The E enzyme is the apex for downstream enzymatic cascades
The E1 enzyme is the apex for downstream enzymatic cascades and signaling pathways mediated by Ub and Ub-like proteins (Ubls) (Pickart, 2001). Studying characteristics of E1 and its catalytic functions may throw light to the role of ubiquitination in cell development. All known eukaryotic E1 are monomeric 110–120kDa proteins, although the E1 of either SUMO or NEDD8 (Ub-like proteins – Ubl) present heterodimer complexes and all have three common domains (Fig. 1). They have one domain with two MoeB or ThiF adenylation sequence repeats in the N-terminal for Ub E1. One MoeB or ThiF repeat binds ATP and Ub as an adenylate and the other provides structural stability (Walden et al., 2003, Lois and Lima, 2005, Lee and Schindelin, 2008). These two MoeB or ThiF repeats are separated in SUMO and NEDD8 E1 (one in each subunit). The second one is the catalytic domain having the cysteine residue involved in covalent thioester linkage with Ub. The third domain is the ubiquitin folding domain (UFD) at the carboxy-terminal end of E1 that binds to the E2 (Komatsu et al., 2001, Lee and Schindelin, 2008, Schulman and Harper, 2009).
A characterization of G. intestinalis E1 is reported here. It is shown that the enzyme is time-specifically expressed during encystation and that it undergoes an unique post-translational processing generating two fragments of 68kDa (N-terminal) and 47kDa (C-terminal). It was found, using polyclonal carboxypeptidase a against E1 N- or C-terminal regions, that E1 localizes in the trophozoites in a precise pattern bound to small granules or vesicles. The lethality of RNA antisense silencing of Giardia E1 indicates that E1 is essential. On the other hand, the over-expression of E1 greatly increases the encystation rate, thereby demonstrating a direct relationship between E1 and G. intestinalis differentiation. These results show that Giardia E1 has a different behavior to that found in other eukaryotes and this could be a clue for the evolution of these enzymes. This is the first report of a direct involvement of E1 (and maybe of the ubiquitination pathway) in the differentiation of this early-branching eukaryote.
Materials and methods
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 cell cycle 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.