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Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process.

Merino MC, Zamponi N, Vranych CV, Touz MC, Rópolo AS - PLoS Negl Trop Dis (2014)

Bottom Line: With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome.These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades.Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

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Phylogeny of DHHC proteins.(A) Phylogenetic relationships between DHHC proteins from Giardia and several other species. Phylogenetic tree of DHHC proteins inferred from ML analyses is depicted in the left panel. Symbols correspond to aLRT values >0.7. Sequence taxonomic identity is displayed with colors (outer circle around the tree), as shown in the upper right panel. MCs are labeled as A, B, C, D, E and F. Giardia DHHC proteins are colored in red and indicated in black in the inner circle around the tree. Each Giardia DHHC protein position in the tree (MC) is indicated in the table (lower right panel). (B) Trichomonas duplicated DHHC sequences accumulate mutations. Giardia DHHC proteins are indicated in light blue, and Trichomonas DHHC proteins in yellow. Variations in the HC, C, and DHHC portions of the DHHC-CRD domain were mapped in the tree using a green-to-black-to-red color code. Full conservation is depicted in light green, while lack of conservation is shown in red. A clade of highly mutated Trichomonas sequences is displayed in red.
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pntd-0002997-g004: Phylogeny of DHHC proteins.(A) Phylogenetic relationships between DHHC proteins from Giardia and several other species. Phylogenetic tree of DHHC proteins inferred from ML analyses is depicted in the left panel. Symbols correspond to aLRT values >0.7. Sequence taxonomic identity is displayed with colors (outer circle around the tree), as shown in the upper right panel. MCs are labeled as A, B, C, D, E and F. Giardia DHHC proteins are colored in red and indicated in black in the inner circle around the tree. Each Giardia DHHC protein position in the tree (MC) is indicated in the table (lower right panel). (B) Trichomonas duplicated DHHC sequences accumulate mutations. Giardia DHHC proteins are indicated in light blue, and Trichomonas DHHC proteins in yellow. Variations in the HC, C, and DHHC portions of the DHHC-CRD domain were mapped in the tree using a green-to-black-to-red color code. Full conservation is depicted in light green, while lack of conservation is shown in red. A clade of highly mutated Trichomonas sequences is displayed in red.

Mentions: In order to elucidate the phylogenetic relationship among the PATs and to infer the evolutionary history of Giardia DHHC proteins, we retrieved 1034 DHHC-CRD protein sequences from 84 completely sequenced eukaryotic genomes, including the Giardia lamblia genome (Assemblage A, isolate WB), by means of the DHHC PAT HMMer profile from Pfam (zf-DHHC). A Multiple Sequence Alignment was constructed with PROMALS3D [49], and Block Mapping and Gathering with Entropy (BMGE) [51] was used to select columns suitable for Maximum Likelihood (ML) phylogenetic inference. Maximum likelihood phylogenetic trees were calculated using PhyML [52], and Branch support was evaluated by approximate likelihood-ratio test (aLRT) [84]. The resultant phylogenetic tree can be divided in six monophyletic clades (MC), three of which together contain almost 90% of all sequences (MC D, E and F). Four MC have Giardia DHHC proteins: MC A and D contain one DHHC sequence each, while MC E and F contain five and two Giardia sequences respectively (Figure 4A and figures S2, S3, S4, S5). Without any further consideration than the topology of the tree and the early divergent phylogenetic status of Giardia, it can be argued that the Most Recent Common Ancestor of Giardia and the rest of the eukaryotic lineage (MRCA) had a minimum of four and a maximum of six groups of PATs. However, of the two Giardia-lacking MC one is almost entirely composed of Plant paralogues (MC C). Moreover, many MC contain subclades composed mostly or even only by Plant paralogues, suggesting that gene duplication have largely taken place in this group. All these can be seen as an indication of functional diversification among Plants, which also constitutes a plausible evolutionary mechanism for the origin of the MC C.


Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process.

Merino MC, Zamponi N, Vranych CV, Touz MC, Rópolo AS - PLoS Negl Trop Dis (2014)

Phylogeny of DHHC proteins.(A) Phylogenetic relationships between DHHC proteins from Giardia and several other species. Phylogenetic tree of DHHC proteins inferred from ML analyses is depicted in the left panel. Symbols correspond to aLRT values >0.7. Sequence taxonomic identity is displayed with colors (outer circle around the tree), as shown in the upper right panel. MCs are labeled as A, B, C, D, E and F. Giardia DHHC proteins are colored in red and indicated in black in the inner circle around the tree. Each Giardia DHHC protein position in the tree (MC) is indicated in the table (lower right panel). (B) Trichomonas duplicated DHHC sequences accumulate mutations. Giardia DHHC proteins are indicated in light blue, and Trichomonas DHHC proteins in yellow. Variations in the HC, C, and DHHC portions of the DHHC-CRD domain were mapped in the tree using a green-to-black-to-red color code. Full conservation is depicted in light green, while lack of conservation is shown in red. A clade of highly mutated Trichomonas sequences is displayed in red.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109852&req=5

pntd-0002997-g004: Phylogeny of DHHC proteins.(A) Phylogenetic relationships between DHHC proteins from Giardia and several other species. Phylogenetic tree of DHHC proteins inferred from ML analyses is depicted in the left panel. Symbols correspond to aLRT values >0.7. Sequence taxonomic identity is displayed with colors (outer circle around the tree), as shown in the upper right panel. MCs are labeled as A, B, C, D, E and F. Giardia DHHC proteins are colored in red and indicated in black in the inner circle around the tree. Each Giardia DHHC protein position in the tree (MC) is indicated in the table (lower right panel). (B) Trichomonas duplicated DHHC sequences accumulate mutations. Giardia DHHC proteins are indicated in light blue, and Trichomonas DHHC proteins in yellow. Variations in the HC, C, and DHHC portions of the DHHC-CRD domain were mapped in the tree using a green-to-black-to-red color code. Full conservation is depicted in light green, while lack of conservation is shown in red. A clade of highly mutated Trichomonas sequences is displayed in red.
Mentions: In order to elucidate the phylogenetic relationship among the PATs and to infer the evolutionary history of Giardia DHHC proteins, we retrieved 1034 DHHC-CRD protein sequences from 84 completely sequenced eukaryotic genomes, including the Giardia lamblia genome (Assemblage A, isolate WB), by means of the DHHC PAT HMMer profile from Pfam (zf-DHHC). A Multiple Sequence Alignment was constructed with PROMALS3D [49], and Block Mapping and Gathering with Entropy (BMGE) [51] was used to select columns suitable for Maximum Likelihood (ML) phylogenetic inference. Maximum likelihood phylogenetic trees were calculated using PhyML [52], and Branch support was evaluated by approximate likelihood-ratio test (aLRT) [84]. The resultant phylogenetic tree can be divided in six monophyletic clades (MC), three of which together contain almost 90% of all sequences (MC D, E and F). Four MC have Giardia DHHC proteins: MC A and D contain one DHHC sequence each, while MC E and F contain five and two Giardia sequences respectively (Figure 4A and figures S2, S3, S4, S5). Without any further consideration than the topology of the tree and the early divergent phylogenetic status of Giardia, it can be argued that the Most Recent Common Ancestor of Giardia and the rest of the eukaryotic lineage (MRCA) had a minimum of four and a maximum of six groups of PATs. However, of the two Giardia-lacking MC one is almost entirely composed of Plant paralogues (MC C). Moreover, many MC contain subclades composed mostly or even only by Plant paralogues, suggesting that gene duplication have largely taken place in this group. All these can be seen as an indication of functional diversification among Plants, which also constitutes a plausible evolutionary mechanism for the origin of the MC C.

Bottom Line: With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome.These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades.Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

Show MeSH
Related in: MedlinePlus