Gonen H, Stancovski I, Shkedy D, Hadari T, Bercovich B, Bengal E, Mesilati S, Abu-Hatoum O, Schwartz A L, Ciechanover A
Department of Biochemistry, Carmel Medical Center, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
J Biol Chem. 1996 Jan 5;271(1):302-10. doi: 10.1074/jbc.271.1.302.
Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase approximately 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. The native enzyme has an apparent molecular mass of approximately 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an -SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-kappa B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.
通过泛素系统降解蛋白质涉及两个不同的步骤，即泛素与底物的缀合以及加合物的降解。缀合遵循三步机制。首先，泛素被泛素激活酶E1激活。激活后，几种E2酶（泛素载体蛋白或泛素缀合酶，UBCs）之一将泛素从E1转移到与几种泛素 - 蛋白连接酶E3之一结合的蛋白质底物上。这些酶催化该过程的最后一步，即泛素与蛋白质底物的共价连接。底物与E3的结合是特异性的，这意味着E3在识别和选择用于缀合及后续降解的蛋白质中起主要作用。到目前为止，仅鉴定出少数连接酶，显然还有更多尚未被发现。在此，我们描述了一种新型连接酶，它参与非“N端规则”蛋白质底物（如肌动蛋白、肌钙蛋白T和MyoD）的缀合和降解。这种底物特异性表明该酶可能参与肌肉蛋白的降解。该连接酶与先前描述的非N端规则UBC E2 - F1协同作用。有趣的是，它还参与靶向溶菌酶，溶菌酶是一种真正的N端底物，可被E3α和E2 - 14 kDa识别。这种新型连接酶通过与蛋白质N末端残基不同的信号识别溶菌酶。因此，似乎某些蛋白质可以通过多种识别基序和不同的缀合酶对进行靶向。我们已将该连接酶纯化了约200倍，并证明它与其他已知的E3不同，包括E3α/UBR1、E3β和E6 - AP。天然酶的表观分子量约为550 kDa，似乎是一个同二聚体。由于其不寻常的大小，我们将这种新型连接酶命名为E3L（大）。E3L含有一个对其活性至关重要的 - SH基团。与最近描述的几种E3酶一样，包括E6 - AP和参与NF - κB前体p105加工的连接酶，这种新型连接酶存在于哺乳动物组织中，但不存在于小麦胚芽中。
