ENTH PROTEIN CHARACTERIZATION IN GIARDIA LAMBLIA

FELIZIANI C; MIRAS S; ZAMPONI N; TOUZ MC

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias.; 2011

The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are located underneath the plasma membrane of the trophozoites. To accomplish both functions, Giardia requires a set of coordinate events involving the minimized secretory machinery of the cell including adaptor proteins and clathrin. The adaptor protein Epsin (Eps 15 Interacting protein) was originally discovered based on its binding to the accessory protein Eps15. The epsin family members have an important role as monomer adaptor proteins involved in the clathrinmediated transport, contributing to membrane deformation prior to vesicle formation. These proteins are classified into two groups: epsin, involved in endocytosis, or epsin-related proteins (epsinR), involved in anterograde transport. So far, the presence of monomeric adaptor proteins has not been reported in Giardia. However, by searching in Giardia Genome Database, we found a protein that contains an ENTH domain characteristic of the epsin family. The analysis of the participation of this ENTH protein (ENTHp) in the trafficking towards the PVs was initiated by cloning and expression of HA-fusion protein and its mutants in Giardia trophozoites. Using IFA and confocal microscopy we determined its subcellular localization showing that this protein localize mainly in the cytosol and somehow around the nuclei. Further, we observed a partial colocalization of ENTHp with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein), with a PV marker and with clathrin. The development of monoclonal antibodies directed against N-terminal region of clathrin heavy chain allowed us to test ENTHp-clathrin interaction by IPP. Interactions of the ENTHp with related adaptor subunits are currently underway. Further analysis using knock-down techniques and specific inhibitors will allow us to characterize this protein in the context of its role in lysosomal trafficking and function. These studies will learn the fundamental biochemical and genetics mechanisms that will help us fully understand the evolution of cellular adaptation mechanisms and the minimum of machinery involved in endocytosis and exocytosis in eukaryotic cells. In addition, we might contribute to the discovery of new therapeutic targets and the development of new diagnostic tools for detecting the parasite in biological fluids and the environment.Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are located underneath the plasma membrane of the trophozoites. To accomplish both functions, Giardia requires a set of coordinate events involving the minimized secretory machinery of the cell including adaptor proteins and clathrin. The adaptor protein Epsin (Eps 15 Interacting protein) was originally discovered based on its binding to the accessory protein Eps15. The epsin family members have an important role as monomer adaptor proteins involved in the clathrinmediated transport, contributing to membrane deformation prior to vesicle formation. These proteins are classified into two groups: epsin, involved in endocytosis, or epsin-related proteins (epsinR), involved in anterograde transport. So far, the presence of monomeric adaptor proteins has not been reported in Giardia. However, by searching in Giardia Genome Database, we found a protein that contains an ENTH domain characteristic of the epsin family. The analysis of the participation of this ENTH protein (ENTHp) in the trafficking towards the PVs was initiated by cloning and expression of HA-fusion protein and its mutants in Giardia trophozoites. Using IFA and confocal microscopy we determined its subcellular localization showing that this protein localize mainly in the cytosol and somehow around the nuclei. Further, we observed a partial colocalization of ENTHp with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein), with a PV marker and with clathrin. The development of monoclonal antibodies directed against N-terminal region of clathrin heavy chain allowed us to test ENTHp-clathrin interaction by IPP. Interactions of the ENTHp with related adaptor subunits are currently underway. Further analysis using knock-down techniques and specific inhibitors will allow us to characterize this protein in the context of its role in lysosomal trafficking and function. These studies will learn the fundamental biochemical and genetics mechanisms that will help us fully understand the evolution of cellular adaptation mechanisms and the minimum of machinery involved in endocytosis and exocytosis in eukaryotic cells. In addition, we might contribute to the discovery of new therapeutic targets and the development of new diagnostic tools for detecting the parasite in biological fluids and the environment.Giardia requires a set of coordinate events involving the minimized secretory machinery of the cell including adaptor proteins and clathrin. The adaptor protein Epsin (Eps 15 Interacting protein) was originally discovered based on its binding to the accessory protein Eps15. The epsin family members have an important role as monomer adaptor proteins involved in the clathrinmediated transport, contributing to membrane deformation prior to vesicle formation. These proteins are classified into two groups: epsin, involved in endocytosis, or epsin-related proteins (epsinR), involved in anterograde transport. So far, the presence of monomeric adaptor proteins has not been reported in Giardia. However, by searching in Giardia Genome Database, we found a protein that contains an ENTH domain characteristic of the epsin family. The analysis of the participation of this ENTH protein (ENTHp) in the trafficking towards the PVs was initiated by cloning and expression of HA-fusion protein and its mutants in Giardia trophozoites. Using IFA and confocal microscopy we determined its subcellular localization showing that this protein localize mainly in the cytosol and somehow around the nuclei. Further, we observed a partial colocalization of ENTHp with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein), with a PV marker and with clathrin. The development of monoclonal antibodies directed against N-terminal region of clathrin heavy chain allowed us to test ENTHp-clathrin interaction by IPP. Interactions of the ENTHp with related adaptor subunits are currently underway. Further analysis using knock-down techniques and specific inhibitors will allow us to characterize this protein in the context of its role in lysosomal trafficking and function. These studies will learn the fundamental biochemical and genetics mechanisms that will help us fully understand the evolution of cellular adaptation mechanisms and the minimum of machinery involved in endocytosis and exocytosis in eukaryotic cells. In addition, we might contribute to the discovery of new therapeutic targets and the development of new diagnostic tools for detecting the parasite in biological fluids and the environment.Giardia Genome Database, we found a protein that contains an ENTH domain characteristic of the epsin family. The analysis of the participation of this ENTH protein (ENTHp) in the trafficking towards the PVs was initiated by cloning and expression of HA-fusion protein and its mutants in Giardia trophozoites. Using IFA and confocal microscopy we determined its subcellular localization showing that this protein localize mainly in the cytosol and somehow around the nuclei. Further, we observed a partial colocalization of ENTHp with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein), with a PV marker and with clathrin. The development of monoclonal antibodies directed against N-terminal region of clathrin heavy chain allowed us to test ENTHp-clathrin interaction by IPP. Interactions of the ENTHp with related adaptor subunits are currently underway. Further analysis using knock-down techniques and specific inhibitors will allow us to characterize this protein in the context of its role in lysosomal trafficking and function. These studies will learn the fundamental biochemical and genetics mechanisms that will help us fully understand the evolution of cellular adaptation mechanisms and the minimum of machinery involved in endocytosis and exocytosis in eukaryotic cells. In addition, we might contribute to the discovery of new therapeutic targets and the development of new diagnostic tools for detecting the parasite in biological fluids and the environment.Giardia trophozoites. Using IFA and confocal microscopy we determined its subcellular localization showing that this protein localize mainly in the cytosol and somehow around the nuclei. Further, we observed a partial colocalization of ENTHp with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein), with a PV marker and with clathrin. The development of monoclonal antibodies directed against N-terminal region of clathrin heavy chain allowed us to test ENTHp-clathrin interaction by IPP. Interactions of the ENTHp with related adaptor subunits are currently underway. Further analysis using knock-down techniques and specific inhibitors will allow us to characterize this protein in the context of its role in lysosomal trafficking and function. These studies will learn the fundamental biochemical and genetics mechanisms that will help us fully understand the evolution of cellular adaptation mechanisms and the minimum of machinery involved in endocytosis and exocytosis in eukaryotic cells. In addition, we might contribute to the discovery of new therapeutic targets and the development of new diagnostic tools for detecting the parasite in biological fluids and the environment.