Mechanisms of action for the IBD-risk gene INAVA: an epithelial guard receptor for inflammation and integrity of the intestinal barrier

我们的目标是阐明 IBD 风险基因 INAVA（以前称为 C1ORF106）如何在人类肠道上皮细胞中发挥作用 管理环境引起的细胞应激、炎症和粘膜表面的完整性。 我们最近发现 INAVA 表现出双重活性，在机制上将上皮屏障功能和 IL1β 的炎症信号传导 (eLife 2018)。这是由 INAVA 的签名未知函数域驱动的 DUF3338，我们新定义为 TRAF6 依赖性多泛素化的增强子。 DUF3338也稳定 结合 GTP 交换因子 (GEF) 细胞粘附素-2 (ARNO)，在一种情况下阻断蛋白质中的 INAVA 活性 泛素化，在另一种情况下，作用于侧膜，INAVA-ARNO 复合物影响皮质 F-肌动蛋白动力学和上皮屏障功能。我们现在知道 INAVA 通过形成多种压力途径发挥作用 胞质斑点增强信号转导中的蛋白质泛素化并影响细胞蛋白质稳态。 因此，我们提出 INAVA 作为一种保护受体来固有地感知肠道功能障碍 上皮细胞并恢复肠道稳态以应对危险。阐明 INAVA 的功能将是 有关屏障上皮细胞如何与腔内和上皮下微环境相互作用的信息，以及 粘膜宿主防御生物学。 在目标 1 中，我们将解释 INAVA 如何在细胞外 ROS (H2O2) 诱导的蛋白质泛素化中发挥作用， IL1b，首先关注 ROS 感应 E3 连接酶 KEAP-1 和 SCF 复合物。不同的组成 压力诱发的泪点将通过基于我们自己的和之前的两项假设驱动的研究进行测试 已发表的研究（Monahan Science 2018 和 Huttlin Nature 2017），并通过公正的蛋白质组学分析 泛素修饰的蛋白质将识别 INAVA 作用的效应子和底物 - 并被 ARNO 阻断 确认特异性。我们将像 eLife 2018 论文中那样对关键结果进行机械研究，并在初级阶段得到确认 人类肠道肠类（对于所有目标）。 在 Aim2 中，我们将阐明 INAVA puncta 的结构，它作为分子的组装机制 冷凝物，以及多核糖体或泛素链是否形成起始支架。我们还将调查 punta 通过激活蛋白酶体或自噬进行分解，从而描述 INAVA puncta 的机制 下调。 在目标 3 中，我们将测试 INAVA C 端和 CUPID 域的结构功能，包括 IBD-SNP Y33F，定义侧膜和泪点的靶向以及对上皮屏障组件和功能的影响。 为了进一步阐明诱发这些事件的生理刺激，我们将跟踪我们的化学物质的主要“命中” 筛选 INAVA 泪点形成和侧膜靶向（分别为 HSP90 和 ROCK 抑制剂）

Our goal is to elucidate how the IBD-risk gene INAVA (previously C1ORF106) acts in human intestinal epithelia to manage environmentally-induced cell stress, inflammation, and the integrity of mucosal surfaces. We recently found that INAVA exhibits dual activities that mechanistically link epithelial barrier function and inflammatory signaling by IL1β (eLife 2018). This is driven by INAVA’s signature Domain of Unknown Function DUF3338, which we newly define as an enhancer of TRAF6-dependent polyubiquitination. DUF3338 also stably binds the GTP-exchange factor (GEF) cytohesin-2 (ARNO), in one case blocking INAVA activity in protein ubiquitination, and in another case acting at lateral membranes where the INAVA-ARNO complex affects cortical F-actin dynamics and epithelial barrier function. We now know INAVA acts in multiple stress pathways by forming cytosolic puncta to enhance protein ubiquitination in signal transduction and affecting cellular proteostasis. As such, we have proposed that INAVA acts as a guard receptor to innately sense dysfunction in the intestinal epithelium and restore intestinal homeostasis in response to danger. Elucidating the function of INAVA will be informative for how barrier epithelial cells interact with the lumenal and sub-epithelial microenvironment, and the biology of mucosal host defense. In Aim 1 we will explain how INAVA functions in protein ubiquitination induced by extracellular ROS (H2O2) and IL1b, focusing first on the ROS-sensing E3-ligases KEAP-1 and SCF complex. The composition of the different stress-induced puncta will be tested by hypothesis-driven studies based upon our own and two previously published studies (Monahan Science 2018 and Huttlin Nature 2017), and by unbiased proteomic analysis of ubiquitin-modified proteins that will identify effectors and substrates of INAVA action - and blocked by ARNO to confirm specificity. Key results will be studied mechanistically as in our eLife 2018 paper and confirmed in primary human intestinal enteroids (as for all Aims). In Aim2 we will elucidate the structure of the INAVA puncta, it’s mechanism of assembly as a molecular condensate, and if polysomes or ubiquitin chains form the initiating scaffold. We will also investigate puncta disassembly by activation of the proteasome or autophagy, thus delineating mechanism(s) of INAVA puncta down-regulation. In Aim 3 we will test structure-function of the INAVA C-terminal and CUPID domains, including the IBD-SNP Y33F, to define lateral membrane and puncta targeting and effects on epithelial barrier assemblies and function. To further elucidate physiologic stimuli inducing these events, we will follow up on top “hits” of our chemical screen for INAVA puncta formation and lateral membrane targeting (HSP90 and ROCK inhibitors respectively)