Molecular mechanisms of E3 ubiquitin ligase - E2 ubiquitin-conjugating enzyme pairings

泛素化（或泛素化）是一个复杂的中心过程，参与大多数细胞过程的调节，它是由三种酶介导的。E2泛素偶联酶在很大程度上被忽视了，因为它们被认为只是辅助的。然而，E2s负责从泛素激活酶E1接收泛素，与E3泛素连接酶相互作用，最终催化泛素附着在底物上。因此，e2是泛素化途径的核心，也负责泛素介导过程的多样性，导致蛋白质降解，重新定位或活性调节。到目前为止，还没有关于植物中E2-E3对的生理学报道。提出的项目旨在确定形成泛素化的E2-E3分子网络。为此，我们将使用植物U-box型E3泛素连接酶（PUB）22，这是一种对生物和非生物胁迫反应的负调节因子，以及相关的连接酶。我们的结果表明，PUB22-E2对在不同的层上被指定。配对特异性可以由结构域组成施加，也可以由细胞信号传导调节。此外，我们的数据表明E2-E3对的细胞定位既可以由E3决定，也可以由E2决定。在这项工作的过程中，我们计划阐明E2-E3相互作用特异性的决定因素，调控机制，并确定E2-E3对介导的泛素化类型（连接部分的数量和连锁类型）。详细的功能分析也将深入了解所选E2s的生物学作用。此外，我们将生成工具和开发方法，将能够识别真正的E2-E3对，从而也能够深入分析E3泛素连接酶。

Ubiquitination (or ubiquitylation) is a central process intricately involved in the regulation of most cellular processes and it is mediated by three enzymes. The E2 ubiquitin conjugating enzymes have largely been neglected because they were considered to be only auxiliary. However, E2s are responsible for receiving ubiquitin from the ubiquitin activating enzyme E1, interacting with the E3 ubiquitin ligase and finally, catalysing the attachment of ubiquitin to the substrate. Therefore, the E2s are at the heart of the ubiquitination pathways and also accountable for the diversity of ubiquitin-mediated processes leading to protein degradation, relocalization or modulation of activity. To date no physiological E2-E3 pair has been reported in plants. The proposed project aims to identify molecular E2-E3 networks that shape ubiquitination. To do so we will use the plant U-box type E3 ubiquitin ligase (PUB)22, a negative regulator of responses to both biotic and abiotic stresses, as well as related ligases. Our results suggest that PUB22-E2 pairs are specified at different layers. Pairing specificities can be imposed by domain composition and also be regulated by cellular signalling. Furthermore, our data indicates that the cellular localization of E2-E3 pairs can be both dictated by the E3, as well as, the E2. In the course of this work we plan to shed light on the determinants of the E2-E3 interaction specificities, regulatory mechanisms and identify the type of ubiquitination mediated by E2-E3 pairs (number of attached moieties and linkage type). A detailed functional analysis will also give insight into the biological roles of selected E2s. In addition, we will generate tools and develop methods that will enable the identification of true E2-E3 pairs and thus also enable the in-depth analysis of E3 ubiquitin ligases.