Detection and Characterization of the In Vitro E3 Ligase Activity of the Human MID1 Protein
Detection and Characterization of the In Vitro E3 Ligase Activity of the Human MID1 Protein
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DOI：
10.1016/j.jmb.2011.01.048
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发表时间：
2011-04-08
影响因子：
5.6
通讯作者：
Massiah, Michael A.
中科院分区：
文献类型：
--
作者：
Han, Xiaofeng;Du, Haijuan;Massiah, Michael A.
人MID 1（midline-1）是一种微管相关蛋白，被认为靶向蛋白磷酸酶2A的催化亚基进行降解。它结合α 4，然后招募蛋白磷酸酶2A的催化亚基。作为TRIM家族的成员，MID 1有三个连续的锌结合结构域RING（真正有趣的新基因），Bbox 1和Bbox 2-具有相似的β β α折叠。在这里，我们描述了在体外表征这些域单独和串联。我们观察到，RING结构域表现出更大的泛素（Ub）E3连接酶活性相比，Bbox结构域。与RING-Bbox 1和RING-Bbox 1-Bbox 2结构域串联的自聚泛素化产物的量显著大于单个结构域的自聚泛素化产物的量。然而，没有多泛素化的产品中观察到的Bbox 1-Bbox结构域串联。使用Ub的突变体，我们观察到这些MID 1结构域构建体通过Ub的Lys 63促进Ub链延伸。此外，我们观察到，高分子量的蛋白质产物主要是由于在一个网站（赖氨酸154）上的Bbox 1结构域的RING-Bbox 1和RING-Bbox 1-Bbox 2结构。我们观察到MID 1 E3结构域可以与多种E2结合酶相互作用。最后，在测定中观察到源自与Bbox 1紧密结合的α 4的C末端的45个氨基酸的肽被单泛素化，并且似乎下调形成的多泛素化产物的量。这些研究揭示了生物多样性。E3连接酶活性，并显示其三个锌结合结构域如何有助于MID 1的整体功能。(C)2011爱思唯尔有限公司保留所有权利。
Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains RING (really interesting new gene), Bbox1, and Bbox2-that have similar beta beta alpha-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MIDI. E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function. (C) 2011 Elsevier Ltd. All rights reserved.