Characterisation of de-ubiquitylating enzymes in the regulation of apoptosis

在果蝇中，细胞存活的调节关键依赖于凋亡抑制蛋白DIAP1。DIAP1的缺失会导致半胱氨酸天冬氨酸酶的激活和细胞的死亡。DIAP1调控启动子caspase DRONC(caspase-9的同源物)和效应器caspase Drice和DCP-1(caspase-3和-7的常见同源物)。为此，DIAP1需要一个环状结构域，为其提供泛素和泛素样蛋白Nedd8的E3-连接酶活性。泛素不仅可以作为一个单一的单元连接(单一泛素化)，还可以通过自身序列中的内部赖氨酸(K)残基形成链(多泛素化)。连接点(泛素序列中存在的7个赖氨酸)在很大程度上决定了泛素化的结果。研究最多的链类型是K48连接链和K63连接链，前者导致泛素化蛋白质的蛋白酶体降解，后者主要导致蛋白质复合体的形成和信号事件。为了从蛋白质中去除泛素，已经进化出去泛素化酶(DUB)。研究表明，DIAP1介导的caspase泛素化有助于DIAP1的抗凋亡作用。我们和其他人已经证明了DIAP1泛素化的起始者caspase DRONC和效应者caspase DCP-1和Drice。最近的数据表明，caspase失活的非降解机制，而不是泛素介导的蛋白酶体降解。然而，在caspase必须被激活的情况下，需要移除这些修改以允许caspase激活。为了确定参与细胞凋亡调控的去泛素化酶，我们进行了体内RNAi筛选。为此，所有预测的果蝇解偶联酶的RNAi在果蝇发育中的眼睛中与促凋亡IAP拮抗剂Reaper(RPR)和头部退化缺陷(HID)结合表达。这导致了酶的鉴定，当被击倒时，抑制或增强IAP拮抗剂诱导的小眼睛表型。在这项研究中，我们计划描述这一体内筛选的两个命中，以阐明去泛素化酶在调节细胞凋亡中的作用。这将导致确定决定细胞生存或死亡的新机制。

In Drosophila, the regulation of cell survival depends critically on the Inhibitor of Apoptosis (IAP) protein DIAP1. Loss of DIAP1 leads to immediate caspase activation and apoptotic cell death. DIAP1 regulates the initiator caspase DRONC (homologue of caspase-9) and the effector caspases drICE and Dcp-1 (common homologues of caspase-3 and -7). For this purpose, DIAP1 requires a RING domain, providing it with E3-ligase activity for ubiquitin and the ubiquitin-like protein Nedd8.Attachment of the small modifier ubiquitin to lysine residues of the target protein can have various effects. Ubiquitin cannot only be attached as a single unit (mono-ubiquitylation) but can also form chains (poly-ubiquitylation) through internal lysine (K) residues in its own sequence. The linkage point (7 lysines present within the ubiquitin sequence) determines largely the consequence of the ubiquitylation. The best-studied chain types are K48-linked chains, which lead to proteasomal degradation of the ubiquitylated protein, and K63-linked chains, which mainly lead to formation of protein complexes and signalling events. To remove ubiquitin from proteins, de-ubiquitylating enzymes (DUBs) have evolved. DIAP1-mediated ubiquitylation of caspases has been shown to contribute to DIAP1s anti-apoptotic potential. We and others have demonstrated that DIAP1 ubiquitylates the initiator caspase DRONC and the effector caspases Dcp-1 and drICE. Recent data indicate non-degradative mechanisms by which the caspases get inactivated, rather than ubiquitin-mediated proteasomal degradation. However, under conditions where caspases have to be activated, these modifications need to be removed to allow caspase activation. To identify de-ubiquitylating enzymes that are involved in the regulation of apoptosis, we have performed an in vivo RNAi screen. To this end, RNAi fly lines of all predicted Drosophila de-conjugating enzymes were expressed in the developing eye of flies in combination with the proapoptotic IAP-antagonists reaper (rpr) and head involution defective (hid). This led to the identification of enzymes that, when knocked-down, either suppress or enhance the IAP-antagonist induced small eye phenotype. In this study, we plan to characterise two hits of this in vivo screen to elucidate the role of de-ubiquitylating enzymes in the regulation of apoptosis. This will lead to the identification of new mechanisms that determine whether a cell lives or dies.