Untersuchungen der Ubiquitin-vermittelten Regulation von Apoptose in Drosophila - Ubiquitin-mediated regulation of apoptosis

执行细胞死亡程序的半胱氨酸天冬氨酸酶的激活是一个必须严格调控的事件。细胞凋亡抑制蛋白(IAP)家族的成员通过阻断caspase活性来确保细胞存活。虽然人们普遍认为IAP由于空间阻断而抑制caspase，但最近来自果蝇的数据表明，IAP介导的caspase失活需要泛素结合。然而，关于泛素介导的caspase抑制的分子机制还知之甚少。因此，以果蝇为模型系统，我提出了一种研究泛素介导的caspase调控的策略：我将检测Dronc泛素化的结果，Dronc是果蝇中的一个关键启动子，它是多泛素化的，并被DIAP1灭活。我将确定泛素修饰的类型和位置。将产生非泛素化的Dronc-突变体，并与野生型Dronc进行比较，分析Dronc？S的催化活性、亚细胞定位和与蛋白质伙伴的结合能力。同时，我将阐明DIAP1和S E3泛素蛋白连接酶的活性是如何被调节的。特别是，我将重点介绍E2、UEV和E3连接酶在促进DIAP1？S E3活性中的作用。综上所述，这些研究可能有助于揭示带环的IAP如何阻止半胱氨酸天冬氨酸酶，从而阻止细胞凋亡。

Activation of caspases, which execute the cell death programme, is an event that has to be tightly regulated. Members of the inhibitor of apoptosis (IAP) protein family ensure cell survival by blocking caspase activity. While it is generally believed that IAPs inhibit caspases due to a steric blockade, recent data from Drosophila indicate that IAP-mediated inactivation of caspases requires ubiquitin-conjugation. However, little is known regarding the molecular mechanism underlying ubiquitin-mediated inhibition of caspases. Thus, using Drosophila melanogaster as a model system, I propose a strategy to investigate ubiquitin-mediated regulation of caspases: I will examine the consequence of ubiquitylation of Dronc, a key initiator caspase in Drosophila that is poly-ubiquitylated and inactivated by DIAP1. I will determine the type and position of ubiquitin modification made. Non-ubiquitylatable Dronc-mutants will be generated and analysed in comparison to wild-type Dronc, regarding Dronc¿s catalytic activity, subcellular localization and ability to bind to protein partners. In parallel, I will elucidate how DIAP1¿s E3 ubiquitin-protein ligase activity is regulated. In particular, I will focus on the role of E2s, UEVs and E3 ligases in contributing to DIAP1¿s E3 activity. Together, these studies are likely to shed light into how RING-bearing IAPs keep caspases in abeyance and hence block apoptosis.