Ubiquitin-conjugating ligases

Ubiquitin tags proteins for protein degradation. The ubiquitination requires three different enzymatic activities, a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2 or Ubc) and a ubiquitin ligase (E3). The action of all three enzymes leads to the establishment of a poly-ubiquitin chain on target proteins which are then recognized and proteolyzed by the 26S proteasome. 

Ardley, H. C., et al., Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7. / Biol Chem, 2001, 276(22), 19640-7. 

Fig. 5.1. The ubiquitin-conjugation pathway. Steps in ubiquitin activation and substrate modification. El, ubiquitin activating enzyme E2, ubiquitin-conjugating enzyme E3, ubiquitin-protein ligase. Atoms involved in the thiol ester and amide bonds are shown.

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

The essential components of the SCF ubiquitin E3 ligase include Skpl, Cul-1/ Cdc53, one of many F-box proteins, and the RINC-H2-finger protein Rod (Rbxl or Hrtl) (Figure 6.2). Although initial studies did not reveal the presence of a fourth component of the SCF complex [14, 15], later work showed that a RINC-H2-finger protein, Rod, is an essential subunit of the SCF complex [3]. The SCF complex thus contains three invariable components (Rod, Cull, and Skpl) which provide a core structure to which one of the many substrate-specific subunits (F-box proteins) binds. The Rocl-Cull-Skpl core also independently interacts with the ubiquitin E2-conjugating enzyme to couple ubiquitin transfer to the substrates [3]. One of the E-box proteins binds directly to a specific substrate and such interaction facilitates the polyubiquitination of the substrate by ubiquitin... 

Ubiquitin ligases largely control the substrate specificity of ubiquitin-conjugation reaction. The temporal specificity of ubiquitin conjugation to substrates by these enzymes is provided by regulation of the ligase activity. Activity of ubiquitin ligases can be modulated by posttranslational modification such as phosphorylation and by allosteric modification of the enzyme, or by attachment to UbL proteins. 

Since the enzyme that is most specific in the ubiquitin-conjugation reaction, E3, is also highly regulated, temporal regulation of ubiquitination is easily achieved. For example, if a given E3 is activated by a protein kinase, activation of the E3 is tied to the stimulation of the kinase. For example, phosphorylation activates the ARC ubiquitin ligase. An interesting example is that of serum-inducible kinase (SNK). SNK stimulates... 

Orian, A., Whiteside, S., Israel, A., Stancovski, I., Schwartz, A.L., and Ciechanover, A. (1995). Ubiquitin-mediated processing of the NF-kB transcriptional activator precursor pl05 Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. J. Biol. Chem. 270,21707-21714. 

Fig. 1. A schematic diagram outlining the hierarchic structure of the ubiquitin system. In an ATP-dependent manner a thioester bond is formed between the C-terminus of ubiquitin and an internal cystein residue of the ubiquitin-activating enzyme. Subsequently, ubiquitin is transferred to a member of the family of ubiquitin-conjugating enzymes, which are also able to form a thioester bond with ubiquitin. The third class of enzymes, the ubiquitin ligases, direct ubiquitin to the proteolytic substrates. Different families of this class of enzymes are known, some of which are also able to form a thioester intermediate with ubiquitin (HECT-domain ligases). The final ubiquitin-substrate linkage is an isopeptide bond between the C-terminus of ubiquitin and internal lysine residues in the substrate proteins...

Ubiquitin-Activating Enzyme, Ubiquitin-Conjugating Enzymes and Ubiquitin-Ligases... 

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