Mechanism of polyubiquitin chain assembly by an ER-associated ubiquitin ligase

用76个残基的泛素(Ub)共价修饰蛋白质，从而改变目标蛋白质的稳定性、定位或活性，几乎调控真核细胞功能的方方面面。这个反应需要三种酶的顺序作用；激活酶(E1)在其催化的半胱氨酸和泛素中的Gly76的羧基之间形成硫酯键以激活它；结合酶(E2)，从E1接收泛素；泛素连接酶(E3)，将泛素分子从E2转移到底物。对于每个真核生物物种，都有两个E1酶，而大约有几十个E2酶(表1)和数千个E3连接酶被发现。E3连接酶可分为两大类，Hect结构域(与E6相关蛋白C末端同源)和那些携带催化环(真正有趣的新基因)结构域的连接酶。 我们用纯化的gp78c和UBE2G2建立了体外泛素化系统。gp78c是ER相关环连接酶的胞浆结构域。该实验允许我们组装Lys48连接的多泛素链。利用这个系统，我们已经证明了多泛素链可以在UBE2G2的催化半胱氨酸上预先组装，然后转移到底物上。

Covalent modification of a protein with the 76-residue protein ubiquitin (Ub), thereby changing the stability, localization, or activity of the target protein, regulates almost all aspects of eukaryotic cellular function. This reaction requires the sequential actions of three types of enzymes; an activating enzyme (E1) forms a thioester linkage between its catalytic cysteine and the carboxyl group of Gly76 in ubiquitin to activate it; a conjugating enzyme (E2) that receives ubiquitin from the E1; a ubiquitin ligase (E3) that transfers the ubiquitin molecule from the E2 to a substrate. For every eukaryotic species, there are two E1 enzymes, whereas approximately dozens of E2 enzymes (Table 1) and thousands of E3 ligases are found. The E3 ligases can be classified into two major catagories, the HECT domain (homologous to E6-associated protein C-Terminus)-containing ligases and those carrying a catalytic RING (really interesting new gene) domain. We have established an in vitro ubiquitination system using purified gp78c, a cytosolic domain of an ER-associated RING ligase and ube2g2. The assay allows us to assemble Lys48 linked polyubiquitin chains. Using this system, we have demonstrated that polyubiquitin chains can be preassembled on the catalytic cysteine of Ube2g2 before being transferred to a substrate.