br Author Contributions Statement br Conflicts of
Author Contributions Statement
Conflicts of interest
Acknowledgments We thank T. Ishii, H. Inaba, and S. Nakamura (Department of Cell Biology, TMDU) for kindly gifting BACCS and technical assistance; S. Kaneko, Y. Ishida, and R. Usumi (Department of Orthodontic Science, TMDU) for experimental advice; K. Nakahama (Department of Cellular Physiological Chemistry, TMDU) for technical advice; and M. Akiyama (URA, Research Administration Division, TMDU) for statistical advice. This work was supported by JSPS KAKENHI [grant numbers JP16K01351 to T.A., JP16K11778 to J.H., JP18K19328 to T.N.] and a grant from the Takeda Science Foundation to T.A.
Introduction Giardia intestinalis is a pathogenic microorganism inhabiting the upper small intestine of humans and many other vertebrates, and is one of the most frequent diarrhoea-causing parasites worldwide (Adam, 2001). G. intestinalis is a binucleated, flagellated protozoan which is considered one of the earliest branching eukaryotes (Svard et al., 2003). The complete G. intestinalis genome has been reported, showing that this parasite have less complex molecular machineries than other eukaryotes (Morrison et al., 2007). Additionally G. intestinalis lacks some of the typical organelles of higher eukaryotic cells, such as mitochondria, peroxisomes and a classical Golgi apparatus (Adam, 2001). Giardia possesses an interesting life cycle that may be a primitive Sildenafil mesylate clinical to different environmental conditions, permitting Giardia to survive both within and outside hosts, alternating between its two stages: trophozoites and cysts (Lujan et al., 1998). The trophozoite is the motile and vegetative stage, which lives and colonizes the upper small intestine by attaching to the epithelial cells. The quadrinucleated cyst is the transmissible stage and is extremely resistant to harsh environmental conditions due to the presence of a protective cell wall. The life cycle begins when cysts are ingested by the host from contaminated water, food, or interpersonal contact. The cysts sense the low pH in the stomach and start a process called excystation, which is finally completed in the upper small intestine where the trophozoites rapidly emerge and proliferate. Some trophozoites are forced down with the intestinal flow and are induced to encyst when confronting low cholesterol concentration in the lower parts of the small intestine. Cysts mature in the large intestine and are then released with the feces (Lujan et al., 1998, Adam, 2001). Giardia\'s encystations involves important molecular and cellular processes (DNA replication, nuclear division), turnover of proteins, and a special regulation of synthesis, sorting and transport of cyst wall components (Carranza and Lujan, 2010). 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 cell cycle 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).