Limits...
New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

Romero A, Estévez-Calvar N, Dios S, Figueras A, Novoa B - PLoS ONE (2011)

Bottom Line: Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis.Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells.Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Vigo, Spain.

ABSTRACT
Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

Show MeSH

Related in: MedlinePlus

Modular architecture of initiator and executioner caspase proteins.Initiator and executioner labeled as A and B, respectively. ScanProsite was used to predict the domain features of all caspase proteins. DED domain in mussel caspase-8 was predicted by the SMART software.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037946&req=5

pone-0017003-g001: Modular architecture of initiator and executioner caspase proteins.Initiator and executioner labeled as A and B, respectively. ScanProsite was used to predict the domain features of all caspase proteins. DED domain in mussel caspase-8 was predicted by the SMART software.

Mentions: ScanProsite and SMART software was used to predict the domain features of all translated caspase sequences. Mussel caspase-2 presented one large N-terminal prodomain containing a CARD (92-residue long structural motif). SMART software predicted the presence of a single DED in caspase-8 with a low score value. Except for caspase-2, all caspase proteins presented the two subunits P20 and P10 (Figure 1A). The lack of the P20 domain in caspase-2 was obtained after several different RACE amplifications. Moreover, the structure of this molecule was confirmed by PCR amplification, cloning and sequencing of the complete molecule. The structure of the executioner caspases in mussel was similar to the structures of caspase-3, caspase-7 and caspase-1 described in insects (Figure 1B). The large and small subunits are the result of cleavage of the proenzyme at Asp (D) residues, which are homologous to human Asp316 and Asp330 (based on equivalent sites described in human caspase 2). The caspase proteins possessed four different sites called S1, S2, S3 and S4 pockets involved in the recognition of a core tetrapeptide motif (P4-P3-P2-P1) within the substrate and its catalysis. Conserved residues presented in the S1, S2, S3 and S4 sites were detected. The Arg179 and Gln283 residues from the S1 site were conserved in all caspases except for caspase-2, where the Gln283 residue was substituted. Ser339 and Arg341 characterize S2 and S3 sites, respectively. Both residues are incompletely conserved in mussel caspases. The Ser339 was replaced in caspase-2, caspase-3/7-3 and caspase-8, and the Arg341 was substituted in caspase-2 and caspase-3/7-4. The S4 binding site, which has been described as the key determinant of substrate specificity, varied markedly between the different caspases. Trp348, which helps confer the physical shape of the specificity pocket, was only conserved in caspase-3/7-3 and caspase-8. Proline replaced Trp348 (Pro, like Trp, is a hydrophobic amino acid residue) in caspases-3/7-1, -3/7-2 and -3/7-4 and Ala replaced Trp in caspase-2. The predicted active site (QACRG) was present in all caspases except for caspase-2. In a typical caspase, following substrate binding, catalysis employs a typical cysteine protease mechanism involving a catalytic dyad composed of Cys285 and His237, plus an `oxyanion hole' involving Gly238 and Cys285 (all of which were conserved in mussel except for caspase-2) (Figure 2).


New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

Romero A, Estévez-Calvar N, Dios S, Figueras A, Novoa B - PLoS ONE (2011)

Modular architecture of initiator and executioner caspase proteins.Initiator and executioner labeled as A and B, respectively. ScanProsite was used to predict the domain features of all caspase proteins. DED domain in mussel caspase-8 was predicted by the SMART software.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037946&req=5

pone-0017003-g001: Modular architecture of initiator and executioner caspase proteins.Initiator and executioner labeled as A and B, respectively. ScanProsite was used to predict the domain features of all caspase proteins. DED domain in mussel caspase-8 was predicted by the SMART software.
Mentions: ScanProsite and SMART software was used to predict the domain features of all translated caspase sequences. Mussel caspase-2 presented one large N-terminal prodomain containing a CARD (92-residue long structural motif). SMART software predicted the presence of a single DED in caspase-8 with a low score value. Except for caspase-2, all caspase proteins presented the two subunits P20 and P10 (Figure 1A). The lack of the P20 domain in caspase-2 was obtained after several different RACE amplifications. Moreover, the structure of this molecule was confirmed by PCR amplification, cloning and sequencing of the complete molecule. The structure of the executioner caspases in mussel was similar to the structures of caspase-3, caspase-7 and caspase-1 described in insects (Figure 1B). The large and small subunits are the result of cleavage of the proenzyme at Asp (D) residues, which are homologous to human Asp316 and Asp330 (based on equivalent sites described in human caspase 2). The caspase proteins possessed four different sites called S1, S2, S3 and S4 pockets involved in the recognition of a core tetrapeptide motif (P4-P3-P2-P1) within the substrate and its catalysis. Conserved residues presented in the S1, S2, S3 and S4 sites were detected. The Arg179 and Gln283 residues from the S1 site were conserved in all caspases except for caspase-2, where the Gln283 residue was substituted. Ser339 and Arg341 characterize S2 and S3 sites, respectively. Both residues are incompletely conserved in mussel caspases. The Ser339 was replaced in caspase-2, caspase-3/7-3 and caspase-8, and the Arg341 was substituted in caspase-2 and caspase-3/7-4. The S4 binding site, which has been described as the key determinant of substrate specificity, varied markedly between the different caspases. Trp348, which helps confer the physical shape of the specificity pocket, was only conserved in caspase-3/7-3 and caspase-8. Proline replaced Trp348 (Pro, like Trp, is a hydrophobic amino acid residue) in caspases-3/7-1, -3/7-2 and -3/7-4 and Ala replaced Trp in caspase-2. The predicted active site (QACRG) was present in all caspases except for caspase-2. In a typical caspase, following substrate binding, catalysis employs a typical cysteine protease mechanism involving a catalytic dyad composed of Cys285 and His237, plus an `oxyanion hole' involving Gly238 and Cys285 (all of which were conserved in mussel except for caspase-2) (Figure 2).

Bottom Line: Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis.Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells.Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas, Vigo, Spain.

ABSTRACT
Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

Show MeSH
Related in: MedlinePlus