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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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The expression of cyst wall protein transcripts and the amount of cysts are different among dhhc transgenic encysting parasites.(A) qRT-PCR analysis of cwp1, cwp2, and cwp3 transcripts expression in dhhc transgenic parasites after 48 h of encystation (white bars), relative to the expression in wild-type encysting cells (control) (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. (B) Percentage of water-resistant cysts in dhhc transgenic parasites determined by flow cytometry after 48 h of encystation. The results are presented as the percentage (mean ± SEM) of cysts in three independent experiments. The asterisks indicate that there was significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001).
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pntd-0002997-g009: The expression of cyst wall protein transcripts and the amount of cysts are different among dhhc transgenic encysting parasites.(A) qRT-PCR analysis of cwp1, cwp2, and cwp3 transcripts expression in dhhc transgenic parasites after 48 h of encystation (white bars), relative to the expression in wild-type encysting cells (control) (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. (B) Percentage of water-resistant cysts in dhhc transgenic parasites determined by flow cytometry after 48 h of encystation. The results are presented as the percentage (mean ± SEM) of cysts in three independent experiments. The asterisks indicate that there was significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001).

Mentions: When CWP expression was analyzed in dhhc transgenic parasites by qRT-PCR, we observed that, except for gla_2116 transgenic cells, which displayed similar levels or even moderate decrease in the mRNA expression of CWPs compared to the control, the other dhhc-ha transgenic parasites showed increased expression of CWP1, CWP2, and CWP3 (Figure 9A). Several transcription factors have been described as involved in the regulation of cwp gene transcription [91], [92], [93], [94], [95], [96], [97]. However, the mechanisms underlying transcription control in this parasite have not been completely elucidated. It has always been assumed that the mobilization mechanism for transcription factors in many organisms is based on proteolytic processing [98], [99], [100], [101]. Nevertheless, there is a group of lipid-modified transcription factors whose mobilization mechanism to the nucleus is not based on proteolytic processing but on reversible palmitoylation [102]. If that were the case for the transcription factors involved in Giardia encystation, DHHC proteins would be palmitoylating different transcriptions factors that, in turn, may regulate CWP expression. It would be interesting to explore the molecular architecture of Giardia transcription factors to find out whether palmitoylation is involved in regulating their shuttling between the cytoplasm and the nuclei.


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)

The expression of cyst wall protein transcripts and the amount of cysts are different among dhhc transgenic encysting parasites.(A) qRT-PCR analysis of cwp1, cwp2, and cwp3 transcripts expression in dhhc transgenic parasites after 48 h of encystation (white bars), relative to the expression in wild-type encysting cells (control) (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. (B) Percentage of water-resistant cysts in dhhc transgenic parasites determined by flow cytometry after 48 h of encystation. The results are presented as the percentage (mean ± SEM) of cysts in three independent experiments. The asterisks indicate that there was significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002997-g009: The expression of cyst wall protein transcripts and the amount of cysts are different among dhhc transgenic encysting parasites.(A) qRT-PCR analysis of cwp1, cwp2, and cwp3 transcripts expression in dhhc transgenic parasites after 48 h of encystation (white bars), relative to the expression in wild-type encysting cells (control) (black bars). The data are the means and SEM of three separate experiments, and each experiment was carried out in triplicate. (B) Percentage of water-resistant cysts in dhhc transgenic parasites determined by flow cytometry after 48 h of encystation. The results are presented as the percentage (mean ± SEM) of cysts in three independent experiments. The asterisks indicate that there was significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001).
Mentions: When CWP expression was analyzed in dhhc transgenic parasites by qRT-PCR, we observed that, except for gla_2116 transgenic cells, which displayed similar levels or even moderate decrease in the mRNA expression of CWPs compared to the control, the other dhhc-ha transgenic parasites showed increased expression of CWP1, CWP2, and CWP3 (Figure 9A). Several transcription factors have been described as involved in the regulation of cwp gene transcription [91], [92], [93], [94], [95], [96], [97]. However, the mechanisms underlying transcription control in this parasite have not been completely elucidated. It has always been assumed that the mobilization mechanism for transcription factors in many organisms is based on proteolytic processing [98], [99], [100], [101]. Nevertheless, there is a group of lipid-modified transcription factors whose mobilization mechanism to the nucleus is not based on proteolytic processing but on reversible palmitoylation [102]. If that were the case for the transcription factors involved in Giardia encystation, DHHC proteins would be palmitoylating different transcriptions factors that, in turn, may regulate CWP expression. It would be interesting to explore the molecular architecture of Giardia transcription factors to find out whether palmitoylation is involved in regulating their shuttling between the cytoplasm and the nuclei.

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