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)