Palmitoylation and its role in the protozoan parasite Giardia lamblia

MERINO MC; VRANYCH CV; MIRAS S; TOUZ MC; ROPOLO AS

Nueva York

Conferencia; Gordon Research Conferences; 2012

Introduction. Palmitoylation refers to the addition of palmitate to a cysteine residue of proteins. Despite palmitoylation has been involved in protein trafficking, cell signaling and localization to lipid rafts, its biological function has not been completely elucidated. Previous work by our lab showed that variant-specific surface proteins from G. lamblia are palmitoylated and revealed the site of palmitoylation. Three palmitoyl transferases (PATs) of G. lamblia have been identified and some of the possible biological consequences of palmitoylation in this parasite have been determined. The goals of the present work are to characterize other PATs and the target proteins. Proteins that promote palmitoylation have been identified in S. cerevisiae including effector of Ras function (Erf2) and the SNARE protein Ykt6 which have a common Asp-His-His-Cys-cysteine-rich domain (DHHC-CRD) motif that likely confers PAT activity. Materials and Methods. WB 1267 G. lamblia trophozoites were axenically grown in modified TYI-S-33 medium enriched with 10% heat-inactivated fetal bovine serum supplemented with 0.1% bovine bile at 37°C. Induction of encystation was performed as previously described. DNA from trophozoites was isolated and PCR was performed using specific primers. Plasmidic DNA was sequenced and trophozoites were then transfected. Inhibition of palmitoylation was carried out by adding 2-Fluoropalmitic acid (2-FPA). Results. Looking at Giardia genome database, we found other possible PATs. These proteins display the typical DHHC-CRD. By PCR we determined that four possible enzymes are present in G. lamblia genome. PCR products were then cloned in pTub-ApaH7HApac or pINDG-V5 vectors. Immunofluorescense analysis of the epitope-tagged proteins showed that they localize mainly in the cytoplasm or around the nuclei in trophozoites transfected with each PAT. To know the substrates of palmitoylation, we are carrying out experiments with palmitic acid labeled with tritium. When we inhibited palmitoylation during encystation, the percentage of cells expressing Cyst wall protein 1+ decreased (CWP 1+). Moreover, we observed an increase of CWP1+ parasites in encysting Giardia trophozoites that overexpressed a PAT compared to wild type G. lamblia. Main Conclusions. Due to the many intracellular signals involved in encystation it is possible that palmitoylation participates in that process. Further studies are needed to find out the molecular mechanisms in which palmitoylation may be involved. The characterization of proteins which may act as PATs and target proteins of palmitoylation in Giardia would contribute to clarify the mechanisms used by the parasite in key biological events such as antigenic variation and/or encystation.