Ubiquitin Ubiquitinylation Enzymes

Fig. 2.9 Ubiquitinylation of proteins and degradation in the proteosome. Ubiquitin (Ub) is initially activated by an enzyme El, whereby the C-terminal carboxyl group of ubiquitin becomes attached to an SH group of El via a thioester bond. The activated ubiquitin is then transferred from El-Ub to the ubiquitin-conjugating enzyme, E2. Finally, the ubiquitin is covalently attached to the target protein in a reaction catalyzed by the E3 ubiquitin ligase. Re-...

Ubiquitinylating enzymes localized to the cytosolic face of the ER add ubiquitin to misfolded ER proteins as they exit the ER. This reaction, which is coupled to hydrolysis of ATP,... 

The third step of ubiquitinylation, the transfer of ubiquitin to the target protein, is catalyzed by a ubiquitin-protein-ligase, or E3 enzyme. In this reaction ubiquitin is linked by its C-terminal glycine in an amide isopeptide linkage to an e-NH2-group of the substrate proteins Lys residues. 

In other families of E3 enzymes, no intermediate E3-ubiquitin linkage can be demonstrated. In this case ubiquitin is transferred directly from E2 to the substrate protein. The E3 enzymes are nevertheless required for ubiquitinylation since the E3 enzymes are responsible for substrate selection and are foimd in tight complexes with the cognate E2 proteins. 

The discovery of this family of E3 enzymes started from the studies on the targeted degradation of the p53 tumor suppressor protein. Ubiquitinylation and degradation of p53 can be mediated by the papilloma virus E6 oncoprotein (see below) in collaboration with a further protein, E6-AP (E6 associated protein). E6-AP was the first member of a large family of E3 enzymes, the Hect (homologous to E6-AP C-ter-minus ) domain family. These proteins form a ubiquitin-E3 intermediate and then transfer the ubiquitin to lysine side chains of the substrate protein. They contain an essential active site Cys residue near the C-terminus and one or several WW domains (see Chapter 8.2.6). Fig. 2.11 illustrates schematically the function of Hect -type E3 ligases on the example of the p53 ubiqutiniylation. 

Fig. 2.11 Ubiquitinylation of the tumor suppressor protein p53. The attachment of ubiquitin molecules to p53 is catalyzed by the Hect-domain E3 ubiquitin ligase E6-AP which receives the activated ubiquitin from an E2 enzyme. An intermediate is formed where the ubiquitin is linked via a thioester bond to an active site cysteine of E6-AP. The E6 protein binds both to E6-AP and p53 and confers substrate specificity. C. cysteine K lysine.

Sumoylation is a covalent modification of proteins that is related to, but functionally distinct from ubiquitination (review Wilson and Rangasami, 2001). As in ubiquitinylation, sumoylation involves the covalent attachment of a small protein moiety, termed SUMO, to target proteins. The reactions leading to sumoylation of substrate proteins are related to those involved in ubiquitination. El- and E2 like enzymes are responsible for the attachment of the SUMO moiety to lysine residues of the target protein. As compared to ubiquitination, sumolyation is more sequence specific and requires a particular amino acids in the neighbourhood of the lysine to be modified. 

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