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

获得E3泛素连接酶底物识别机制的详细结构信息是非常重要的。然而，X射线结晶学和核磁共振分析不适合于在体外容易聚集的蛋白质和在细菌或其他生物中不能很好表达的重组蛋白质。酵母ER中的一种这样的E3连接酶是Doa10。Doa10有14个跨膜区，被认为既识别错误折叠的蛋白质，也识别含有N-degron的蛋白质。为了开始分析Doa10识别这些蛋白质的机制，我们试图引入体内定位交联法。以前的研究表明，Doa10特异性识别Matalpha2的N端序列Deg1。事实上，在N端与Deg1融合的模型蛋白会被蛋白酶体以Doa10依赖的方式降解。我们首先在Deg1中引入了琥珀色密码子，并试图引入光活性氨基酸。然而，在这项研究过程中，另一个小组报告了Deg1的N-乙酰化在Doa10识别中的重要作用。目前，我们正试图通过体内定位交联法观察N-乙酰化酶的耗尽或过表达对Doa10识别Deg1的影响。我们还开始分析其他ER膜E3连接酶Hrd1。我们已经证明，Hrd1的第二个跨膜区对于与Hrd1的底物识别亚单位Hrd3结合至关重要。我们目前正在分析Hrd1和Hrd3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。

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.