Zebrafish Caspase-3: Molecular Cloning, Characterization, Crystallization and Phylogenetic Analysis

Chakraborty, Chiranjib; Nandi, Shyam Sundar; Sinha, Surajit; Gera, V. K.

doi:10.2174/092986606777145850

Cited by 19 publications

(12 citation statements)

References 30 publications

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“…Caspase-3 is the most well characterized caspase in fish, including zebrafish [174,175], salmon [176] and sea bass [177]. BLAST search revealed a close relation between fish caspase-3 and other vertebrate caspase-3 molecules, which is supported by the relatively high percentage of aminoacid identities (50-70%) reported between fish and non-fish vertebrate caspase-3 molecules [174,177].…”

Section: Fish Homologs Of Mammalian Executioner Caspasesmentioning

confidence: 90%

Fish and Apoptosis: Molecules and Pathways

Santos¹,

Vale²,

Reis³

et al. 2008

CPD

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Apoptosis is a genetically controlled and evolutionarily conserved form of active cell death, albeit with an increase in complexity with continuing development. A high conservation at the functional and molecular level has been described between the players of the apoptotic machinery in invertebrates (Caenorhabditis elegans and Drosophila) and mammals. However, fish represent an excellent and advantageous model for the study of vertebrate development and disease, bridging the gap between the C. elegans/Drosophila and mouse/human models. 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. Last but not less important, both the environment and human health will obviously gain by using the knowledge generated through the use of fish models, for developing better prophylactic and therapeutic measures with impact on the aquaculture industry. In the present article, structural and functional data on the most important apoptosis related molecules, namely death-receptor, Bcl-2 and caspase families, and mechanisms are reviewed. The data point to the existence in fish of apoptotic pathways equivalent to those of mammals, making fish useful animal models for studying apoptosis, which may have great applicability for the advance of the knowledge on the role of apoptotic cell death in human apoptosis-related disorders as well as in pharmaceutical design.

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Section: Fish Homologs Of Mammalian Executioner Caspasesmentioning

confidence: 90%

Fish and Apoptosis: Molecules and Pathways

Santos¹,

Vale²,

Reis³

et al. 2008

CPD

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“…Activation of caspase-3 in zebrafish following c-irradiation and overexpression of caspase3a, caspase-9, and pro-apoptotic Bcl-2 family members has been confirmed by immunohistochemistry using a crossreactive monoclonal antibody against the cleaved, active form of caspase-3 [24, 31]. Structural information for caspase-3a has been obtained by X-ray crystallography, providing a unique level of detail for this important component of the zebrafish apoptotic machinery [91]. In addition to the canonical effector caspases, two novel zebrafish caspases (caspase a and caspase b) containing N-terminal pyrin domains and sharing greatest homology with mammalian caspase-1 have been reported to induce apoptosis following overexpression [92].…”

Section: The Zebrafish Apoptosome and Effector Caspasesmentioning

confidence: 99%

The zebrafish as a model organism for the study of apoptosis

2009

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Apoptosis plays important roles in embryogenesis, tissue homeostasis, and immune system regulation. The zebrafish (Danio rerio) is a powerful vertebrate model organism that has been extensively used to study apoptotic cell death during normal development and under conditions of cellular stress. In the past 5 years, a detailed picture has begun to emerge of the molecular underpinnings of the cell-intrinsic and the cell-extrinsic apoptosis signaling pathways in zebrafish. We begin this review with an introduction to the techniques and experimental approaches that are used to study apoptosis in zebrafish. We follow with a general overview of developmental apoptosis during zebrafish embryogenesis. Finally, we present a comprehensive review of the intrinsic and extrinsic apoptosis pathways in zebrafish, focusing on the high degree of conservation with humans and other mammals. Recent publications that draw upon the unique advantages of the zebrafish system to study novel aspects of apoptosis regulation and function are highlighted throughout.

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“…This cell death process is evolutionarily conserved and some of its components have stayed through animal evolution, including in fish. In this case, a caspase-3 gene has been identified in zebrafish (Yabu et al 2001;Chakraborty et al 2006) and sea bass (Reis et al 2007), retaining the motifs that are functionally important, such as the active site and the cleavage site at the aspartic residue; nevertheless, phylogenetic analysis shows that fish corresponds to a divergent evolving group, hindering the detection of fish caspase through commercial antibodies developed against mammalian caspases. In order to solve this problem, we have developed an antibody that allows the detection of a salmonid caspase-3.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, molecular weights determined for pro-caspase and active caspase-3 in RTS11 infected cells are according with those predicted in zebrafish and sea bass. In the first species caspase-3 was cloned fused to a His tag sequence at the N-terminus, the western blotting using an anti-His-tag antibody reported the precursor form (31.5 kDa), and an active subunit of 13 kDa (Yabu et al 2001;Chakraborty et al 2006). In sea bass, caspase-3 shows an ORF of 281 amino acid residues with a predicted molecular mass of 31.1 kDa; in this case, a human caspase-3 antibody can recognizes only the large subunit of the activated enzyme but not the precursor form (Reis et al 2007).…”

Section: Discussionmentioning

confidence: 99%

“…The caspase-3 gene has been sequenced and characterized in zebrafish (Danio rerio) (Yabu et al 2001;Chakraborty et al 2006;Eimon and Ashkenazi, 2010), sea bass (Dicentrarchus labrax L.) (Reis et al 2007) and gudgeon (Gobio gobio) (Nadzialek et al 2010), though sequences are available in the databases for other teleostei species, such as trout (Oncorhynchus mykiss), salmon (Salmo salar), seabream (Sparus auratus), Japanese medaka (Oryzias latipes), channel catfish (Ictalurus punctatus), mandarin fish (Siniperca chuatsi) and tiger puffer (Takifugu rubripes).…”

Section: Introductionmentioning

confidence: 99%

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Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)

Rojas¹,

Guzmán²,

Valenzuela³

et al. 2012

Electron. J. Biotechnol.

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Background:Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays.

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Zebrafish Caspase-3: Molecular Cloning, Characterization, Crystallization and Phylogenetic Analysis

Cited by 19 publications

References 30 publications

Fish and Apoptosis: Molecules and Pathways

Fish and Apoptosis: Molecules and Pathways

The zebrafish as a model organism for the study of apoptosis

Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)

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