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In vivo site-sp ecific cross-linking analysis of substrate recognition mechanisms of membrane-associated E3 ligase enzymes

膜相关 E3 连接酶底物识别机制的体内位点特异性交联分析

基本信息

批准号：
22657034
负责人：
NAKATSUKASA Kunio
金额：
$ 2.19万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22657034/
关键词：
膜タンパク質小胞体分解光架橋構造

项目摘要

It is important to obtain detailed structural insight into substrate recognition mechanisms by E3 ubiquitin ligases. However, X-ray crystallography and NMR analysis are not suitable for proteins that are prone to aggregate in vitro and recombinant proteins that are not expressed well in bacteria or other organisms. One such E3-ligase enzyme is Doa10 in the yeast ER. Doa10 has 14 transmembrane regions and are thought to recognize both misfolded proteins and N-degron containing proteins. To begin to analyze the mechanism by which Doa10 recognizes these proteins, we tried to introduce the in vivo site-specific crosslinking method. Previous studies have suggested that Doa10 specifically recognizes Deg1, an N-terminal sequence of Matalpha2. In fact, model proteins that are fused with Deg1 at their N-terminus are degraded by the proteasome in Doa10 dependent manner. We first introduced amber codons in Deg1 and tried to introduce photoreactive amino acids. However, in the course of this study, other group reported an essential role of N-acetylation of Deg1 in recognition by Doa10. Currently we are trying to see the effects of depletion or overexpression of N-acetylation enzymes on the recognition of Deg1 by Doa10 using in vivo site-specific crosslinking method. We also started to analyze other ER membrane E3 ligase Hrd1. We have shown that the second transmembrane region of Hrd1 is critical for binding to Hrd3, a substrate recognition subunit of Hrd1. We are currently analyzing how the interaction between Hrd1 and Hrd3 changes during substrate recognition and ubiquitination.

重要的是获得详细的结构洞察底物识别机制的E3泛素连接酶。然而，X射线晶体学和NMR分析不适合于在体外易于聚集的蛋白质和在细菌或其他生物体中表达不好的重组蛋白质。一种这样的E3-连接酶是酵母ER中的Doa 10。Doa 10具有14个跨膜区，并且被认为识别错误折叠的蛋白质和含有N-降解决定子的蛋白质。为了开始分析Doa 10识别这些蛋白质的机制，我们尝试引入体内位点特异性交联方法。以前的研究表明，Doa 10特异性识别Deg 1，这是Matalpha 2的N-末端序列。事实上，在其N-末端与Deg 1融合的模型蛋白以Doa 10依赖性方式被蛋白酶体降解。我们首先在Deg 1中引入琥珀密码子，并尝试引入光反应性氨基酸。然而，在这项研究的过程中，其他小组报告了Deg 1的N-乙酰化在Doa 10识别中的重要作用。目前，我们正在尝试使用体内位点特异性交联方法观察N-乙酰化酶的缺失或过表达对Doa 10识别Deg 1的影响。我们还开始分析其他ER膜E3连接酶Hrd 1。我们已经表明，Hrd 1的第二个跨膜区是关键的结合Hrd 3，Hrd 1的底物识别亚基。我们目前正在分析Hrd 1和Hrd 3之间的相互作用如何在底物识别和泛素化过程中发生变化。