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

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 diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMO CoZi-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.

Structural basis for the simultaneous recognition of NEMO and acceptor ubiquitin by the HOIP NZF1 domain

“…Mutations on the surface of NZF1 that binds to NEMO significantly reduce the level of linear ubiquitination of NEMO and, subsequently, NF-κB activation 14 . Moreover, it is suggested that the priming step of NEMO ubiquitination is independent of HOIP catalytic activity 15 , proposing the involvement of HOIL-1L in priming he linear ubiquitination process. HOIL-1L is also shown to regulate LUBAC activities by catalyzing mono-ubiquitination of the LUBAC components, followed by the attachment of linear ubiquitin chains catalyzed by HOIP 16 .…”

Chemical Protein Synthesis in Medicinal Chemistry

“…Ubiquitinylated proteins and proteins modified by ubiquitin-like modifiers such as SUMO are another class of modified proteins that are actively pursued by protein chemists due to the difficulty in obtaining them recombinantly. Although the role of ubiquitinylation in protein degradation by the proteasome is well established, there is increasing evidence that reversible ubiquitinylation also regulates cell cycle events through nonproteolytic mechanisms.…”

“…Another recent report from Haj-Yahya and Lashuel also used a semisynthetic approach for installing a pSer residue in the C-terminal part of the Tau protein and for deciphering the role of this and other PTMs in healthy and diseased states. 19 Ubiquitinylated proteins 20 and proteins modified by ubiquitin-like modifiers such as SUMO 21 are another class of modified proteins that are actively pursued by protein chemists due to the difficulty in obtaining them recombinantly. Although the role of ubiquitinylation in protein degradation by the proteasome is well established, there is increasing evidence that reversible ubiquitinylation also regulates cell cycle events through nonproteolytic mechanisms.…”

The life and work of Robert (Bob) C. Sheppard

“…63,64 They have also been adapted to mitigate solubility issues and to aid the assembly of complex, unique targets. 65,66 More recently, there has been a renaissance in flow methods of Fmoc-SPPS and in their automation, both in academia and industry. New instruments and more efficient protocols have been developed that enable cycle times of a remarkable 5 min or less.…”