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

剖析果蝇 TNF 和 TNF 受体在调节神经元形态中的作用

• 批准号: RGPIN-2019-05621
• 负责人: Barker, Philip
• 金额: $ 2.62万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686599
• 关键词: Dissecting; Roles; Drosophila; TNF; Receptors

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.***

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1。由TNFR调节的关键细胞事件之一是半胱天冬酶的激活。在一些生理环境中，TNFR激活引起总是驱动细胞凋亡的“核小体”的组装。在其他情况下，TNFR激活JNK信号级联，其可以诱导半胱天冬酶依赖性细胞凋亡或激活半胱天冬酶以发挥关键的亚致死作用2。导致溶酶体组装和活化的信号传导途径已被充分理解，但控制JNK依赖性胱天蛋白酶活化的TNFR驱动机制仍在很大程度上未知。事实上，哺乳动物TNFR超家族成员的复杂性和由它们激活的下游信号通路已经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程序，使用果蝇来识别受体诱导的JNK依赖性caspase激活所需的基本信号事件。果蝇只有一个TNF样配体Eiger 2，3和一个TNFR受体Wengen 4。果蝇中Eiger诱导的半胱天冬酶激活依赖于JNK激活;因此，它是TNF-TNFR信号转导的简化模型，可用于表征TNF和JNK激活之间的信号转导途径2，3. * Eiger蛋白过度表达甚至在缺乏Wengen蛋白的果蝇中也产生显著的细胞死亡。因此，我们进行了筛选，以确定额外的Eiger受体，我们发现Sidekick在Eiger诱导的细胞死亡中起着至关重要的作用。Sidekick是1型跨膜蛋白，最近被描述为粘附分子5。一个独立的小组已经确定Grindenwald是这种受体复合物的另一个潜在成分，我们已经证实Grindenwald在Eiger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信号传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Eiger-Wengen信号中起作用，并发现E2 bendless对于Eiger诱导的细胞死亡是必不可少的。在独立的筛选中，我们（1）鉴定了18种蛋白质，当Eiger途径被激活时，这些蛋白质变得高度泛素化，（2）鉴定了11种E3连接酶，这些酶是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H2 A的泛素化和性梳额外（Sce），E3连接酶负责单泛素化的H2 A7，8，是必需的Eiger诱导的细胞死亡和依赖半胱天冬酶修剪果蝇ddaC感觉神经元。在目标2中，我们将检验这样的假设：Sex Combs Extra的艾格峰依赖性激活以及随后的组蛋白H2 A的遍在化在艾格峰诱导的caspase激活中发挥关键作用。*