Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of protein palmitoylation yields a low amount of Giardia cysts.(A) Growth curves displaying optimal concentrations of 2-BP (left panel) or 2-FP (right panel) that do not affect Giardia growth. Giardia trophozoites were cultured with different concentrations of 2-BP (10, 20, 40, 50, 75 or 100 µM), 2-FP (100, 150 or 200 µM), or DMSO (control) for 48 h. The parasites were then analyzed by staining them with Trypan blue to distinguish live from dead cells and by counting them in a Neubauer chamber. The graph displays the number (mean ± SEM) of parasites counted in three independent experiments. (B) Percentage of encysting parasites and cysts after inhibition of protein palmitoylation. Giardia trophozoites were induced to encyst and 2-BP (10, 20 or 40 µM), 2-FP (100 uM) or DMSO (Control) added to the encysting media. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and analyzed by fluorescence microscopy. One representative cell of each encystation state (encysting I, encysting II, cyst) is shown in the upper panel. The graph in the lower panel represents the percentage (mean + SEM) of the cells counted in each state in three independent experiments. The asterisks indicate significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001). (C) Number of nuclei in encysting II parasites treated with palmitoylation inhibitors. Trophozoites were induced to encyst and 2-BP (20 or 40 µM), 2-FP (100 µM) or DMSO (Control) added to the encystation media as described above. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and DAPI, and analyzed by fluorescence microscopy. One representative encysting II cell is shown. Scale bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109852&req=5

pntd-0002997-g002: Inhibition of protein palmitoylation yields a low amount of Giardia cysts.(A) Growth curves displaying optimal concentrations of 2-BP (left panel) or 2-FP (right panel) that do not affect Giardia growth. Giardia trophozoites were cultured with different concentrations of 2-BP (10, 20, 40, 50, 75 or 100 µM), 2-FP (100, 150 or 200 µM), or DMSO (control) for 48 h. The parasites were then analyzed by staining them with Trypan blue to distinguish live from dead cells and by counting them in a Neubauer chamber. The graph displays the number (mean ± SEM) of parasites counted in three independent experiments. (B) Percentage of encysting parasites and cysts after inhibition of protein palmitoylation. Giardia trophozoites were induced to encyst and 2-BP (10, 20 or 40 µM), 2-FP (100 uM) or DMSO (Control) added to the encysting media. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and analyzed by fluorescence microscopy. One representative cell of each encystation state (encysting I, encysting II, cyst) is shown in the upper panel. The graph in the lower panel represents the percentage (mean + SEM) of the cells counted in each state in three independent experiments. The asterisks indicate significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001). (C) Number of nuclei in encysting II parasites treated with palmitoylation inhibitors. Trophozoites were induced to encyst and 2-BP (20 or 40 µM), 2-FP (100 µM) or DMSO (Control) added to the encystation media as described above. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and DAPI, and analyzed by fluorescence microscopy. One representative encysting II cell is shown. Scale bars = 5 µm.

Mentions: The fact that Giardia encysting cells displayed a large amount of palmitoylated proteins prompted us to find out whether inhibition of protein palmitoylation would influence Giardia encystation. Several compounds have been reported to block protein palmitoylation [73]. The 2-bromopalmitate (2-BP) [74] and the 2-fluoropalmitate (2-FP) [73] inhibitors are non-metabolizable palmitate analogs that block palmitate incorporation into proteins using a still unclear mechanism. These two compounds have been widely used, act as broad inhibitors of palmitate incorporation and do not appear to selectively inhibit the palmitoylation of specific protein substrates. To test the effect of these inhibitors during encystation, Giardia wild-type trophozoites were induced to encyst together with the addition of either 2-BP or 2-FP. It has been reported that 2-BP is not well tolerated by in vitro cultured cells and causes cell death even after a brief exposure to 100 µM of 2-BP [75]. Thus, a growth curve was performed to determine the optimal concentrations that do not affect Giardia growth (10, 20 or 40 µM for 2-BP and 100 µM for 2-FP), observing that trophozoites died under concentrations higher than 50 µM of 2-BP or 150 µM of 2-FP (Figure 2A). After 48 h of encystation, treated or control parasites were harvested, permeabilized, stained with anti-CWP1 mAb and analyzed by fluorescence microscopy (Figure 2B). Wild-type encysting trophozoites were classified as encysting I (EI) (corresponding to 6 h of encystation [76]), encysting II (EII) (corresponding to 12 h of encystation [76]), and cysts (corresponding to 24–48 h of encystation [76]) (Figure 2B, upper panel), based on the following features: cell shape, membrane staining, and number and size of the ESVs. As shown in figure 2B (lower panel), there was a significant reduction in the amount of cysts when parasites were treated with 2-BP (20 µM or 40 µM) or 2-FP (100 µM).


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)

Inhibition of protein palmitoylation yields a low amount of Giardia cysts.(A) Growth curves displaying optimal concentrations of 2-BP (left panel) or 2-FP (right panel) that do not affect Giardia growth. Giardia trophozoites were cultured with different concentrations of 2-BP (10, 20, 40, 50, 75 or 100 µM), 2-FP (100, 150 or 200 µM), or DMSO (control) for 48 h. The parasites were then analyzed by staining them with Trypan blue to distinguish live from dead cells and by counting them in a Neubauer chamber. The graph displays the number (mean ± SEM) of parasites counted in three independent experiments. (B) Percentage of encysting parasites and cysts after inhibition of protein palmitoylation. Giardia trophozoites were induced to encyst and 2-BP (10, 20 or 40 µM), 2-FP (100 uM) or DMSO (Control) added to the encysting media. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and analyzed by fluorescence microscopy. One representative cell of each encystation state (encysting I, encysting II, cyst) is shown in the upper panel. The graph in the lower panel represents the percentage (mean + SEM) of the cells counted in each state in three independent experiments. The asterisks indicate significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001). (C) Number of nuclei in encysting II parasites treated with palmitoylation inhibitors. Trophozoites were induced to encyst and 2-BP (20 or 40 µM), 2-FP (100 µM) or DMSO (Control) added to the encystation media as described above. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and DAPI, and analyzed by fluorescence microscopy. One representative encysting II cell is shown. Scale bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002997-g002: Inhibition of protein palmitoylation yields a low amount of Giardia cysts.(A) Growth curves displaying optimal concentrations of 2-BP (left panel) or 2-FP (right panel) that do not affect Giardia growth. Giardia trophozoites were cultured with different concentrations of 2-BP (10, 20, 40, 50, 75 or 100 µM), 2-FP (100, 150 or 200 µM), or DMSO (control) for 48 h. The parasites were then analyzed by staining them with Trypan blue to distinguish live from dead cells and by counting them in a Neubauer chamber. The graph displays the number (mean ± SEM) of parasites counted in three independent experiments. (B) Percentage of encysting parasites and cysts after inhibition of protein palmitoylation. Giardia trophozoites were induced to encyst and 2-BP (10, 20 or 40 µM), 2-FP (100 uM) or DMSO (Control) added to the encysting media. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and analyzed by fluorescence microscopy. One representative cell of each encystation state (encysting I, encysting II, cyst) is shown in the upper panel. The graph in the lower panel represents the percentage (mean + SEM) of the cells counted in each state in three independent experiments. The asterisks indicate significant difference compared with the control (Student's t test: * p<0.05; **p<0.01; ***p<0.001). (C) Number of nuclei in encysting II parasites treated with palmitoylation inhibitors. Trophozoites were induced to encyst and 2-BP (20 or 40 µM), 2-FP (100 µM) or DMSO (Control) added to the encystation media as described above. After 48 h, the encysting parasites were stained with anti-CWP1 mAb and DAPI, and analyzed by fluorescence microscopy. One representative encysting II cell is shown. Scale bars = 5 µm.
Mentions: The fact that Giardia encysting cells displayed a large amount of palmitoylated proteins prompted us to find out whether inhibition of protein palmitoylation would influence Giardia encystation. Several compounds have been reported to block protein palmitoylation [73]. The 2-bromopalmitate (2-BP) [74] and the 2-fluoropalmitate (2-FP) [73] inhibitors are non-metabolizable palmitate analogs that block palmitate incorporation into proteins using a still unclear mechanism. These two compounds have been widely used, act as broad inhibitors of palmitate incorporation and do not appear to selectively inhibit the palmitoylation of specific protein substrates. To test the effect of these inhibitors during encystation, Giardia wild-type trophozoites were induced to encyst together with the addition of either 2-BP or 2-FP. It has been reported that 2-BP is not well tolerated by in vitro cultured cells and causes cell death even after a brief exposure to 100 µM of 2-BP [75]. Thus, a growth curve was performed to determine the optimal concentrations that do not affect Giardia growth (10, 20 or 40 µM for 2-BP and 100 µM for 2-FP), observing that trophozoites died under concentrations higher than 50 µM of 2-BP or 150 µM of 2-FP (Figure 2A). After 48 h of encystation, treated or control parasites were harvested, permeabilized, stained with anti-CWP1 mAb and analyzed by fluorescence microscopy (Figure 2B). Wild-type encysting trophozoites were classified as encysting I (EI) (corresponding to 6 h of encystation [76]), encysting II (EII) (corresponding to 12 h of encystation [76]), and cysts (corresponding to 24–48 h of encystation [76]) (Figure 2B, upper panel), based on the following features: cell shape, membrane staining, and number and size of the ESVs. As shown in figure 2B (lower panel), there was a significant reduction in the amount of cysts when parasites were treated with 2-BP (20 µM or 40 µM) or 2-FP (100 µM).

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