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(以前的C1ORF106)在人类肠道上皮细胞中的作用 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，即机械连接上皮屏障功能和 IL1β的炎症信号(eLife 2018年)。这是由INAVA的未知函数的签名域驱动的 DUF3338，我们新定义它是TRAF6依赖的多泛素化的增强子。DUF3338也稳定 结合GTP交换因子(Global)细胞内毒素-2(Arno)，在一种情况下阻断蛋白质中的INAVA活性 泛素化，以及在另一种情况下作用于侧膜，其中INAVA-Arno复合体影响皮质 F-肌动蛋白动力学与上皮屏障功能。我们现在知道INAVA通过形成 胞浆斑点蛋白在信号转导中增强蛋白质泛素化作用，影响细胞蛋白稳定。 因此，我们提出INAVA作为一种守卫受体，先天感觉肠道功能障碍。 上皮细胞和恢复肠道动态平衡，以应对危险。解释INAVA的功能将是 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，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(H_2O_2)和 IL1b，首先关注感受ROS的E3连接酶Keap-1和SCF复合体。不同的构成 压力诱导的点状效应将通过假设驱动的研究进行测试，这些研究基于我们自己和之前的两个研究 发表的研究(Monahan Science 2018和Huttlin Nature 2017)，以及对 泛素修饰的蛋白质，将识别INAVA作用的效应物和底物-并被Arno to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，并在初选中得到确认 人类肠道肠样病变(就所有目标而言)。 在AIM2中，我们将阐明INAVA斑点的结构，它作为一个分子的组装机制 凝结物，如果多聚体或泛素链形成引发支架。我们还将调查Sitta 蛋白酶体激活或自噬的解体，从而描述了INAVA点状病毒的机制(S) 下调监管。 在目标3中，我们将测试INAVA C末端和Cupid结构域的结构和功能，包括IBD-SNP Y33F，以确定侧膜和点状靶向以及对上皮屏障组装和功能的影响。 为了进一步阐明导致这些事件的生理刺激，我们将追踪我们的化学物质的最高“命中率”。 INAVA斑点形成和侧膜靶向(分别为HSP90和ROCK抑制剂)的筛选

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)