br Materials and methods br Results br

Materials and methods
Results
Discussion Ubiquitination is an important eukaryotic process that defines the fate of proteins and their functionality. It has multiple roles in cell survival, differentiation and development (Glickman and Ciechanover, 2002). Ubiquitination is usually known as a protein degradation signal, but many examples have shown its role as an important signal, being regulated by E3-ligase/deubiquitinating enzymes similary to that occuring by kinases/phosphatases during phosphorylation/dephosphorylation (Woelk et al., 2007). Ubiquitin is present in eukaryotes but not in prokaryotes. Ubiquitination consists of an enzymatic cascade that begins with ubiquitin activation by the E1 ubiquitin-activating enzyme (Pickart, 2001, Glickman and Ciechanover, 2002). E1 enzymes are thus considered the apex for downstream enzymatic cascades and ubiquitin-mediated signaling pathways. E1 manipulation leads to defining ubiquitination\'s role in different cells. An E1-deficient strain dramatically reduces Ub conjugation in yeast, producing TASIN-1 receptor arrest (Ghaboosi and Deshaies, 2007); E1 has been shown to have several functions during different development stages in Caenorhabditis elegans and Drosophila melanogaster (Kulkarni and Smith, 2008, Lee et al., 2008). This work focused on studying G. intestinalis E1 for ascertaining the relevance of ubiquitination in this primitive eukaryote\'s development and to explore ubiquitination\'s primordial roles in the eukaryotic linage. The results showed that E1 was an essential gene for G. intestinalis survival and differentiation. E1 enzymes have characteristic domains that are important for their function (Schulman and Harper, 2009), Giardia\'s E1 showed the typical domains of this family (Fig. 1). It was found that the e1 gene was expressed in proliferating trophozoites and its expression was differently regulated during encystations (Fig. 3). E1 mRNA was constitutively transcribed in growing cells but, when differentiation to cyst was induced, the expression increased, reaching its highest level 12h after induction and then progressively decreasing its expression until the cyst was formed (Fig. 3A). Such behavior was also observed at the protein level (Fig. 3D) and represent the first evidence that E1 and ubiquitination might have an important role during G. intestinalis encystation. E1 protein was studied using specific anti-E1 antibodies in wild type cells and in transfected cells overexpressing the enzyme. The results revealed several unusual characteristics, which are unique to this ubiquitin-activating enzyme. The E1 was translated as a full-length protein and then quickly proteolytically processed to generate two major peptides: E1-68 (N-terminal) and the E1-47 (C-terminal) (the names according with their approximate molecular weight) (Fig. 2, Fig. 3). These proteins were observed in immunoblots from trophozoites, cyst, all encystation stages and in cells transformed with the tagged protein. In transformed trophozoites, that express a recombinant E1, the proteolytic event occurs as in normal cells since the full length protein was not detected, but an enhanced E1 activity was observed (reflected as a much higher Ub-conjugates production). The ubiquitination in vitro assay showed that E1 functionality was not affected by the proteolytic process of the enzyme. This processing of Giardia E1 in two polypeptides E1-68 and E1-47 strongly resembles the heterodimeric E1 proteins in the ubiquitination-like SUMO and NEED8 pathways. Members of the E1 family as SUMO- and NEED8-activating enzymes (E1s) are heterodimers as shown in Fig. 1 (Schulman and Harper, 2009) and have to be associated to generate the active E1. SAE1 (for SUMO) and NAE1 (for NEED8) are homologous to the N-terminal half of the E1 for Ub described here, and on the other hand UBA2 (for SUMO) and UBA3 (for NEED8) are homologous to the C-terminal half. Both subunits (MoeB or ThiF repeats) are important in generating the adenylation domain responsible for the Ub-like protein (Ubl) recognition (Lee and Schindelin, 2008, Schulman and Harper, 2009). By analogy to these heterodimeric E1 enzymes, and based in our results that show the absence of the complete protein both in normal and in transformed cells over-expressing E1, we suggest the possibility that Giardia\'s E1 peptides: E1-68 and E1-47, associate to generate the active enzyme.