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

通过E3泛素连接酶获得对底物识别机制的详细结构洞察是很重要的。然而，x射线晶体学和核磁共振分析不适用于在体外容易聚集的蛋白质和在细菌或其他生物中表达不佳的重组蛋白质。其中一个这样的e3连接酶是酵母内质网中的Doa10。Doa10有14个跨膜区域，被认为可以识别错误折叠的蛋白质和含有N-degron的蛋白质。为了开始分析Doa10识别这些蛋白质的机制，我们尝试引入体内位点特异性交联方法。先前的研究表明，Doa10特异性识别Matalpha2的n端序列Deg1。事实上，在其n端与Deg1融合的模型蛋白以依赖于Doa10的方式被蛋白酶体降解。我们首先在Deg1中引入琥珀色密码子，并尝试引入光反应性氨基酸。然而，在本研究过程中，另一组报道了Deg1的n -乙酰化在Doa10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Doa10识别Deg1的影响。我们也开始分析其他ER膜E3连接酶Hrd1。我们已经证明Hrd1的第二个跨膜区域对于Hrd1的底物识别亚基Hrd3的结合至关重要。我们目前正在分析Hrd1和Hrd3之间的相互作用在底物识别和泛素化过程中是如何变化的。

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.