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天冬酶的激活是一个必须受到严格调控的事件。凋亡抑制剂（IAP）蛋白家族成员通过阻断半胱天冬酶活性来确保细胞存活。虽然一般认为IAPs通过空间阻断抑制caspases，但最近来自果蝇的数据表明，IAPs介导的caspases失活需要泛素偶联。然而，关于泛素介导的半胱天冬酶抑制的分子机制知之甚少。因此，以黑腹果蝇为模型系统，我提出了一种研究泛素介导的半胱天冬酶调控的策略：我将研究Dronc泛素化的后果，Dronc是果蝇中一个关键的半胱天冬酶启动物，它被DIAP1多泛素化并失活。我将确定所做泛素修饰的类型和位置。与野生型的Dronc相比，将产生非普遍的Dronc突变体，并分析其催化活性、亚细胞定位和与蛋白质伴侣结合的能力。同时，我将阐明如何调节DIAP1的E3泛素蛋白连接酶活性。我将特别关注E2s、UEVs和E3连接酶在促进DIAP1的E3活性中的作用。总之，这些研究很可能揭示了携带ring的内联蛋白是如何使半胱天冬酶处于暂停状态，从而阻止细胞凋亡的。

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.