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

泛素化（或泛素化）是一个复杂参与大多数细胞过程调节的中心过程，它由三种酶介导。E2泛素结合酶在很大程度上被忽视，因为它们被认为只是辅助性的。然而，E2负责从泛素活化酶E1接收泛素，与E3泛素连接酶相互作用，并最终催化泛素与底物的连接。因此，E2是泛素化途径的核心，也是导致蛋白质降解、重新定位或活性调节的泛素介导过程的多样性的原因。到目前为止，还没有在植物中报道生理E2-E3对。该项目旨在确定形成泛素化的分子E2-E3网络。为此，我们将使用植物U盒型E3泛素连接酶（PUB）22，其是对生物和非生物胁迫以及相关连接酶的响应的负调节剂。我们的研究结果表明，PUB 22-E2对指定在不同的层。配对特异性可以通过结构域组成来施加，也可以通过细胞信号传导来调节。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，调节机制，并确定由E2-E3对介导的泛素化的类型（连接部分的数量和连接类型）。详细的功能分析也将深入了解所选E2的生物学作用。此外，我们将开发工具和方法，使真正的E2-E3对的鉴定，从而也使E3泛素连接酶的深入分析。

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.