The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. We synthesized ubiquitin thioester and NEMOCoZi and conjugated them together via a native isopeptide bond using a ligation auxiliary. The synthesis of the new hdmb auxiliary is achieved in two easy steps, and it is easily installed onto the lysine side-chain by reductive amination, making this technology more accessible to the non-specialists. We showed for the first time that ubiquitylated NEMOCoZi has similar affinity to linear di-ubiquitin chains as unmodified NEMOCoZi. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the HOIP component of LUBAC alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step, requiring different LUBAC components.
References:
Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP
F. Burlina, A.-B. M. Abdel-Aal, R. Raz, I. Pinzuti, G. Papageorgiou, J. Li, R. Antrobus, S. R. Martin, S. Kunzelmann, B. Stieglitz, J. Offer.
Communications Chemistry, 2019, 2(1),111.
DOI : 10.1038/s42004-019-0211-7