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，在许多细胞事件中起关键作用1。tnfr调控的关键细胞事件之一是半胱天冬酶的激活。在某些生理环境中，tnfr激活引起凋亡细胞的组装，而凋亡细胞总是驱动细胞凋亡。在其他情况下，tnfr激活JNK信号级联，可以诱导caspase依赖性细胞凋亡或激活caspase发挥关键的亚致死作用2。导致凋亡细胞组装和激活的信号通路已经被很好地理解，但tnfr驱动的控制jnk依赖性caspase激活的机制仍然很大程度上未知。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，我们启动了一个新的项目，使用果蝇来识别受体诱导的jnk依赖性半胱天冬酶激活所需的基本信号事件。果蝇只有一种tnf样配体Eiger2,3和一种tnf受体Wengen4。iger诱导的果蝇caspase激活依赖于JNK激活；因此，它是TNF- tnfr信号传导的简化模型，可用于表征TNF和JNK激活之间的信号传导途径2,3。****Eiger过表达即使在缺乏Wengen的果蝇中也产生显著的细胞死亡。因此，我们进行了筛选，以确定其他艾格受体，我们发现Sidekick在艾格诱导的细胞死亡中起着至关重要的作用。Sidekick是一种1型跨膜蛋白，最近被描述为一种粘附分子5。一个独立的小组已经确定了格林登瓦尔德是这种受体复合物的另一个潜在成分，我们已经证实了格林登瓦尔德在艾格信号传导中起着重要作用。在目标一中，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翻译后修饰。我们进行了一系列的实验来阐明K63泛素化是否在艾格尔-温根信号传导中起作用，并发现E2复合物对艾格尔诱导的细胞死亡至关重要。在独立筛选中，我们(1)鉴定了18种在艾格尔通路被激活时被大量泛素化的蛋白，(2)鉴定了11种艾格尔诱导死亡所需的E3连接酶。有趣的是，从这个数据集中，我们发现艾格信号导致组蛋白H2A泛素化，而负责H2A7、8单泛素化的E3连接酶Sex Combs Extra （Sce）是艾格诱导的细胞死亡和果蝇ddaC感觉神经元caspase依赖性修剪所必需的。在目标2中，我们将验证艾格蛋白依赖性的Sex Combs Extra激活以及随后的组蛋白H2A泛素化在艾格蛋白诱导的caspase激活中发挥关键作用的假设

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.***