The formation and readout of linear M1-linked ubiquitin chains play a central role in NF-κB inflammatory signaling. This M1 ubiquitin linkage is synthesized by the Linear Ubiquitin Assembly Complex (LUBAC), which is comprised of three proteins: HOIP, HOIL-1L, and SHARPIN. HOIP and HOIL-1L are both RBR E3 ligases, with HOIP acting as the main enzyme that creates M1-linked chains. HOIL-1L has broader activity and can attach ubiquitin to hydroxyl groups on small polar sidechains and even sugar molecules. NEMO (NF-κB Essential Modulator) is immediately downstream of LUBAC, and it has been shown to phase separate when bound to long M1-linked chains, leading to activation of the IKK complex.
Despite substantial progress in understanding how LUBAC and NEMO work, key questions persist. In this work, we aim to further characterize both LUBAC and NEMO using protein biochemistry and solution biophysics, with the long-term goal of resolving unique structures by cryo-electron microscopy. Our objectives are to better understand how LUBAC assembles, to define the chain types it can generate, and to elucidate how NEMO engages with longer M1-linked ubiquitin chains.