Analysis Of The Nuclear Translocation Of Arginine Deiminase During Giardia Differentiation

GONZALO F. MAYOL; NATACHA ZLOCOWSKI; CECILIA V. VRANYCH; MARÍA C. TOUZ; ANDREA S. RÓPOLO

Mar Del Plata

Congreso; X Congreso de Protozoología y Enfermades Parasitarias; 2014

SUMOylation, a posttranslational modification of proteins, is vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. During the encystations process, ADI translocates to the nuclei. The first signal that drives ADI to the nuclei is SUMOylation which probably exposes nuclear localization signals (NLSs) promoting the entry of the enzyme to the nuclei. Once inside, ADI acts as a peptidyl arginine deiminase and induces the downregulation of cwp genes, allowing the encystation process to end. In this work we found that in trophozoites overexpressing the SUMO protein, ADI is highly observed inside the nuclei and also the production of cyst is lower than in wild type trophozoites. Moreover, in immunofluorescence assays, ADI and SUMO co-localize in the nuclear membrane. In other eukaryotic cells, proteins with NLSs are transported through the nuclear envelope by a family of transport proteins called karyopherins like importins and exportines. In the Giardia genome data base, only the importin β-3 subunit (GL50803_15106) is described. It shows the HEAT domain involved in intracellular transport and contains a domain with high probability of SUMO interaction. The entire open reading frame of the importin gene was amplified, cloned in the pTubHA pac vector and sequenced. After stable trophozoite transfection, immunofluoresce assays were performed. Co-localization studies with the enzyme ADI and with the SUMO protein are a matter of ongoing experiments. An important subject for future studies will be to analyze if there is an association of the SUMOylated form of ADI with importins, or with other cargo proteins, in order to crisscross the nuclear membrane during Giardia differentiation.