen) in frame with a C-terminal His-tag. pET-23proIL1β, for producing proIL-1β: the histidine tag from pET-23proIL1β[His] was removed by site-directed mutagenesis (QuickChange® Site-Directed Mutagenesis Kit, Stratagene) using primers listed in Table SII. pET-23proIL1β[D60A], pET-23proIL1β[D100A], pET-23proIL1β[D252A], and pET23proIL1β[D100A/D252A], for producing mutated forms of proIL-1β in D60, D100, D252, or D100/D252: each aspartate was mutated to alanine by site-directed mutagenesis using pET23proIL1β as template and primers listed in Table SII. pET-23matureIL1β, for producing mature sea bass IL-1β (from S101 to Q261 and nontagged): produced using pET-23proIL1β[His] as template and primers listed in Table SII. pET-23matureIL1β[D252A], for producing mature sea bass IL-1β mutated at D252: aspartate 252 was mutated to alanine by site-directed mutagenesis using pET23matureIL1β as template and primers listed in Table SII. Chicken The plasmids harboring the coding region of proIL-1β from chicken (GGpGEX-6P1proIL1β), duck (APpET28proIL1β), goose (AApET28proIL1β and turkey Chapter VI - Sea bass Caspase-1 85 (MGpET28proIL1β) were kindly provided by Dr. Hsien-Sheng Yin and Dr. Long Huw Lee. The coding region of chicken proIL-1β was first amplified by PCR with primers listed in Table SII using the plasmid GGpGEX-6P-1proIL1β as template. The PCR product was purified and cloned into the pGEM-T Easy vector. After digestion with NdeI and XhoI, the chicken proIL-1β was cloned into the expression vector pET-23a (Novagen) rendering GGpET23proIL1β. GGpET23proIL1β[D77A], GGpET23proIL1β[D80A] and GGpET23proIL1β[D82A] for producing mutated forms of chicken proIL-1β in D77, D80 or D82: each aspartate was mutated to alanine by site-directed mutagenesis using GGpET23proIL1β as template and primers listed in Table SII. The cDNAs encoding mature IL-1β (starting at S101) with or without a C-terminal Histag were amplified from pET23proIL1β[His] with the forward primer DLIL1BFWNde4 and the reverse primers DLIL1BRVXho4 or DLIL1BRVXho1, respectively (Table SII) and cloned in pET-23a. Human HSpCMV-XL5proIL1β[D116A], HSpCMV-XL5proIL1β[D128A] and HSpCMVXL5proIL1β[D116A/D128A], for producing mutated forms of human proIL-1β in D116, D128 or D116/D128: each aspartate was mutated to alanine by site-directed mutagenesis using an IL-1β Human cDNA clone (Origene #SC122566) as template and the primers listed in Table SII. Regions coding for mature human IL-1β from A117 or from S129 were amplified by PCR using the IL-1β Human cDNA clone as template and primers described in Table SII and cloned into the NcoI/XhoI restriction sites of pET28a (Novagen), yielding plasmids HSpET28mature1IL1β and HSpET28mature2IL1β, respectively. The above mentioned plasmids, harboring different IL-1β forms, were used to synthesize the respective proteins using the TNT® T7 Quick Coupled Transcription/Translation kit (Promega) in the presence of [35S] methionine (specific activity >1000 Ci/mmol; PerkinElmer Life Sciences) following the manufacturer's instructions. Production of recombinant sea bass IL-1β His-tagged proIL-1β (proIL-1β[His]) was expressed in E. coli BL21 (DE3) CodonPlus at 37 ºC with 1 mM IPTG for 4 h, extracted from bacterial cells as inclusion bodies, solubilised as described for recombinant caspase-1 and purified under denaturing conditions using His-selectTM Nickel Affinity Gel (Sigma). The protein was then refolded by dialysis in three steps against 50 volumes of decreasing concentrations of urea (4 M for Chapter VI - Sea bass Caspase-1 92 FIGURE 4. In vitro auto-processing and processing of sea bass caspase1 isoforms 2, 3 and 4 by isoform 1. (A) Coomassie brilliant blue stained SDS-PAGE of sea bass DilaCASP1iso2 (iso2), DilaCASP1iso3 (iso3) or DilaCASP1iso4 (iso4) incubated alone or in combination with active (p24/p10) DilaCASP1iso1 (iso1), in the presence or absence of caspase1 inhibitor Ac-YVAD-CHO (inhib). Nterminal sequencing revealed that the indicated fragments 1 and 2 correspond to p36 (GSSHHHHHHM1ADK) and p10 (T305LHFVH), respectively. Fragment 3 corresponds to an N-terminal fragment of p45 (GSSHHHHHHM1ADK). (B) Western blotting of the in vitro processing of caspase-1 isoforms as in A, using the anti-p10 polyclonal antibody that detects all polypeptides containing the p10 domain. (C) Western blotting of the in vitro processing of caspase-1 isoforms as in A, using the anti-p20 polyclonal antibody that detects only caspase-1 fragments that have been processed at the p20 cleavage site, i.e. that have released the linker plus the p10 domain. Numbers on the left indicate the mass of the molecular weight markers in kDa. Chapter VI - Sea bass Caspase-1 93 In vivo-generated IL-1β is an 18 kDa polypeptide Peritoneal leukocytes harvested from stimulated sea bass were incubated ex vivo in the presence or absence of a caspase-1 inhibitor. Western blotting analysis with an antisea bass IL-1β antibody showed a specific band with an apparent molecular mass of 18 kDa in culture supernatants and total extracts of cells incubated ex vivo without caspase-1 inhibitor (Fig. 5). This band co-migrates with a recombinant putative mature sea bass IL1β staring after aspartate D100 (MS101-Q261), an aspartate phylogenetically conserved in all available IL-1βs, from sharks to humans (Fig S3). FIGURE 5. Sea bass IL-1β processing in vivo. (A) Western blotting using the anti-sea bass IL-1β polyclonal antibody. (B) Ponceau staining showing the protein loading. Inhibitor: specific caspase-1 inhibitor Ac-YVADCHO. Recombinant putative mature sea bass IL1β (MS101-Q261) was loaded as control. Supernatant and Cells denote TCA precipitates of the supernatants and cell pellet corresponding to 1 ml of cell suspension after incubation and centrifuging, respectively. Numbers on the left indicate the mass of the molecular weight markers in kDa. Chapter VI - Sea bass Caspase-1 94 Evolutionary divergence of the caspase-1 cleavage site in proIL-1β As several attempts to identify the caspase-1 cleavage site in native mature IL-1β have failed, in vitro approaches were used. As shown in figure 6, in vitro synthesized sea bass proIL-1β was processed by sea bass active caspase-1 in a time-dependent manner giving rise to a band with an apparent molecular mass of 18 kDa that, as the in vivo obtained band, co-migrates with the putative mature sea bass IL-1β MS101-Q261. Mutation of D100 abolished the generation of the 18 kDa fragment, with accumulation of higher molecular mass species (Fig. 6), whereas mutating D60, an aspartate residue previously suggested as a possible caspase-1 cleavage site in fish [31], did not affect proIL-1β processing even when incubated with a caspase-1 buffer that promotes IL-1β cleavage (Fig. S4A). This suggests that cleavage of sea bass proIL-1β by caspase-1 occurs C-terminal to D100. FIGURE 6. Time-course analysis of in vitro synthesized bass proIL-1β and proIL-1β[D100A] by sea bass caspase-1. The same volume of in vitro synthesized proIL1β and proIL-1β[D100A] were used and loaded on the gel. Fragments 1, 2 and 3 are labelled. Putative mature in vitro synthesized sea bass IL-1β (MS101-Q261) has been loaded as control. Numbers on the left indicate the mass of the molecular weight markers in kDa. The duple bands corresponding to the highest molecular weight form (fragment 1) suggested cleavage at the C-terminal end of proIL-1β. This interpretation was supported by N-terminal sequencing of fragment 1’ (Fig. S4A), which revealed that this fragment has the N-terminal sequence of proIL-1β (M1ESEMKC). Furthermore, mutation of D252 results in a proIL-1β protein that no longer yields fragment 1 upon incubation with caspase-1 (Fig S4C). Chapter VI - Sea bass Caspase-1 95 An 18 kDa fragment was also generated from recombinant proIL-1β (Fig. S4B), together with a shorter fragment (fragment 3’) which intensity markedly increased with time. N-terminal sequencing of fragments 2’ and 3’ (Fig. S4B) revealed that they start at S101EKRSLVLVP, confirming that they originated from a cleavage after D100. Fragment 3’ was also processed after D252, as mutating D252 abolished its formation (Fig. S4C). Using in vitro synthesized proIL-1β it was found that the appearance of fragment 3’ was time-, bufferand caspase-1 concentration-dependent (Fig. S4A,C,D). Furthermore, only few fish species present a slightly longer C-terminal region including potential cleavable aspartates (Fig. S3). Together with the lack of detection of fragment 3´ in vivo, these data suggest that its appearance may be a consequence of the in vitro conditions, which do not completely mimic the in vivo environment. As the caspase-1 cleavage site described for mammalian proIL-1β is only present in mammals (Fig. S3), it is tempting to speculate that the phylogenetically conserved aspartate identified as the caspase-1 cleavage site in sea bass proIL-1β is also the residue targeted by caspase-1 in the proIL-1β of non-mammalian vertebrates. To address this issue, the processing of in vitro synthesized avian pro-IL1β was investigated. Since avian caspase-1 is not available, sea bass or human caspase-1 were used. Both sea bass and human caspase-1 processed chicken proIL-1β into a mature IL-1β running in SDSPAGE above the 21 kDa marker (Fig. 7A and S5A). A similar fragment was obtained when sea bass caspase-1 was incubated with duck, goose or turkey proIL-1β (Fig. S5B). Due to its apparent size, this suggested that avian proIL-1β could have been cleaved at D77/D80/D82 (sea bass D60), which also align with other phylogenetically conserved aspartates (Fig. S3). Site-directed mutagenesis confirmed that D80 is the preferential aspartate cleavage site (Fig. 7A). Despite the concerns associated with the use of heterologous caspases, both human and sea bass caspase-1 showed cleavage specificity when incubated with heterologous proIL-1βs, as supported by the fact that sea bass caspase-1 processed human proIL-1β at the specific mammalian caspase-1 cleavage site, originating the same mature form obtained by incubation with the human enzyme (Fig. 7B). Moreover, despite the presence of several aspartates in the sea bass proIL-1β including D60 (putative cleavage site in chicken), D100 and D252 (both targeted by sea bass caspase-1 in vitro), the human caspase-1 was unable to cleave sea bass proIL-1β (Fig. S5C), suggesting a higher specificity acquired during phylogeny. Chapter VI - Sea bass Caspase-1 96 FIGURE 7. Processing of in vitro synthesized chicken and human proIL-1β by sea bass caspase-1. (A) Processing of in vitro synthesized chicken proIL-1β and mutants by sea bass caspase-1. The same volume of in vitro translated chicken proIL-1β, proIL1β[D77A], proIL1β[D80A] and proIL1β[D82A] were loaded on the gel. In vitro translated putative mature chicken IL-1β forms (MI119-R267, MI122-R267 and MS81R267) were loaded as controls. (B) Processing of in vitro synthesized human proIL-1β and mutants by sea bass caspase-1. (C) Processing of in vitro synthesized human proIL-1β and mutants by human caspase-1. The same volume of in vitro synthesized proIL-1β, proIL-1β[D116A], proIL-1β[D128A] and proIL-1β[D116A/D128A] was loaded on the gel. In vitro translated mature human IL-1β (MA117-S269) and human IL-1β form (MS129-S269) starting at S129, homologue to sea bass S101, were loaded as controls. Mature forms are highlighted by arrow heads. Numbers on the left indicate the mass of the molecular weight markers in kDa. Chapter VI - Sea bass Caspase-1 97 Discussion Caspase-1 primary structure has since long been known in humans [17, 18, 66] and mice [55, 64], but was only recently determined for two fish, sea bass (GenBank: DQ198376 and DQ198377) and sea bream [59]. Comparative analyses of the gene and of the primary structure of sea bass caspase-1 indicate that a strikingly conserved structure has been kept throughout evolution, suggesting that its function, specificity and processing mechanisms are highly conserved from at least teleost fish to mammals. In humans, in addition to the caspase-1 isoform α, four mRNA isoforms (β, γ, δ and ε) resulting from one or more alternative splicing events have been described [67]. In the present work, besides sea bass caspase-1 isoform 1 (DilaCASP1iso1), three alternatively spliced mRNAs encoding DilaCASP1iso2, DilaCASP1iso3, and DilaCASP1iso4 were identified (Figs. 1 and S1B). Contrary to some of the human isoforms, where the prodomain (isoform γ) or both the pro-domain and the region containing the active site (isoforms δ and ε) have been deleted [67], all sea bass isoforms contain the proand p20 domains, including the fully conserved active site QACRG sequence. The differences introduced by the alternative splicing events in sea bass caspase-1 isoforms are all located downstream from the active site, with DilaCASP1iso2 and DilaCASP1iso3 displaying a shorter p10 domain with a different amino acid sequence at the C-terminal end. DilaCASP1iso4 has a longer p20 domain but no p10 (Fig. S1B). The presence of alternative transcript variants of caspase-1 in sea bass and humans [68, 69] suggests that their existence has been conserved throughout evolution and therefore they are likely to play a functional role, as described for other caspase isoforms (see below). Similarly to what has been described for human caspase-1 [17, 56, 57, 70], DilaCASP1iso1 is autocatalytically converted in vitro into two active heterodimeric complexes consisting of p10 paired either with p24 or p20 (Figs. 2A and 3). Strikingly, as for Drosophila melanogaster DRONC [71], sea bass caspase-1 p24 is generated by cleavage after a glutamate residue (P1 position) instead of the aspartate described in mammalian caspases as an absolute requirement for enzyme specificity [13, 17, 72]. Conversion of sea bass recombinant p45 to p20/p10 and p24/p10 heterodimers occurs in a time-dependent manner through a series of intermediates resembling the autoprocessing of the human caspase-1 precursor, in which the first step is the cleavage of p10 and linker from the p45 form, followed by proteolytic conversion of the resulting p36 into smaller and more active p20 and p24 forms [56, 57]. Thus, these results suggest that processing of pro-caspase-1 through several intermediates has been evolutionarily maintained at least from teleost fish to humans. Chapter VI - Sea bass Caspase-1 98 Sea bass caspase-1 isoforms 2 and 3 display some auto-processing activity, although in a much lesser degree than isoform 1. Of notice is that none of the fragments resulting from auto-processing of isoforms 2 and 3 correspond to p24, p20 or p10 (Fig. 4B and C), explaining why no enzymatic activity could be detected. However, contrary to the human isoforms [67], both sea bass isoforms 2 and 3 are processed into their p36, p20 and shorter p10 subunits by isoform 1. These data do not allow to conclude whether isoforms 2 and 3 participate in IL-1β processing or isoform 1 activation in vivo. On the other hand, being largely inactive, they may participate in a direct inhibitory mechanism of caspase-1, together with DilaCASP1iso4, by competing through CARD-CARD interactions for the inflammasome complex that activates caspase-1, as has been proposed for other CARD-containing proteins such as COP, INCA, ICEBERG and caspase-12 (reviewed in [73]). Alternative isoforms have been implicated in the inhibition of caspase-9 [74-76], and more recently also of caspase-3, where a short isoform (caspase-3S) antagonizes its apoptotic activity [77], thereby playing a physiological role in regulating cell death. Therefore, sea bass caspase-1 isoforms could also play a role in the regulation of the inflammatory response. Moreover, processing of isoforms 2 and 3 by isoform 1 released shorter p10 fragments, which could compete with normal p10 to generate inhibitory p20/short p10 heterodimers, as suggested for human ICEε [67]. In mammals, IL-1β is a pro-inflammatory cytokine produced as a 31 kDa inactive precursor [9, 10] that is processed by caspase-1 cleavage after D116 in humans and D117 in mice, yielding a C-terminal secreted active form of 17.5 kDa [11, 12, 14-18]. The primary structure of fish proIL-1β is similar to that of the mammalian cytokine [20-30]. However, the caspase-1 cleavage site present in all mammalian proIL-1β molecules is absent in the proIL-1β sequences reported so far for non-mammalian vertebrates (Fig. S3 and [39-41]), and the actual cleavage site has been greatly debated since the identification of the first non-mammalian IL-1β [22]. Cleavage of in vitro synthesized and recombinant sea bass proIL-1β by caspase-1 suggests that mature sea bass IL-1β results from processing of its pro-form at D100 giving rise to an 18 kDa polypeptide, in accordance with the form detected in vivo (Fig. 5). In view of the above, it would be tempting to speculate that mammalian IL-1β is the exception, rather than the norm, in what concerns proteolytic activation, since an aspartate residue corresponding to sea bass D100 is strictly conserved in all other vertebrate IL-1β sequences (Fig. S3). Particularly in chicken, this position could correspond to the activation site, rather than the alanine residue that was considered for the putative mature protein that was structurally characterised [36], as that constitutes an improbable caspase-1 cleavage site. However, the results presented here suggest that Chapter VI - Sea bass Caspase-1 99 avian proIL-1βs may not be cleaved at the equivalent sea bass D100, but instead preferentially cleaved after D80 (Fig. 7 and S5B), present in another proIL-1β stretch where aspartates are phylogenetically well conserved (Fig. S3). In other fish species, fragments with different molecular masses have been proposed as putative active IL-1β [31, 37, 38, 78, 79], and aspartate D60 in sea bream, equivalent to chicken D80, has even been suggested as a possible cleavage site, because processing at D60 would generate a fragment with a molecular mass consistent with the apparent size (22 kDa) of the IL-1β fragment detected by Western blotting in that species [31]. However, not only none of the reported studies included amino acid sequencing of the processed IL-1β fragments, but mutation of D60 did not affect at all sea bass IL-1β processing (Fig. S4A), in opposition to mutation of D100 where IL-1β processing to an 18 kDa form was abrogated (Fig. 6, S4A,C). To which extent processing of proIL-1β at this position is the norm among fish remains to be elucidated. However, cleavage at D100, here reported for sea bass proIL-1β, will most certainly have a significant structural impact in the mature molecule. Homology models of mature sea bass IL-1β produced using the available experimental models as templates (PDB entries 1IOB (human), 8I1B (mouse) or 2WRY (chicken)) indicate that cleavage at D100 would result in the ablation of the N-terminal beta strand, with concomitant destabilization of the other beta sheet-forming segment at the C-terminus of the molecule (Fig. 8). Therefore, sea bass proIL-1β is certainly structurally distinct in this region, although the existing differences cannot be inferred by homology modelling with the available templates. FIGURE 8. Three-dimensional homology model of sea bass Il-1β (left), and chicken (pdb entry: 2wry; center) and human (pdb entry: 1iol; right) 3D structures. All molecules are displayed in the same relative orientation. The last β-strand is absent in the sea bass model, destabilizing the pairing C-terminus of the molecule. The sea bass molecule was modelled with the Swiss Model server (http://swissmodel.expasy.org/) using the experimental coordinates of human IL-1β as template and default parameters. The resulting model had good quality indicators. Figure prepared with PyMol (http://www.pymol.org/). Chapter VI - Sea bass Caspase-1 100 Notably, in the mammalian proIL-1β  caspase-1 cleavage site the P1’ position is almost always occupied by a small hydrophobic amino acid, which has been described as one of the preferential features of the peptide substrates of mammalian caspase-1 [13, 17]. On the contrary, most of the aspartates in the putative caspase-1 cleavage sites of non-mammalian vertebrates, sequence-wise to sea bass D100 or chicken D80, are not followed by a small hydrophobic amino acid. Also of notice is the fact that a small hydrophobic amino acid is present in the P1’ position after the phylogenetically conserved sea bass caspase-1 cleavage sites in monkeys (A129 in P1’), raising questions about the evolutionary driving force that resulted in the appearance of the mammalian cleavage site, as the mammalian aspartate at position P1 is only conserved in mammalian IL-1β sequences. In conclusion, the present work shows that in a teleost fish, pro-caspase-1 autoprocessing occurs through a series of intermediates, yielding active p24/p10 and p20/p10 heterodimers in a similar way to that described previously for human caspase-1. Moreover, the existence of alternative spliced variants of caspase-1 in sea bass is reported, suggesting that caspase-1 isoforms have been evolutionarily maintained and therefore likely play a regulatory role in the inflammatory response, as shown for isoforms of other caspases. Finally, it is shown that sea bass IL-1β is cleaved by caspase-1 at a phylogenetically conserved aspartic acid present in all known IL-1β sequences. However, in avian proIL-1βs another well phylogenetically conserved aspartate, only absent in humans, chimpanzees and zebrafish, may correspond to the cleavage site, suggesting that IL-1β processing may be class and/or species specific. Taken together, these results confirm that proIL-1β from non-mammalian vertebrates possess a specific caspase-1 cleavage site. Chapter VI - Sea bass Caspase-1 101 Acknowledgements We are grateful to Dr. R. Mentele (MPI fuer Biochemie, Martinsried, Germany) for Nterminal protein sequencing of the IL-1β polypeptides. We wish to acknowledge Frederico Ferreira-da-Silva for providing technical support from the Protein Production and Purification Unit (UP3) at IBMC (Porto). This work was supported by a grant (PTDC/CVT/69086/2006; FCOMP-01-0124-FEDER-007173) from the Portuguese Science and Technology Foundation (FCT). MIRR was the recipient of FCT fellowship SFRH/BD/37717/2007. AdV was supported by Programa Ciência – financed by POPH - QREN - Tipologia 4.2 - Promoção do Emprego Científico, co-funded by Fundo Social Europeu and National funding from MCTES. Chapter VI - Sea bass Caspase-1 109 SUPPORTING INFORMATION Cloning and sequencing of caspase-1 Messenger RNA was purified with the MicroPoly(A)PureTM kit (Ambion®) and transcribed to cDNA using the SuperscriptTM II First-Strand Synthesis System (Invitrogen). PCRs were done according to standard procedures. For amplifying full length sequences Pfu DNA polymerase was used in the reactions. DNA sequencing was performed using primers detailed in Table SII. Complementary DNAs used for 5’ Rapid Amplification of cDNA Ends (5’RACE) experiments were purified using High Pure PCR Product Purification Kit reagents (Roche), and used with the 5´RACE System from Invitrogen (Version 2.0). The purified cDNA was dATP tailed using Recombinant Terminal Transferase (Roche). Messenger RNA (~340 ng) extracted from the head kidney of a fish 1 h after stimulation with LPS was reverse transcribed using primer APv (Table SII). Degenerate primers were designed based on conserved regions of caspase-1 amino acid sequences from different vertebrates. The cDNA was first PCR amplified using primers CASP1FW1/CASP1RV1 followed by a semi-nested amplification using primers CASP1FW1/CASP1RV2 (Table SII). A third amplification with primers CASP1FW1/CASP1RV2 was carried out and a PCR product with the expected size (168 bp) was purified, cloned and sequenced. This sequence was used to design the specific reverse primers DLCASP1RV1, DLCASP1RV2 and DLCASP1RV3 (Table SII), in order to obtain the 5’ untranslated region (5´-UTR) using the 5’RACE strategy. The obtained PCR product was purified, cloned and sequenced and, because it did not include the start codon, the 5’RACE strategy was repeated using again the specific reverse primers DLCASP1RV1 and DLCASP1RV2, plus the reverse primer DLCASP1RV5, designed based on the sequence obtained from the previous 5’RACE (Table SII). The fragment obtained was cloned, sequenced and used to design the specific primer DLCASP1FW7 (Table SII) in the 5’-UTR. This primer was combined with primer AUAP (Table SII), in a PCR aiming to obtain the complete sequence of sea bass caspase-1. Four PCR products ranging from 1500 to 1680 bp were purified, cloned into the pGEM-T Easy Vector (Promega) and sequenced. Chapter VI - Sea bass Caspase-1 110 FIGURE S1. Caspase-1 is encoded by a single gene in sea bass and encodes at least four different transcripts. (A) Southern blotting analysis. Restriction enzymes EcoRI, Spe I, BamHI, NcoI, NdeI, ApaLI (zero cutters in sea bass caspase-1 gene), and HindIII and XbaI (single and double cutter in intron 1 of sea bass caspase-1 gene, respectively) were used to digest sea bass gDNA from 4 fish, according to the manufacturer’s instructions (Fermentas). Digestion products were subjected to 0.8% agarose gel Chapter VI - Sea bass Caspase-1 111 electrophoresis and Southern blotting [2]. A fragment of the sea bass caspase-1 coding region was amplified with primers DLCASP1FW4 and DLCASP1RV6 (Table SII). The PCR product (367 bp) was purified as previously described, labelled using the Gene ImagesTM AlhPhos DirectTM Labelling and Detection System (Amersham Biosciences), and used as probe to detect the caspase-1 gene. For signal generation and detection the Chemiluminescent Signal Generation and Detection with CDP-StarTM protocol from the same kit was followed. Numbers on the left indicate kbp. (B) Multiple sequence alignment of the predicted primary structures of sea bass caspase-1 isoforms and human capase-1α (HosaCASP1alpha). The sequences were aligned with CLUSTAL W [1] using default parameters. Domain boundaries were determined with PROSITE (www.expasy.org/prosite) and comparison with human capase-1α. Sea bass caspase-1 comprises a prodomain (Met1-Asp108), followed by a p20 domain (Gln109-Asp278), a short connecting peptide (Ser279-Asp304), and a p10 domain (Thr305-Leu394), and signaled on top of the alignment. The active-site pentapeptide QACRG is boxed. The aspartic acid cleavage sites are shaded in gray. Asterisks and “:” and “.” denote identity and chemical similarity between amino acids according to the default CLUSTAL W scoring matrix. The amino acid residues in the sea bass caspase-1 isoforms 2, 3 and 4 that differ from the isoform 1 are in bold. DilaCASP1iso1 (GenBank accession number: DQ198376) has 1663 bp including, as in human and mouse caspase-1 mRNA [3, 4], two polyadenylation signals within the 3’-UTR. The ORF encodes a 394 aa long protein that, as in the human and mouse sequences, has no hydrophobic signal sequence. Sequence analysis revealed that the sea bass protein contains both caspase family p20 and p10 domain profiles and a caspase family active site signature (K257PKIIIIQACRG). A pro-domain containing a caspase recruitment domain (CARD), typical of pro-inflammatory caspases, is also present. DilaCASP1iso2 (GenBank accession number: HQ398873) results from an alternative splicing event involving the utilization of an alternative splicing site located within intron 7, which leads to the retention of 31 nucleotides from this intron (Fig. 1). This creates a putative caspase-1 isoform containing the complete proand p20 domains but having a shorter p10 domain (72 aa compared to 90 aa of the full length p10). DilaCASP1iso3 (GenBank accession numbers: HQ398874) is generated by an exon skipping event, in which exon 7 is lost (Fig. 1). This creates a putative caspase-1 isoform containing the complete proand p20 domains but having a much shorter p10 domain (25 aa compared to 90 aa of the full length p10). DilaCASP1iso4 (GenBank accession number: HQ398875) results from two alternative splicing events. One results in the retention of intron 5, generating a stop codon located 62 nucleotides downstream the beginning of this intron. The other is generated by an exon skipping event, in which exon 7 is spliced out of the transcript, as in DilaCASP1iso3 (Fig. 1). This creates a putative caspase-1 isoform containing a complete pro-domain; however, the splicing originates a shift in the reading frame that generates a different coding region after the active center. Although there is an aspartate residue within this alternative coding region, it is apparently not used as cleavage site, as this isoform is not auto-processed nor processed by active DilaCASP1iso1 (see below), therefore rendering a longer p20 domain (11 aa longer and 21 aa different) and no p10. (C) Expression analysis of caspase-1 isoforms in sea bass spleen. Complementary DNA was synthesized from total RNA extracted from spleens of 9 non-stimulated fish as described in supporting information section “cloning and sequencing of caspase-1”. The cDNAs were amplified using the forward primer DLCASP1FW11 (designed in an exon/intron boundary) for all isoforms and a reverse primer specific for different isoforms (Table SII): isoform1, DLCASP1RV18 (between exon 7 e 8); isoform 2, DLCASP1RV16 (intron 7); isoform 3/4m, DLCASP1RV19 (between exon 6 and 8); and isoform 4, DLCASP1RV17 (intron 5). The reverse primer DLCASP1RV19 amplifies isoforms 3 and 4 but, in combination with the primer DLCASP1FW11, different size products are obtained (989 bp for isoform 3 and 1076 bp for isoform 4). The PCR products were purified and sequenced. Chapter VI - Sea bass Caspase-1 112 FIGURE S2. Neighbour-joining tree (MEGA version 3.1) [5] with p-distance and complete deletion of gaps of inflammatory (caspase-1, -4, -5, -11 and -12) and apoptogenic (caspase-3 and -9) caspases. The amino acid sequences were aligned with CLUSTAL W [1] using the default parameters. The numbers in branching nodes INFLAMMATORY CASPASES Homo sapiens caspase-4 alpha Bos taurus caspase-4 Homo sapiens caspase-5a Macaca mulatta caspase-5 Felis catus caspase-1 Canis lupus familiaris caspase-1/4 Mus musculus caspase-12 Rattus norvegicus caspase-12 Macaca mulatta caspase-12 Canis lupus familiaris caspase-12 Felis catus caspase-12a Mus musculus caspase-11 Rattus norvegicus caspase-4 Rattus norvegicus caspase-11 Homo sapiens caspase-1 Rattus norvegicus caspase-1 Mus musculus caspase-1 Gallus gallus caspase-1 Xenopus laevis caspase-1 Dicentrarchus labrax caspase-1 Sparus aurata caspase-1 Danio rerio caspase-a Danio rerio caspase-b Mus musculus caspase-9 Rattus norvegicus caspase-9 Homo sapiens caspase-9 Dicentrarchus labrax caspase-9 Tetraodon nigroviridis putative caspase-9 Dicentrarchus labrax caspase-3 Takifugu rubripes caspase-3 Danio rerio caspase-3 Homo sapiens caspase-3 Rattus norvegicus caspase-3 Mus musculus caspase-3 100 100 100 96 100 100 93 100 100 100 100 100 100 100 100 88 99 83 49 99 99 100 49 88 88 96 47 100 100 62 86 APOPTOGENIC CASPASES Chapter VI - Sea bass Caspase-1 113 denote the bootstrap percentages for 1000 replicates. The different branches are supported by high bootstrap values. GenBank accession numbers: for caspase-1 see Table SI; for caspase1/4: ABX79372 (Canis lupus familiaris); for caspase-3: ABC70996 (Dicentrarchus labrax), AAM43816 (Takifugu rubripes), BAB32409 (Danio rerio), CAC88866 (Homo sapiens), AAH81854 (Rattus norvegicus), AAH38825 (Mus musculus); for caspase-4: NP_001216 (H. sapiens), NP_788811 (Bos taurus), NP_446188 (R. norvegicus); for caspase-5: ABB58698 (H. sapiens), XP_001100375 (Macaca mulatta); for caspase-9: BAA87905 (H. sapiens), AAK26235 (R. norvegicus), AAH56447 (M. musculus), ABC70998 (D. labrax); the sequence of Tetraodon nigroviridis putative caspase-9 (CAG01765) was obtained by Blast search of the Genbank database with the sequence of sea bass caspase-9 as query; for caspase-11: CAA73531 (M. musculus), AAK38735 (R. norvegicus); for caspase-12: AAT91067 (M. musculus), EDL78548 (R. norvegicus), ABG21363 (M. mulatta), NP001070704 (C. lupus familiaris), ABX79369 (Felis catus). Chapter VI - Sea bass Caspase-1 114 FIGURE S3. Alignment of IL-1β amino acid sequences from vertebrates belonging to different Classes. The regions having aspartate cleavage sites are shown (aspartates are shaded black). The sequences were aligned with CLUSTAL W [1] using the default parameters. G enBank accession numbers are shown within brackets. Aspartate residues are blue colour and bold type. The caspase-1 cleavage site in mammalian IL-1β sequences is shaded yellow. The conserved aspartic acids homologous to the caspase-1 cleavage site in sea bass IL-1β sequence are shaded green. The conserved aspartic acids homologous to the putative caspase-1 cleavage site in avian IL1β sequence are shaded red. Mutated aspartate residue in the sea bass and chicken IL-1β sequences are shaded black. Chapter VI - Sea bass Caspase-1 115 FIGURE S4. Processing of in vitro synthesized and recombinant sea bass proIL-1β by caspase-1. (A) Timecourse processing in vitro synthesized sea bass proIL-1β proIL1β[D60A] and proIL1β[D100A] by sea bass caspase-1, in the presence or absence of caspase-1 inhibitor AcYVAD-CHO, using caspase-1 buffer. In vitro synthesized putative mature sea bass IL-1β (MS101-Q261) was loaded as control. (B) Timecourse processing of recombinant sea bass proIL-1β by caspase-1 using caspase-1 buffer. proIL-1β[His] (proIL-1β) and DilaCASP1iso1 (Casp1) were loaded as controls. On the right, a schematic illustration of the fragments obtained, as concluded from Nterminal sequencing, is shown; capital letters denote amino acid residues and the superscript numbers the amino acid position within the sea bass IL-1β sequence. (C) Time-course processing of in vitro synthesized sea bass proIL-1β proIL1β[D60A] and proIL1β[D100A] using caspase-1 buffer. In vitro synthesized mature sea bass IL-1β (MS101Q261) was loaded as control. (D) Processing of in vitro synthesized sea bass proIL-1β with or without caspase1 buffer and with different amounts of caspase-1 (relative amount of caspase-1 added to the reaction). In vitro synthesized mature sea bass IL-1β (MS101-Q261) was loaded as control. ProIL-1β fragment 1, 2 and 3 are indicated in (C) and (D). Numbers on the left (right in D) indicate the mass of the molecular weight markers in kDa. The same volume of each in vitro synthesized proIL-1β forms were used and loaded on the gel. Chapter VI - Sea bass Caspase-1 116 FIGURE S5. Processing of in vitro synthesized sea bass and chicken proIL-1β by human and sea bass caspase-1, respectively. (A) Chicken proIL-1β is processed by human caspase-1. Putative mature chicken IL1β forms (MS81R267, MI119R267 and MI122R267) were loaded as controls. (B) Processing of in vitro synthesized chicken, duck, goose and turkey proIL-1β by sea bass caspase-1. Putative mature chicken IL-1β forms (MS81R267, MI119R267 and MI122R267) were loaded as controls. (C) Sea bass proIL-1β is not processed by human caspase-1. Numbers on the left indicate the mass of the molecular weight markers in kDa. The same volume of in vitro synthesized proIL-1β forms for each species was used and loaded on the gel. Chapter VI - Sea bass Caspase-1 117 Table SI. Amino acid sequence conservation for caspase-1 of different species. The percentages of similarity and identity were calculated by pair-wise alignments with the program needle [6] as implemented at EBI web site (www.ebi.ac.uk) with first and extending gap penalties of 10 and 0.5, respectively. Species Dicentrarchus labrax Accession number Identity Similarity Gaps Sparus aurata 62.7% 76.7% 4.7% CAM32183 Danio rerio caspy 38.5% 53.4% 19.7% NP_571580 Danio rerio caspy2 30.7% 45.0% 23.8% NP_690840 Rattus norvegicus 37.0% 52.3% 10.0% NP_036894 Mus musculus 37.0% 51.2% 12.1% NP_033937 Xenopus laevis XICE-a 36.9% 51.1% 9.3% BAA14017 Homo sapiens isoform alpha percursor 36.0% 51.1% 8.6% NP_150634 Felis catus 35.4% 51.6% 11.3% NP_001009365 Gallus gallus 26.4% 39.1% 31.6% AAC69917 Chapter VII – Concluding remarks 124 IL-1β in fish has not been disclosed. Since the sequencing of the first non-mammalian IL1β, 13 years ago [11], it became evident that non-mammalian IL-1βs do not have the mammalian caspase-1 cleavage site, therefore raising the question of how this cytokine is activated in non-mammalian species. Considering this, the sequence of sea bass caspase-1 and the study of its involvement in the processing of IL-1β appeared as an important issue to be addressed. I. Sea bass apoptotic caspases As mentioned in the introduction section, the initiator caspases have a critical role of transmitting the apoptotic signal through the activation of the executioner caspases and of generating the caspase cascade. The initiator caspases of the extrinsic and intrinsic pathways, caspase-8 and -9 respectively, as well as the central executioner of apoptosis, caspase-3, were identified in sea bass. Although some fish caspase genes have two copies (Annex 1), resulting from the early whole-genome duplication in the teleost lineage after the divergence from tetrapods about 450 million years ago [12-14], Southern blotting analysis suggest that the caspase genes identified in this thesis are present as single copies in the sea bass genome. At the molecular level, the obtained sequences of sea bass caspase-8, -9 and -3 retain the motifs that are functionally important, such as the pentapeptide active-site motif (QACXG) and the cleavage sites at the aspartic acids that give rise to the characteristic organisation of caspases in NH2-terminal pro-domain, large and small subunits. Moreover, a short pro-domain typical of executioner caspases was identified in caspase-3, while long pro-domains characteristic from initiator caspases were present in caspases-8 and -9. In the case of sea bass caspase-8, two death effector domains (DED) were predicted by bioinformatics analysis. The same approach identified a caspase recruitment domain (CARD) in the NH2-terminal pro-domain of sea bass caspase-9. As reviewed in the introduction, both DED and CARD are important domains involved in protein-protein interactions necessary to mediate dimerization and consequent activation of caspase-8 and -9, respectively. The sea bass caspases possess significant degrees of similarity to the corresponding sequences from several vertebrates of different taxonomic groups. Furthermore, it was shown that sea bass caspase-8, -9 and -3 were expressed at low levels in several organs from non-stimulated sea bass and that their expressions are up-regulated in the spleen in response to apoptotic stimuli. The data gathered from the molecular characterisation of sea bass apoptotic caspases allowed to demonstrate that caspase-8, -9 and -3 are activated in peritoneal macrophages and neutrophils treated ex vivo with AIP56 and that both the extrinsic and intrinsic pathways Chapter VII – Concluding remarks 125 are involved in the AIP56-induced apoptosis [15]. Thereby, this work provided information that contributed to a better understanding of the biology of fish apoptotic caspases and allowed the development of tools that may be used in the study of the mechanisms of apoptosis in fish. II. Sea bass caspase-1 and its involvement in IL-1β processing The work described in this thesis identified, in the sea bass genome, a caspase-1 gene which primary structure and gene organisation have been conserved through evolution. Sea bass caspase-1 pro-form is auto-processed through several intermediates in a similar way as its human homolog, resulting in active p24/p10 and p20/p10 heterodimers. However, similarly to what has been reported for Drosophila melanogaster DRONC [16], the exclusive preference for cleavage at an aspartic acid at P1 position described for mammalian caspases is not an absolute requirement for sea bass caspase1. Also of notice, is the presence of alternative splice variants of sea bass caspase-1 that may have a role in the regulation of the inflammatory response as described for other caspase isoforms in mammals [17-22]. The important role of the pro-inflammatory cytokine IL-1β as a mediator of inflammatory responses has been documented in several species of fish [23-26]. In the present thesis (Chapter VI), significant advances in the understanding of fish IL-1β processing are presented, through the identification of a cleavage site for caspase-1 at an aspartic acid absolutely conserved in all the IL-1β sequences reported so far. However, the equivalent sequence-wise aspartate in avian IL1βs is not used as cleavage site in birds. Instead, in birds, another phylogenetically conserved aspartate, only absent in humans, chimpanzees and zebrafish, is used, suggesting that IL-1β processing may be class and/or species specific. These results clearly show that proIL-1β from non-mammalian vertebrates have a specific caspase-1 cleavage site. Moreover, the cleavage at D100, in sea bass proIL-1β, implies significant structural changes in the mature sea bass IL-1β when compared to that of mammals and chicken. Solving the 3D structure of sea bass IL-1β would help to define the functional implications, if any, of a different mature IL-1β structure. In conclusion, the sea bass counterparts of mammalian caspases-3, -8 and -9 were sequenced and characterised, and shown to be expressed/activated upon an apoptotic stimulus. The data obtained allowed the production of monospecific polyclonal antibodies against peptides form each identified caspase that were shown to be valuable tools for monitoring the active forms of the caspases, as exemplified for caspase-1 in Chapter VII – Concluding remarks 126 Chapter VI. The genetic information as well as the antibodies produced in this work may be used to follow the involvement of sea bass caspases in biological processes at genetic and proteomic level, under physiological or pathological conditions. Among many questions that have been raised by the results obtained during this thesis, four main aspects must be highlighted: (i) the cleavage at D100 in sea bass proIL-1β leads to the question of whether other fish proIL-1β are also cleaved at an equivalent aspartate residue. Cleavage at this aspartate implies significant structural differences in the mature sea bass/fish IL-1β, when compared to that of mammals and chicken. Solving the 3D structure of sea bass IL-1β would help defining the functional implications of such structural differences and would give insights into the evolutionary driving force that has led to the appearance of different caspase-1 cleavage sites in proIL-1βs from different classes/species of vertebrates. (ii) The involvement of caspase-1 isoforms in the processing/regulation of caspase-1 and pro-IL-1β have been neglected. It is very likely that, as shown for other caspases, caspase-1 isoforms play an important role in the regulation of the inflammasome and consequent secretion of pro-inflammatory cytokines; these is an issue that is worth to investigate. (iii) The increasing significance of the inflammasomes in different pathologies, such as auto-inflammatory and infectious diseases, has been uncovered in recent years. In an attempt to limit the pathogen replication, the activation of inflammasomes is accompanied by the induction of pyroptosis during infection by several pathogens. However, in fish there are no studies regarding inflammasomes or pyroptosis. Thereby, dissecting the molecular mechanisms for caspase-1 activation at the inflammasomes as well as to study the interference of pathogens with inflammasome function in fish are research fields worth to be pursued. Since it is known that there is an increased expression of IL-1β, as well as other inflammatory cytokines, during the initial phase of the Phdp infection, it would be interesting to investigate the involvement of the inflammasome and the occurrence of pyroptosis in the Phdp infections. (iv) The emerging roles of caspases, including in embryonic development, monocyte differentiation and regulation of T cell activation as well as their participation in the balance between survival and death, show the importance of these molecules. Whether fish caspases possess non-apoptotic and non-inflammatory functions remains to be investigated. Chapter VII – Concluding remarks 127 References 1. Thompson, C.B., Apoptosis in the pathogenesis and treatment of disease. Science, 1995. 267(5203): p. 1456-62. 2. Saikumar, P., et al., Apoptosis: definition, mechanisms, and relevance to disease. Am J Med, 1999. 107(5): p. 489-506. 3. Strowig, T., et al., Inflammasomes in health and disease. 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Jaillon, O., et al., Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature, 2004. 431(7011): p. 946-57. Chapter VII – Concluding remarks 128 15. Costa-Ramos, C., et al., The bacterial exotoxin AIP56 induces fish macrophage and neutrophil apoptosis using mechanisms of the extrinsic and intrinsic pathways. Fish Shellfish Immunol, 2011. 30(1): p. 173-81. 16. Snipas, S.J., et al., Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC. Cell Death Differ, 2008. 15(5): p. 938-45. 17. Huang, Y., et al., Molecular Cloning and Characterization of a Novel Caspase-3 Variant That Attenuates Apoptosis Induced by Proteasome Inhibition. Biochem Biophys Res Commun, 2001. 283(4): p. 762-769. 18. Seol, D.-W. and T.R. Billiar, A Caspase-9 Variant Missing the Catalytic Site Is an Endogenous Inhibitor of Apoptosis. J. Biol. Chem., 1999. 274(4): p. 2072-2076. 19. Srinivasula, S.M., et al., Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis. Cancer Res, 1999. 59(5): p. 999-1002. 20. Boldin, M.P., et al., Involvement of MACH, a Novel MORT1/FADD-Interacting Protease, in Fas/APO-1and TNF Receptor Induced Cell Death. Cell, 1996. 85(6): p. 803-815. 21. Muzio, M., et al., FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell, 1996. 85(6): p. 817-27. 22. Alnemri, E.S., T. Fernandes-Alnemri, and G. Litwack, Cloning and expression of four novel isoforms of human interleukin-1 beta converting enzyme with different apoptotic activities. J Biol Chem, 1995. 270(9): p. 4312-7. 23. Fujiki, K., et al., Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1 beta, high affinity immunoglobulin E Fc receptor gamma subunit and serum amyloid A. Fish Shellfish Immunol, 2000. 10(3): p. 229-42. 24. Jiang, S., et al., Molecular characterization, recombinant expression and bioactivity analysis of the interleukin-1[beta] from the yellowfin sea bream, Acanthopagrus latus (Houttuyn). Fish Shellfish Immunol, 2008. 24(3): p. 323-336. 25. Lu, D.-Q., et al., Interleukin-1[beta] gene in orange-spotted grouper, Epinephelus coioides: Molecular cloning, expression, biological activities and signal transduction. Mol Immunol, 2008. 45(4): p. 857-867. 26. Pelegrin, P., et al., Interleukin-1beta isolated from a marine fish reveals upregulated expression in macrophages following activation with lipopolysaccharide and lymphokines. Cytokine, 2001. 16(2): p. 67-72. ANNEX I Fish and Apoptosis: Molecules and Pathways. Nuno M.S. dos Santos, Ana do Vale, Marta I.R. Reis and Manuel T. Silva Current Pharmaceutical Design (2008) 14(2): 148-169. Annex I 131 Annex I 132 Annex I 133