Computational Molecular Biology 2016, Vol.6, No.1, 1-20
5
et al., 2004). Inhibition of NFkB pathway in selective cells can prove to be an attractive therapeutic proposal for
treating or subliming various diseases resulting from neurodegeneration. (Karlsen et al., 2007; Lawrence, 2009;
Maqbool et al., 2013; Qin et al., 2007).
4 Inhibitors of the NFkB Pathway
Owing to the plethora of cellular functions governed by the NFkB pathway, the regulation and inhibition of NFkB
pathway by various levels of the pathway could be useful in various dysfunctions of the c ell. There are more than
700 known inhibitors which regulate the NFkB at various levels (Gilmore and Herscovitch, 2006). The NFkB
pathway in general could be regulated at three points: 1) blocking the signal reception by interfering with the
receptor-ligand binding resulting in the abolition of the signaling cascade at the initial stage. Bluml et al. described
therapeutic consequences of blocking TNF receptor in arthritis (Bluml et al., 2012) 2) by interfering with any of
the factors involved in the cascade in the cytoplasm. Targeting IKK (Luo et al., 2005), IkB (Anchoori et al., 2010)
and 3) targeting the nuclear translocation of the NFkB factors or interfering with their DNAbinding (D’Acquisto,
2002). The blocking of the E3 ligases and targeting the nuclear translocation of the NFkB pathway can confer
specific inhibition and response and hence could discourage cellular toxicity in patients. We here have discussed
about the details of E3 ubiquitin ligases and why they are a potential drug target.
4.1 E3 ubiquitin ligases: activity and inhibition
In the NFkB pathway, ubiquitin-proteosomal system (UPS) plays a major role in selective catabolism of proteins
in the cell (Figure 1). Avram Hershko, Aaron Ciechanover and Irwin Rose were awarded the Nobel Prize in 2004
for deciphering the central role of the UPS. Ubiquitin protein is found only in eukaryotic cells and is ubiquitously
expressed in all cells. It is localized in the nucleus as well as the cytoplasm (Melino, 2005). The ubiquitination
cascade is catalyzed by the interplay of three enzymes: E1 which is an ubiquitin activating enzyme, E2 which acts
as ubiquitin conjugating enzyme and E3 which shows ubiquitin ligase activity. E1 activates ubiquitin by forming
thioester linkages between the Cys residue of E1 active site and C-terminus Gly 76 residue of ubiquitin. The
E1-ubiquitin complex is transferred to E2. The E2 also forms thio-ester linkages via active site Cys residue with
the Gly 76 of ubiquitin. The final step is catalyzed by E3 ligase where it mediates covalent attachment of the
ubiquitin by forming an iso-peptide bond between the internal Lys residue of the target protein and Gly 76 of
ubiquitin. Multiple rounds of the cycle results in K48 linked poly-ubiquitinated conjugates. This polyubiquitin
acts as a marker of protein for identification by 26S proteosome (Ciechanover, 1994; Glickman and Ciechanover,
2002; Landréet al., 2014; Scheffner et al., 1995). Lys 6, 11, 27, 29, 33, 48 and 63 are the specific Lys residues of
the ubiquitin by which it can conjugate to itself and participate in chain formation, Lys 48 and 63 being more
common. Gly residue in the C-terminus of one ubiquitin molecule attaches to any seven Lys of another ubiquitin
(David, Ziv, Admon, and Navon, 2010). The recognition of a substrate by E3 ubiquitin ligase for ubiquitination is
the most important step in conferring specificity of the reaction. The ubiquitination reaction could be reversed, the
process is named as deubiquitination. The enzymes catalyzing these reactions are termed as deubiquitinases
(DUBs) (Amerik and Hochstrasser, 2004). There are also a group of proteins which are similar to ubiquitin and
are termed as ubiquitin- like proteins (UBLs). Some of the known UBLs include SUMO, NEDD8, ISG15 and
FAT10 (Herrmann et al., 2007). The E3 ligases can be classified into three types based on domain, structure and
mode of action: the HECT (homologous to E6-associated protein C-terminus) type, the RING (Really Interesting
New Gene) finger type and the U-box family (modified RING finger without the full Zn2+ binding ligands) E3
ubiquitin ligases. The RING type can be further classified into two groups, RIR (RING in between RING-RING)
and multi-protein complexes CRL (Cullin-RING E3) type. The RING type and the structurally similar U-box
family E3 ligases bind the E2-ubiquitin complex and target protein s imultaneously, acting as scaffolding proteins.
It transfers the ubiquitin from E2 to the target protein by placing the target Lys close to E2-ubiquitin thioester
bond. In the HECT type E3 ligase ubiquitin is first transferred from E2 to the active site Cys residue of the E3
ligase. The thioester linked ubiquitin is then transferred to the target protein (Metzger et al., 2010).
