Ubiquitin is an evolutionary highly conserved 76-residue protein with a molecular weight of 8.6kDa. Linear ubiquitination is a post-translational protein modification essential in innate and adaptive immune signaling. C-terminal of the ubiquitin molecule attaches itself to the ε-amino group of lysine side chains on target proteins, through the actions of activating and conjugating enzymes. Major ubiquitin modification involves the linking of a lysine side chain on the proximal ubiquitin and the C-terminal of the neighboring distal ubiquitin to form lengthened chains of ubiquitin molecule. Linear ubiquitin chains are formed by LUBAC (linear ubiquitin chain assembly complex) which contains HOIP (HOIL-1L interacting protein), HOIL-1L (heme-oxidized IRP2 ubiquitin ligase 1), and SHARPIN SH3 and multiple ankyrin repeat domains protein (SHANK)-associated RBCK1 homology (RH) domain interactor.
Recombinant linear chains of defined length are expressed in Escherichia coli and purified to homogeneity. Amide linkages join the N- and C-terminus of each ubiquitin molecule to each other. This molecule is HIS-tagged at the N-terminus of the most distal ubiquitin.
Ubiquitin is essential in post-translational modifications that regulate a variety of cellular pathways, such as response to viral infections. Ubiquitin molecules link together through lysine residues to form elongated lysine chains, and the ubiquitin lysine residue participating in the formation of these chains is responsible for the complexity linked with ubiquitin modifications. Almost all the seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) on ubiquitin are thought to be capable of forming homotypic chains that drive different linkage-dependent cellular pathways. The most common modification is K48-based chain which drives the modified protein to proteasomal degradation. Another well characterized modification is the K63-based chain which is linked with endocytic process-regulation, DNA-damage response, and innate immune response signaling. Linear ubiquitin chains also play a role in NFκB (nuclear factor) signaling activation when stimulated by various signals, where these chains link with NEMO (NF-κB essential modulator).