Characterisation of de-ubiquitylating enzymes in the regulation of apoptosis

在果蝇中，细胞存活的调节关键取决于凋亡抑制剂（IAP）蛋白DIAP 1。DIAP 1的缺失导致立即的半胱天冬酶活化和凋亡性细胞死亡。DIAP 1调节起始半胱天冬酶DRONC（半胱天冬酶-9的同源物）和效应半胱天冬酶drICE和Dcp-1（半胱天冬酶-3和-7的常见同源物）。为此，DIAP 1需要一个RING结构域，使其具有泛素和泛素样蛋白Nedd 8的E3-连接酶活性。泛素不仅可以作为单个单元连接（单泛素化），而且还可以通过其自身序列中的内部赖氨酸（K）残基形成链（多泛素化）。连接点（存在于泛素序列内的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连接的链，其导致泛素化蛋白的蛋白酶体降解，以及K63连接的链，其主要导致蛋白质复合物和信号事件的形成。为了从蛋白质中去除泛素，已经进化出去泛素化酶（DUBs）。DIAP 1介导的半胱天冬酶泛素化已被证明有助于DIAP 1的抗凋亡潜力。我们和其他人已经证明，DIAP 1泛素化的起始caspase DRONC和效应caspase DCP-1和drICE。最近的数据表明，非降解机制，半胱天冬酶得到失活，而不是泛素介导的蛋白酶体降解。然而，在半胱天冬酶必须被激活的条件下，这些修饰需要被去除以允许半胱天冬酶激活。为了鉴定参与细胞凋亡调节的去泛素化酶，我们进行了体内RNAi筛选。为此，所有预测的果蝇去缀合酶的RNAi果蝇品系在果蝇的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）组合表达。这导致了酶的鉴定，当敲低时，抑制或增强IAP拮抗剂诱导的小眼表型。在这项研究中，我们计划重复两次体内筛选，以阐明去泛素化酶在细胞凋亡调控中的作用。这将导致确定决定细胞存活或死亡的新机制。

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.