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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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Analysis of S-palmitoylated proteins displays a different pattern in Giardia growing and encysting parasites.(A) Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. The gel was treated with (hyd+) or without (hyd−) the thioester cleavage reagent hydroxylamine. Samples were then analyzed by autoradiography. (B) Western blotting performed on palmitoylated proteins purified by ABE from hcncp-V5 transgenic trophozoites (HCNCp T) or hcncp-V5 transgenic encysting trophozoites (HCNCp ET). (C) Western blotting performed on palmitoylated proteins purified by ABE from wild-type trophozoites (T) or encysting parasites (ET). The approximate sizes are indicated on the right in kDa.
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pntd-0002997-g001: Analysis of S-palmitoylated proteins displays a different pattern in Giardia growing and encysting parasites.(A) Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. The gel was treated with (hyd+) or without (hyd−) the thioester cleavage reagent hydroxylamine. Samples were then analyzed by autoradiography. (B) Western blotting performed on palmitoylated proteins purified by ABE from hcncp-V5 transgenic trophozoites (HCNCp T) or hcncp-V5 transgenic encysting trophozoites (HCNCp ET). (C) Western blotting performed on palmitoylated proteins purified by ABE from wild-type trophozoites (T) or encysting parasites (ET). The approximate sizes are indicated on the right in kDa.

Mentions: It has been shown that protein palmitoylation actively participates in cell differentiation in a variety of cells [59], [60], [61]. The analysis of the expression of palmitoylated proteins, using metabolic labeling with [3H] palmitic acid, showed that encysting Giardia parasites displayed a different pattern of total protein palmitoylation than growing parasites (Figure 1A, T-ET/hyd−). The results showed a band of ∼60 kDa in trophozoites that may correspond to the expressed VSPs [31] (Figure 1A, T/hyd−). However, when Giardia encysting cells were analyzed, the assay displayed a larger amount of palmitoylated proteins, as can be judged by the larger number of bands displayed compared to trophozoites (Figure 1A, ET/hyd−). When we performed neutral treatment with hydroxylamine, almost complete removal of the attached palmitates was observed in both growing and encysting parasites (Figure 1A, T-ET/hyd+). This confirms that palmitate is attached through a labile thioester linkage (S-palmitoylation) in Giardia, as has been observed in other cell types including parasites [62], being most common among palmitoylated proteins [63]. Protein S-palmitoylation reversibility makes it a flexible, rapid and precise way of protein activity regulation [64] which may be crucial in the encystation process. The fact that the amount of total S-palmitoylated proteins was higher in encysting cells compared to trophozoites suggested that this PTM may play an important role during Giardia differentiation. This observation is in accordance with previous reports showing an important role of protein S-palmitoylation in controlling several crucial processes in parasites such as invasion or motility [44]. During Giardia encystation, the cyst wall proteins (CWPs) are sorted, concentrated within encystation-specific vesicles (ESVs), and exported to the nascent cyst wall [65], [66], [67]. Thus, the larger amount of palmitoylated proteins observed in encysting parasites (Figure 1A, ET/hyd−) may be explained by this additional requirement of protein sorting and export during this stage. In addition to the CWP1, 2 and 3, another type of cyst wall protein has been identified, a High Cysteine Non-variant Cyst protein (HCNCp) [68]. HCNCp belongs to a large group of cysteine-rich, non-VSPs, Type I integral membrane proteins (HCMp) [68]. The palmitoylation prediction algorithm CSS-Palm 3.0 [69] strongly predicts that HCNCp is palmitoylated at cysteines 1602 (CSS-Palm score 6.57, high stringency cut-off 0.31) and 1603 (CSS-Palm score 4.99, high stringency cut-off 0.31), which are located in the transmembrane region and in the cytosolic tail respectively (HMMTOP, (http://www.enzim.hu/hmmtop/) [70], [71]). In order to find out whether HCNCp is palmitoylated or not, we performed the following approach: first, we expressed full length HCNCp as a fusion protein containing a C-terminal V5-tag and a tubulin promoter [39]. The expression of the ∼169 kDa HCNCp protein was equally observed in hcncp-V5 transgenic growing and encysting parasites, together with fragments of 21, 42 and 66 kDa already reported by Davids et al. [68] (Figure S1). Second, hcncp-V5 transgenic trophozoites (HCNCp T) and encysting (HCNCp ET) parasites were subjected to acyl biotin exchange (ABE) as described in Methods. Parallel plus- and minus-hydroxilamine (hyd) samples were analyzed by Western blotting using an anti-V5 mAb (Figure 1B). Only the samples that were treated with hydroxylamine had free cysteine residues able to be detected by biotin/streptavidin (see Methods). When we assayed HCNCp T purified samples, we observed three bands (169, 66 and 21 kDa) and a weak band of 42 KDa (Figure 1B, HCNCp T/hyd+). Also, the four bands (169, 66, 42, and 21 kDa) were observed for HCNCp ET purified sample compared to the control (hyd−), showing that not only the full length but also the smaller epitope-tagged fragments of the HCNCp protein were palmitoylated in encysting parasites (Figure 1B, HCNCp ET/hyd+). The presence of these four bands may account, at least in part, for the bands shown in figure 1A (Figure 1A, ET/hyd−). Although we showed that the constitutively expressed HCNCp can be palmitoylated during growth and encystation, it was clearly reported that HCNCp is almost exclusively expressed during encystation when its expression was analyzed at the mRNA and protein (expression under its own promoter) levels [68]. Altogether, these results suggest that HCNCp is likely important during encystation, while the machinery necessary for its palmitoylation remains unaltered during growth and differentiation. Despite the need of additional assays to accurately identify additional palmitoylation substrates, it seems that this PTM is more frequently founded in encysting cells compared to trophozoites. In parallel to HCNCp T and HCNCp ET samples, we also performed ABE in wild-type trophozoites and encysting parasites and analyzed the purified samples by Western blotting using anti-VSP1267 mAb (Figure 1C). The results showed the specific protein band of VSP1267 (MW ∼60 KDa), in both growing and encysting parasites, suggesting that this PTM may be important for VSP function during the entire 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)

Analysis of S-palmitoylated proteins displays a different pattern in Giardia growing and encysting parasites.(A) Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. The gel was treated with (hyd+) or without (hyd−) the thioester cleavage reagent hydroxylamine. Samples were then analyzed by autoradiography. (B) Western blotting performed on palmitoylated proteins purified by ABE from hcncp-V5 transgenic trophozoites (HCNCp T) or hcncp-V5 transgenic encysting trophozoites (HCNCp ET). (C) Western blotting performed on palmitoylated proteins purified by ABE from wild-type trophozoites (T) or encysting parasites (ET). The approximate sizes are indicated on the right in kDa.
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Related In: Results  -  Collection

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Show All Figures
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pntd-0002997-g001: Analysis of S-palmitoylated proteins displays a different pattern in Giardia growing and encysting parasites.(A) Giardia trophozoites (T) or encysting trophozoites (ET) were labeled with [3H]-palmitic acid and loaded onto SDS-PAGE. The gel was treated with (hyd+) or without (hyd−) the thioester cleavage reagent hydroxylamine. Samples were then analyzed by autoradiography. (B) Western blotting performed on palmitoylated proteins purified by ABE from hcncp-V5 transgenic trophozoites (HCNCp T) or hcncp-V5 transgenic encysting trophozoites (HCNCp ET). (C) Western blotting performed on palmitoylated proteins purified by ABE from wild-type trophozoites (T) or encysting parasites (ET). The approximate sizes are indicated on the right in kDa.
Mentions: It has been shown that protein palmitoylation actively participates in cell differentiation in a variety of cells [59], [60], [61]. The analysis of the expression of palmitoylated proteins, using metabolic labeling with [3H] palmitic acid, showed that encysting Giardia parasites displayed a different pattern of total protein palmitoylation than growing parasites (Figure 1A, T-ET/hyd−). The results showed a band of ∼60 kDa in trophozoites that may correspond to the expressed VSPs [31] (Figure 1A, T/hyd−). However, when Giardia encysting cells were analyzed, the assay displayed a larger amount of palmitoylated proteins, as can be judged by the larger number of bands displayed compared to trophozoites (Figure 1A, ET/hyd−). When we performed neutral treatment with hydroxylamine, almost complete removal of the attached palmitates was observed in both growing and encysting parasites (Figure 1A, T-ET/hyd+). This confirms that palmitate is attached through a labile thioester linkage (S-palmitoylation) in Giardia, as has been observed in other cell types including parasites [62], being most common among palmitoylated proteins [63]. Protein S-palmitoylation reversibility makes it a flexible, rapid and precise way of protein activity regulation [64] which may be crucial in the encystation process. The fact that the amount of total S-palmitoylated proteins was higher in encysting cells compared to trophozoites suggested that this PTM may play an important role during Giardia differentiation. This observation is in accordance with previous reports showing an important role of protein S-palmitoylation in controlling several crucial processes in parasites such as invasion or motility [44]. During Giardia encystation, the cyst wall proteins (CWPs) are sorted, concentrated within encystation-specific vesicles (ESVs), and exported to the nascent cyst wall [65], [66], [67]. Thus, the larger amount of palmitoylated proteins observed in encysting parasites (Figure 1A, ET/hyd−) may be explained by this additional requirement of protein sorting and export during this stage. In addition to the CWP1, 2 and 3, another type of cyst wall protein has been identified, a High Cysteine Non-variant Cyst protein (HCNCp) [68]. HCNCp belongs to a large group of cysteine-rich, non-VSPs, Type I integral membrane proteins (HCMp) [68]. The palmitoylation prediction algorithm CSS-Palm 3.0 [69] strongly predicts that HCNCp is palmitoylated at cysteines 1602 (CSS-Palm score 6.57, high stringency cut-off 0.31) and 1603 (CSS-Palm score 4.99, high stringency cut-off 0.31), which are located in the transmembrane region and in the cytosolic tail respectively (HMMTOP, (http://www.enzim.hu/hmmtop/) [70], [71]). In order to find out whether HCNCp is palmitoylated or not, we performed the following approach: first, we expressed full length HCNCp as a fusion protein containing a C-terminal V5-tag and a tubulin promoter [39]. The expression of the ∼169 kDa HCNCp protein was equally observed in hcncp-V5 transgenic growing and encysting parasites, together with fragments of 21, 42 and 66 kDa already reported by Davids et al. [68] (Figure S1). Second, hcncp-V5 transgenic trophozoites (HCNCp T) and encysting (HCNCp ET) parasites were subjected to acyl biotin exchange (ABE) as described in Methods. Parallel plus- and minus-hydroxilamine (hyd) samples were analyzed by Western blotting using an anti-V5 mAb (Figure 1B). Only the samples that were treated with hydroxylamine had free cysteine residues able to be detected by biotin/streptavidin (see Methods). When we assayed HCNCp T purified samples, we observed three bands (169, 66 and 21 kDa) and a weak band of 42 KDa (Figure 1B, HCNCp T/hyd+). Also, the four bands (169, 66, 42, and 21 kDa) were observed for HCNCp ET purified sample compared to the control (hyd−), showing that not only the full length but also the smaller epitope-tagged fragments of the HCNCp protein were palmitoylated in encysting parasites (Figure 1B, HCNCp ET/hyd+). The presence of these four bands may account, at least in part, for the bands shown in figure 1A (Figure 1A, ET/hyd−). Although we showed that the constitutively expressed HCNCp can be palmitoylated during growth and encystation, it was clearly reported that HCNCp is almost exclusively expressed during encystation when its expression was analyzed at the mRNA and protein (expression under its own promoter) levels [68]. Altogether, these results suggest that HCNCp is likely important during encystation, while the machinery necessary for its palmitoylation remains unaltered during growth and differentiation. Despite the need of additional assays to accurately identify additional palmitoylation substrates, it seems that this PTM is more frequently founded in encysting cells compared to trophozoites. In parallel to HCNCp T and HCNCp ET samples, we also performed ABE in wild-type trophozoites and encysting parasites and analyzed the purified samples by Western blotting using anti-VSP1267 mAb (Figure 1C). The results showed the specific protein band of VSP1267 (MW ∼60 KDa), in both growing and encysting parasites, suggesting that this PTM may be important for VSP function during the entire 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