Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP

Fabienne Burlina,Richard Raz,Abu-Baker M Abdel-Aal,Jiejin Li,John Offer,Robin Antrobus,Simone Kunzelmann,Irene Pinzuti,Stephen R Martin,Benjamin Stieglitz,George Papageorgiou

doi:10.1038/s42004-019-0211-7

Abstract

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. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear di-ubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) 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.

Highlights

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway
The formation of di-ubiquitylated NEMOCoZi was observed showing that ubiquitin elongation is performed by HOIL-1-interacting protein (HOIP) alone and does not require HOIL-1 and SHARPIN
Our original goal was to demonstrate the practicality of our new ligation auxiliary hdmb on a challenging biological question

Summary

Results

The addition of hdmb auxiliary was first attempted on a small model peptide (for easy monitoring by HPLC) corresponding to a fragment of the CoZi domain of mouse NEMO (NEMO298–308, ADIYKADFQAE) and containing the Lys[302] residue targeted for ubiquitylation (Fig. 3). The product was isolated in a combined yield for the two steps (ligation and auxiliary removal) of 55% This result encouraged us to repeat the reaction with ubiquitin. HPLC purification yielded NEMOCoZi-Ub in 38% yield for combined steps of ligation and auxiliary removal, and gratifyingly MALDI-TOF MS gave the expected mass (Fig. 5b). We set out to define the minimal catalytic complex of LUBAC required for linear ubiquitin chain elongation of NEMO primed with Ub. In order to probe if NEMOCoZi-Ub can be utilised as a substrate we used purified proteins (RBR domain of HOIP, E1 and E2 ligases) from bacterial expression systems to reconstitute the ubiquitylation cascade. The formation of di-ubiquitylated NEMOCoZi was observed showing that ubiquitin elongation is performed by HOIP alone and does not require HOIL-1 and SHARPIN

Discussion

17 KDa 14 KDa

Methods