The ubiquitin proteasome system Ub P is the main
The ubiquitin–proteasome system (Ub–P) is the main cellular machinery for protein turnover in eukaryotic cells (Glickman and Ciechanover, 2002). Degradation by Ub–P involves two steps. Target proteins are selectively, specifically, and covalently attached to ubiquitin (Ub) chains, followed by Ub-tagged proteins becoming unfolded and degraded by the 26S proteasome multiprotein complex. The Ub conjugation to proteins (or ubiquitination) is an enzymatic cascade consisting of three sequential steps, resulting in Ub attachment to the ɛ-amino group of a lysine (Lys) residue in the target protein. Conjugation begins with ATP-dependent Ub activation by the ubiquitin-activating enzyme (E1). The E1 enzyme loads two Ub molecules at two different sites; one Ub is covalently linked by its glycine 76 carboxyl group to a cysteine at the E1 catalytic site by a thioester linkage, and a second Ub is non-covalently associated as an adenylate at the E1 adenylation site. The Ub in the active E1 site is transferred to the ubiquitin-conjugating enzyme (E2). The E2 transiently carries the activated Ub molecule as a thiol ester and this activated Ub is then transferred to the target protein bound to one of the multiple ubiquitin-ligases (E3). Several Ub molecules are then added to the Lys residues in the Ub bound to the substrate protein and these chains are recognized by the 26S proteasome (Pickart, 2001, Glickman and Ciechanover, 2002). The Ub–P system plays important roles in many cell functions, such as Fomepizole australia progression, antigenic presentation, and inflammatory response. Defects in this system in humans are related to several diseases (King et al., 1996, Pickart, 2001). The Ub tagged protein may take different pathways to degradation, depending of the kind of Ub-chains or the amount of Ub molecules attached to it. Ub-chains having at least four Lys48-linked Ub subunits generally trigger tagged protein degradation by the proteasome. Lys63-linked Ub-chains have functions in DNA repair and endocytosis. Monoubiquitination also plays important roles in endocytosis, membrane trafficking, DNA repair and histone regulation. Multiubiquitination (adding multiple monomeric Ub) is involved in endocytosis (Woelk et al., 2007). 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 antibodies 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.