Uncontrolled activation of the IkappaB kinase (IKK), the key cellular regulator of NF-κB, causes a variety of disorders, including susceptibility to pathogenic infection, autoimmunity, inflammation-induced malignancies, and inflammatory syndromes. However, targeting the IKK-NF-kB signaling pathway has not yielded any new strategies for fighting inflammatory illnesses. The lack of understanding of how IKK becomes activated in response to various stimuli is the fundamental reason for our failure to exploit this unambiguous target. The IKK complex is made up of three subunits, catalytic IKK1 (also known as IKKa) and IKK2/b kinases, which form a heterodimer, and the dimeric scaffolding protein NEMO (NF-κB Essential Modulator), which is stably bound to the heterodimer. In vitro and in cells, this fundamental tetrameric unit of the IKK complex (IKK1:IKK2:NEMO2), emerges as significantly higher molecular weight multimers. The catalytic activation of IKK2, referred to as canonical signaling, requires linear (M1-linked), K63-linked, or mixed poly-ubiquitin chains (Ub-chain) that engage noncovalently with the NEMO subunits. The mechanism by which this binding information is transferred from NEMO:Ub-chain interaction to yield IKK2 subunit phosphorylation and subsequent activation of IKK is unknown. We hypothesize that multimerization via dimer-dimer interaction is required for IKK2 activation and that the tetramer interface stabilizes the native IKK complex, allowing NEMO to undergo conformational changes upon binding to the Ub-chain. The kinase domain of the IKK2 subunit is activated as a result of structural alterations in NEMO. Disease-causing NEMO mutations or mutations at the tetramer interface do not support the assembly and Ub-chain-dependent NEMO conformational changes required for IKK activation. In support of our hypothesis, we have already shown that a short peptide segment derived from NEMO interacts with IKK2 in a signal-dependent manner and that IKK multimerization requires short homologous peptide segments within IKK1 and IKK2. Under this proposal, we will achieve the following specific aims: AIM 1. We will characterize the dimer-dimer interface which is required for IKK multimerization. We will use disease-causing NEMO mutants and IKK multimerization-defective mutants to test how the structural plasticity of NEMO is linked to IKK2 activation. AIM 2. We will determine if the IKK1- and IKK2-derived peptides disrupt dimer-dimer interaction and IKK2 activation in cells and in vivo. We will test if the IKK-derived peptides independently and in combination with the NEMO-derived peptide can ameliorate systemic inflammation and collagen-induced arthritis in mouse models.