Dissecting Roles for Drosophila TNF and TNF Receptors in Regulating Neuronal Morphology

在哺乳动物中，肿瘤坏死因子受体(TNFR)超家族由29个成员组成，在许多细胞事件中发挥关键作用。受TNFR调控的关键细胞事件之一是caspase的激活。在某些生理环境中，TNFR的激活导致“凋亡体”的组装，而凋亡体总是驱动细胞凋亡。在另一些情况下，TNFR激活JNK信号级联，可以诱导依赖于caspase的细胞凋亡或激活关键亚致死性角色的caspase 2。导致凋亡体组装和激活的信号通路已经很清楚，但控制JNK依赖的caspase激活的TNFR驱动的机制在很大程度上仍不清楚。事实上，哺乳动物TNFR超家族成员及其激活的下游信号通路的复杂性减缓了这一领域的进展。为了解决这个问题，我们启动了一个使用黑腹果蝇的新程序，以确定受体诱导的JNK依赖的caspase激活所需的基本信号事件。果蝇只有一个类似肿瘤坏死因子的配体Eiger2，3和一个TNFR受体Wengen4。Eiger诱导的果蝇caspase激活依赖于JNK的激活；因此，它是一个简化的TNF-TNFR信号模型，可以用来表征TNF和JNK激活之间的信号通路2，3。Eiger过表达会导致显著的细胞死亡，即使在缺乏Wengen的果蝇中也是如此。因此，我们进行了一次筛选，以确定其他Eiger受体，我们发现SideKick在Eiger诱导的细胞死亡中起着关键作用。SideKick是一种1型跨膜蛋白，最近被描述为黏附分子5。另一个研究小组已经确定Grindenwald是该受体复合体的另一个潜在成分6，我们已经证实Grindenwald在Eiger信号转导中发挥重要作用。在第一个目标中，我们将评估Wengen，Sidekick和Grindenwald在Eiger信号通路中的相互作用。泛素化是TNFR信号转导所需的关键的翻译后修饰。我们进行了一系列实验，以阐明K63泛素化是否在Eiger-Wengen信号转导中发挥作用，并发现E2无止境在Eiger诱导的细胞死亡中是必不可少的。在独立的筛选中，我们(1)鉴定了18种当Eiger途径被激活时变得高度泛素化的蛋白质，(2)鉴定了11种E3连接酶，这些连接酶是Eiger诱导的死亡所必需的。有趣的是，从这个数据集，我们发现Eiger信号导致组蛋白H2A的泛素化，而Eiger诱导的细胞死亡和依赖caspase的果蝇ddAC感觉神经元的修剪需要Sex Combs Extra(SCE)，这是Eiger诱导的细胞死亡和依赖caspase的果蝇ddAC感觉神经元修剪所必需的。在目标2中，我们将检验这一假设，即Eiger依赖的Sex Combs Extra激活以及随后的组蛋白H2A泛素化在Eiger诱导的caspase激活中起关键作用。

In mammals, the Tumour Necrosis Factor Receptor (TNFR) superfamily is composed of 29 members with critical roles in numerous cellular events1. One of the key cellular events regulated by TNFRs is the activation of caspases. In some physiological settings, TNFR-activation causes the assembly of an `apoptosome' that invariably drives apoptosis. In others, TNFRs activate a JNK signaling cascade that can either induce caspase-dependent apoptosis or activate caspases for critical sublethal roles2. The signaling pathways that lead to apoptosome assembly and activation are well understood but the TNFR-driven mechanisms that control JNK-dependent caspase activation remain largely unknown. Indeed, the complexity of mammalian TNFR superfamily members and the downstream signalling paths activated by them has slowed progress in this field. To address this, we initiated a new program using Drosophila melanogaster to identify fundamental signalling events required for receptor-induced JNK-dependent caspase activation. Drosophila have only one TNF-like ligand, Eiger2,3 and only one TNFR receptor, Wengen4. Eiger-induced caspase activation in Drosophila depends on JNK activation; it is therefore a simplified model of TNF-TNFR signaling that can be utilised to characterize the signaling pathway between TNF and JNK activation2,3. Eiger overexpression produces significant cell death even in flies lacking Wengen. We therefore conducted a screen to identify additional Eiger receptors and we have found that Sidekick plays a crucial role in Eiger-induced cell death. Sidekick is a type 1 transmembrane protein recently described as an adhesion molecule5. A separate group has identified Grindenwald as another potential component of this receptor complex6 and we have confirmed that Grindenwald plays an important role in Eiger signalling. In aim one, we will evaluate the interplay between Wengen, Sidekick and Grindenwald in the Eiger signaling pathway. Ubiquitination is a critical post-translational modification required for TNFR signaling. We conducted a series of experiments to elucidate if K63 ubiquitination plays a role in Eiger-Wengen signaling and found that the E2 bendless is essential for Eiger-induced cell death. In independent screens, we (1) identified 18 proteins that became heavily ubiquitinated when the Eiger pathway is activated, and (2) identified 11 E3 ligases require for Eiger-induced death. Intriguingly, from this dataset we found that Eiger signaling results in ubiquitination of histone H2A and that Sex Combs Extra (Sce), the E3 ligase responsible of the mono-ubiquitination of H2A7,8, is required for Eiger-induced cell death and for caspase-dependent pruning of Drosophila ddaC sensory neurons. In aim 2, we will test the hypothesis that Eiger-dependent activation of Sex Combs Extra, and the subsequent ubiquitination of histone H2A, play critical role in Eiger-induced caspase activation.