PROJECT SUMMARY Tumor necrosis factor (TNF) is an important cytokine that coordinates cytokine production, inflammation, cell survival, and cell death. How life and death decisions are made in response to TNF is not completely understood. Several molecular events determine whether TNF stimulation of cells leads to a transcriptional response that promotes cell survival or whether it can lead to cell death. Signaling downstream of the TNF receptor is heavily regulated by post-translation modifications including ubiquitination. LUBAC, a ubiquitin (Ub) E3 ligase consisting of Sharpin, HOIL and HOIP, is the only Ub ligase described to date that can modify proteins with linear Ub chains (Met1-Ub chains). LUBAC activity is essential for NF-kB dependent transcription of prosurvival genes upon TNF stimulation. Cells derived from patients with germline mutations in LUBAC components elicit defective NF-kB- dependent gene transcription and aberrant activation of cell death pathways. Similarly, murine models with defective LUBAC components elicit chronic inflammation and increased apoptosis in multiple organs and tissues. Our published studies indicate that cell death, rather than a deficiency in NF-kB dependent gene transcription, in LUBAC-deficient (sharpincpdm) animals is driving the TNF-dependent chronic inflammation as this phenotype is reversed by a compound deficiency in FADD—an important component of the death inducing signaling complex. This insight led us to the hypothesis that LUBAC activity is directly required for the prevention of cell death. In aim 1, we will address the molecular basis for LUBAC modification of protein targets with Met1-Ub chains. We will address how LUBAC can modify FADD with Met1-Ub chains. In aim 2, we will characterize the functional relevance of FADD modification in cells and use a new Ub-based tool to enrich for Met1-Ub chains. In aim 3, we will investigate the function of LUBAC auto-ubiquitination and how phosphorylation can regulate LUBAC’s linear Ub chain forming activity. Our studies will provide mechanistic insight into the etiology of primary immune deficiencies associated with patients with germline mutations in LUBAC components.