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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761644
• 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着关键作用1。 TNFR 调节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 激活会导致“凋亡体”的组装，从而总是驱动细胞凋亡。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。导致凋亡体组装和激活的信号通路已被充分了解，但控制 JNK 依赖性 caspase 激活的 TNFR 驱动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该领域的进展。 为了解决这个问题，我们启动了一项新计划，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。果蝇只有一种 TNF 样配体 Eiger2,3 和一种 TNFR 受体 Wengen4。果蝇中艾格峰诱导的 caspase 激活取决于 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。 即使在缺乏 Wengen 的果蝇中，艾格峰过度表达也会导致显着的细胞死亡。因此，我们进行了筛选以鉴定其他艾格峰受体，我们发现 Sidekick 在艾格峰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，最近被描述为粘附分子 5。一个单独的小组已将格林登瓦尔德确定为该受体复合物的另一个潜在组成部分6，并且我们已经证实格林登瓦尔德在艾格峰信号传导中发挥着重要作用。在目标一中，我们将评估 Wengen、Sidekick 和 Grindenwald 在艾格峰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。我们进行了一系列实验来阐明 K63 泛素化是否在艾格峰-温根信号传导中发挥作用，并发现 E2 弯曲对于艾格峰诱导的细胞死亡至关重要。在独立筛选中，我们 (1) 鉴定了 18 种在艾格峰通路激活时变得高度泛素化的蛋白质，(2) 鉴定了艾格峰诱导死亡所需的 11 种 E3 连接酶。有趣的是，从这个数据集中，我们发现艾格峰信号传导导致组蛋白 H2A 泛素化，而 Sex Combs Extra (Sce)（负责 H2A7,8 单泛素化的 E3 连接酶）是艾格峰诱导的细胞死亡和果蝇 ddaC 感觉神经元的 caspase 依赖性修剪所必需的。在目标 2 中，我们将检验以下假设：Sex Combs Extra 的艾格依赖性激活以及随后的组蛋白 H2A 泛素化在艾格峰诱导的 caspase 激活中发挥关键作用。