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C1 ORF 106）在人肠上皮细胞中的作用 以管理环境诱导的细胞应激、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，在机制上连接上皮屏障功能， IL 1 β的炎症信号传导（eLife 2018）。这是由INAVA的签名未知功能域驱动的 DUF 3338，我们新定义为TRAF 6依赖性多聚泛素化的增强子。DUF 3338还稳定地 结合GTP-交换因子（GEF）细胞粘连蛋白-2（ARNO），在一种情况下阻断蛋白质中的INAVA活性 在另一种情况下，作用于侧膜，其中INAVA-ARNO复合物影响皮质 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 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作为一个警卫受体，在肠道内先天性感觉功能障碍， 并恢复肠道内环境稳定。解释INAVA的功能将是 提供了屏障上皮细胞如何与管腔和上皮下微环境相互作用的信息， 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（H2 O2）诱导的蛋白质泛素化中发挥作用， IL 1b，首先关注ROS敏感E3连接酶KEAP-1和SCF复合物。不同的组成 基于我们自己的和两个先前的假设驱动的研究将测试压力诱导的斑点 发表的研究（Monahan Science 2018和Huttlin Nature 2017），并通过无偏见的蛋白质组学分析， 泛素修饰的蛋白质，将识别INAVA作用的效应物和底物-并被ARNO阻断， 确认特异性。关键结果将像我们的eLife 2018论文中那样进行机械研究，并在主要研究中得到证实。 人肠类肠（与所有目的相同）。 在Aim 2中，我们将阐明INAVA点的结构，它作为分子组装的机制， 如果多聚体或泛素链形成起始支架，则可以使用聚合物缩合物。我们还将调查点 通过激活蛋白酶体或自噬进行分解，从而阐明INAVA斑点的机制 下调。 在目标3中，我们将测试INAVA C-末端和CUPID结构域的结构-功能，包括IBD-SNP Y33 F，以确定侧膜和斑点靶向以及对上皮屏障组装和功能的影响。 为了进一步阐明诱导这些事件的生理刺激，我们将跟踪我们的化学物质的顶部“命中”， 筛选INAVA斑点形成和侧膜靶向（分别为HSP 90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)