Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO

Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.