Immunofluorescence assays were performed to localize the enz
Immunofluorescence assays were performed to localize the enzyme during growth and differentiation to further characterize Giardia E1. As stated before, the assays showed that the Salirasib generated recognized specific E1 forms (E1-114, E1-90 and E1-67 with anti-gNTE1; and E1-47 with anti-gE1CT); the patterns were very similar when using antibodies against the C or the N terminal fragments. The protein was localized at the cytoplasm in several small spots and not in a diffuse form (Fig. 4). The punctuate pattern may indicate E1 vesicular or membrane association. This specific pattern may also reflect regulation by ubiquitination in specifics compartments within the cell. When Giardia was induced to encyst, the protein presented a more diffuse pattern in the cytoplasm (Fig. 4). Encystation involves important molecular and cellular processes and regulation of the synthesis, sorting and transport of cyst wall components (Carranza and Lujan, 2010). The change in the localization pattern in encysting cells may be caused by association of ubiquitination to events involved in the process. Besides, we observed co-localization between some E1 signals and encystation-specific vesicles (ESVs) in encysting cells (Fig. 4), the ESVs are secretory granules which transport CWPs (cyst wall proteins) to the cell surface for release and assembly into the cyst wall (Carranza and Lujan, 2010). The protein was localized also as a diffuse pattern in the cyst cell body but, interestingly, also in the wall or very close to it. Such localization might be important because the only known antibodies to label the wall are those against CWPs. Other proteins had been localized in the cyst wall: A high cysteine non-variant cyst protein (HCNCp) and a group of (EGF)-like cyst proteins (EGFCPs), in these cases the authors used an epitope-tag approach for detecting those molecules in the cyst wall (Davids et al., 2006, Chiu et al., 2010). Localization in the cyst wall of E1 may show a relationship between ubiquitination and the dynamic formation and disruption of the cyst matrix, which is a poorly understood process.
It was thus found that E1 plays an important role in G. intestinalis biology. Inhibiting E1 expression by RNA antisense in trophozoites was lethal while E1 overexpression induced a five-fold increase in cyst formation during encystation. The changes in E1 location during this process and its localization in the cyst wall might show a role for E1 and ubiquitination in related cellular process. All the results presented here strongly indicated that E1, and ubiquitination itself, is essential for Giardia development and differentiation. The specific proteolytic process observed in E1 might be a clue in the evolution of this family of enzymes.
Acknowledgements This work was supported by the Universidad Nacional de Colombia\'s research division (DIB) projects 8003322 and 8003244, the Colombian Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) project 486-80 and the Argentinian Ministerio de Ciencia y Tecnología (MinCyT) project CO/08/10. C.A.N has been awarded a scholarship from the “Universidad Nacional de Colombia” Outstanding Graduate Students Program.
Introduction The study on pyruvate dehydrogenase complex (PDHc) and Escherichia coli (E. coli) PDHc inhibitors has received increasing attention in many years.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 These studies mainly focus on the protein structure, molecular enzymology, inhibitor design and binding mechanisms between inhibitor and target enzyme in vitro. However, there was little report about the practicality of PDHc inhibitors as agrochemicals. PDHc naturally occurs in microbes, plants and animals, thus, some synthesized PDHc inhibitors are likely to be non-selective. These non-selective PDHc inhibitors may be harmful to the environment or humans. However, there was little report on the selectivity of PDHc inhibitors. Therefore, it is of great interest to develop highly selective microbicides with novel structures as PDHc inhibitors.