BECAS FULBRIGHT / FUNDACIÓN BUNGE Y BORN / FUNDACIÓN WILLIAMS
Exploring the Unique Dual Function and the Evolutionary Relationship of the Giardial Epsin-Like Protein
María Berta Pozzi
The Johns Hopkins University
Endocytosis and lysosomal protein trafficking is essential in pathogenic parasites since it is directly linked to vital parasite-specific processes, e.g. host cell invasion, nutrition, and cell differentiation into resistant stages as in the case of Giardia. Epidemiological studies carried out in Argentina revealed that about 80% of parasite-infested children were positive for G. lamblia. The infection impairs the body’s capacity to absorb fat, lactose, vitamin A and vitamin B12, commonly leading to weight loss, and may cause malnutrition, especially in children. The malnutrition can lead to stunted growth in children and can also delay puberty, negatively affect cognitive development and cause premature death. In this proposal we intend to build on our previous studies of protein trafficking, but also to extend our focus to other components of the lysosome delivery machinery. There are many reasons why studying the biochemistry and cell biology of lysosomal protein trafficking in this parasite is a worthwhile undertaking. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. By searching in the GDB, we found the protein GlENTHp (for Giardia lamblia ENTH protein) contains an ENTH domain that shares the 3D structure with the ENTH domain of human epsin, displaying an alpha-helical structure composed of 7 alpha-helices. This domain is present in the epsin or epsin-related (epsinR) form, which are involved in endocytosis and protein trafficking from the Golgi to the lysosomes, respectively. Both epsin and epsinR possess clathrin-binding motifs, but only epsin incorporates an ubiquitin-interaction motif (UIM). Using Giardia’s specific anti-clathrin and anti-ubiquitin antibodies, we determined that GlENTHp interacts with both proteins. Subcellular localization showed this protein mainly in the cytosol and occasionally in the nuclei. Biochemical analysis showed that GlENTHp binds PI(3,4,5)P3 and PI(4)P, phosphoinositides linked to anchoring to the plasma membrane and Golgi, respectively. Moreover, protein-protein interaction experiments showed that GlENTHp associates with the alpha subunit of the adaptor protein-2 (AP2, involved in endocytosis) and the gamma subunit of AP1 (implicated in the Golgi-to-lysosome trafficking). Altogether, these results suggest that GlENTHp participates in the machinery for clathrin-mediated membrane budding acting as a dual epsin-epsinR protein. The main objective of this project is to functionally characterize GlENTHp and determine whether it might play a dual function working as either epsin or epsinR depending on the trophozoite requirement for nutrition (endocytosis) or digestion (protein sorting and degradation). Because many components of the endocytic machinery are structurally and functionally conserved, we will be able to apply the many powerful tools available in the yeast system to test our hypothesis and gain a greater understanding of endosomal/lysosomal pathway in this parasite. Our discoveries of the function of GlENTHp in yeast might also allow for an evolutionary reconstruction of this pathway and contribute to the understanding of the itinerary followed by lysosomal proteins, the participation of early endosomes vs. late endosomes in transport, and the relative impact of different molecular machineries to both forward and retrograde transport processes. Finally, it will be important to examine further molecular, biochemistry and cell biology of this parasite to gain significant insight into Giardia biology, epidemiology and host–parasite interaction, and to generate a comprehensive approach to giardiasis, the disease caused by this parasite. The results obtained from this project will provide material for scientific per-review publications disseminating these discoveries among researchers and the communities having interest in cell biology, evolution and infection diseases all over the world. The group of Dr. Touz has vast experience in performing dissemination activities and will help me to translate the technical results into articles that communicate to wider professional audiences. Most importantly, by completing this internship, I expected to set up the yeast system in the laboratory of my home institution since the technical and conceptual knowledge obtained in Dr. Wendland’s laboratory will assure this possibility. We are convinced that the ongoing collaboration with Dr. Wendland will be particularly worthy since her laboratory has contributed to elucidating the role of epsin and other related proteins by a combination of genetic, biochemical, cell biological and biophysical approaches unavailable in Argentina. The experience of Dr. Wendland’s group and the infrastructure and equipment available at The John Hopkins University are certainly appropriate for the development of the study proposed.