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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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Localization of DHHC-HA proteins in trophozoites and effect of DHHC-HA overexpression in encystation.Subcellular localization of gla_1908-HA (A), gla_2116-HA (B), gla_16928-HA (C), or gla_8711-HA (D) in trophozoites or encysting parasites. For trophozoites, gla_1908-HA, gla_2116-HA or gla_16928-HA were stained with anti-BiP (ER) mAb, anti-HA mAb and DAPI; gla_8711-HA was stained with anti-AP2 (PVs) mAb, anti-HA mAb and DAPI. For encysting parasites, after 48 h of encystation dhhc-ha transgenic parasites were stained with anti-HA mAb, anti-CWP1 mAb and DAPI. The cells were analyzed by fluorescence microscopy. One representative cell from each stage is shown. Yellow areas in trophozoites indicate co-localization between DHHC-HA and ER (gla_1908-HA, gla_2116-HA or gla_16928-HA), or between DHHC-HA and PVs (gla_8711-HA). Yellow areas in encysting parasites indicate co-localization between DHHC-HA and CWP1. The inset in C (gla_16928 transgenic encysting II parasites) corresponds to the zoomed area indicated by the lined box. Scale bars = 5 µm.
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pntd-0002997-g008: Localization of DHHC-HA proteins in trophozoites and effect of DHHC-HA overexpression in encystation.Subcellular localization of gla_1908-HA (A), gla_2116-HA (B), gla_16928-HA (C), or gla_8711-HA (D) in trophozoites or encysting parasites. For trophozoites, gla_1908-HA, gla_2116-HA or gla_16928-HA were stained with anti-BiP (ER) mAb, anti-HA mAb and DAPI; gla_8711-HA was stained with anti-AP2 (PVs) mAb, anti-HA mAb and DAPI. For encysting parasites, after 48 h of encystation dhhc-ha transgenic parasites were stained with anti-HA mAb, anti-CWP1 mAb and DAPI. The cells were analyzed by fluorescence microscopy. One representative cell from each stage is shown. Yellow areas in trophozoites indicate co-localization between DHHC-HA and ER (gla_1908-HA, gla_2116-HA or gla_16928-HA), or between DHHC-HA and PVs (gla_8711-HA). Yellow areas in encysting parasites indicate co-localization between DHHC-HA and CWP1. The inset in C (gla_16928 transgenic encysting II parasites) corresponds to the zoomed area indicated by the lined box. Scale bars = 5 µm.

Mentions: To further analyze these DHHC proteins, we expressed full-length gla_1908, gla_2116, gla_16928 and gla_8711 as fusion DHHC proteins containing C-terminal HA-tag [39] and evaluated their protein expression profiles by Western blotting using an anti-HA mAb (Figure 7). Analysis by semi-quantitative RT-PCR indicated that the overexpression of these fusion proteins was 2 to 3-times higher in transgenic cells, as reported for protein expression using a similar vector [9]. Immunofluorescence assays showed that HA-tagged gla_1908, gla_2116, and gla_16928 partially co-localized with BiP in the endoplasmic reticulum (ER) or around the nuclei of transgenic trophozoites (Figure 8, trophozoite). Our results confirmed the localization of gla_16928 already shown by Touz et al. [31]. Analysis of intracellular localization of yeast and mammalian DHHC proteins revealed that the majority of these localize to the ER and Golgi [20], [87]. However, there are a few exceptions, including human DHHC5 protein [87] and Giardia DHHC protein (EAA36893) [31], which localize to the plasma membrane. Also, we found that gla_8711 partially co-localized with the adaptor protein AP-2 [57] at the lysosomal-like peripheral vacuoles (PVs) as well as in plasma membrane and flagella (Figure 8, trophozoite). Ongoing experiments intended to knock-down this protein may reveal its importance during the Giardia life cycle.


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)

Localization of DHHC-HA proteins in trophozoites and effect of DHHC-HA overexpression in encystation.Subcellular localization of gla_1908-HA (A), gla_2116-HA (B), gla_16928-HA (C), or gla_8711-HA (D) in trophozoites or encysting parasites. For trophozoites, gla_1908-HA, gla_2116-HA or gla_16928-HA were stained with anti-BiP (ER) mAb, anti-HA mAb and DAPI; gla_8711-HA was stained with anti-AP2 (PVs) mAb, anti-HA mAb and DAPI. For encysting parasites, after 48 h of encystation dhhc-ha transgenic parasites were stained with anti-HA mAb, anti-CWP1 mAb and DAPI. The cells were analyzed by fluorescence microscopy. One representative cell from each stage is shown. Yellow areas in trophozoites indicate co-localization between DHHC-HA and ER (gla_1908-HA, gla_2116-HA or gla_16928-HA), or between DHHC-HA and PVs (gla_8711-HA). Yellow areas in encysting parasites indicate co-localization between DHHC-HA and CWP1. The inset in C (gla_16928 transgenic encysting II parasites) corresponds to the zoomed area indicated by the lined box. Scale bars = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002997-g008: Localization of DHHC-HA proteins in trophozoites and effect of DHHC-HA overexpression in encystation.Subcellular localization of gla_1908-HA (A), gla_2116-HA (B), gla_16928-HA (C), or gla_8711-HA (D) in trophozoites or encysting parasites. For trophozoites, gla_1908-HA, gla_2116-HA or gla_16928-HA were stained with anti-BiP (ER) mAb, anti-HA mAb and DAPI; gla_8711-HA was stained with anti-AP2 (PVs) mAb, anti-HA mAb and DAPI. For encysting parasites, after 48 h of encystation dhhc-ha transgenic parasites were stained with anti-HA mAb, anti-CWP1 mAb and DAPI. The cells were analyzed by fluorescence microscopy. One representative cell from each stage is shown. Yellow areas in trophozoites indicate co-localization between DHHC-HA and ER (gla_1908-HA, gla_2116-HA or gla_16928-HA), or between DHHC-HA and PVs (gla_8711-HA). Yellow areas in encysting parasites indicate co-localization between DHHC-HA and CWP1. The inset in C (gla_16928 transgenic encysting II parasites) corresponds to the zoomed area indicated by the lined box. Scale bars = 5 µm.
Mentions: To further analyze these DHHC proteins, we expressed full-length gla_1908, gla_2116, gla_16928 and gla_8711 as fusion DHHC proteins containing C-terminal HA-tag [39] and evaluated their protein expression profiles by Western blotting using an anti-HA mAb (Figure 7). Analysis by semi-quantitative RT-PCR indicated that the overexpression of these fusion proteins was 2 to 3-times higher in transgenic cells, as reported for protein expression using a similar vector [9]. Immunofluorescence assays showed that HA-tagged gla_1908, gla_2116, and gla_16928 partially co-localized with BiP in the endoplasmic reticulum (ER) or around the nuclei of transgenic trophozoites (Figure 8, trophozoite). Our results confirmed the localization of gla_16928 already shown by Touz et al. [31]. Analysis of intracellular localization of yeast and mammalian DHHC proteins revealed that the majority of these localize to the ER and Golgi [20], [87]. However, there are a few exceptions, including human DHHC5 protein [87] and Giardia DHHC protein (EAA36893) [31], which localize to the plasma membrane. Also, we found that gla_8711 partially co-localized with the adaptor protein AP-2 [57] at the lysosomal-like peripheral vacuoles (PVs) as well as in plasma membrane and flagella (Figure 8, trophozoite). Ongoing experiments intended to knock-down this protein may reveal its importance during the Giardia life cycle.

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