Abstract
Linear ubiquitination is a reversible posttranslational modification, which plays key roles in multiple biological processes. Linear ubiquitin chain assembly complex (LUBAC) catalyzes linear ubiquitination, while the deubiquitinase OTULIN (OTU deubiquitinase with linear linkage specificity, FAM105B) exclusively cleaves the linear ubiquitin chains. However, our understanding of linear ubiquitination is restricted to a few substrates and pathways. Here we used a human proteome microarray to detect the interacting proteins of LUBAC and OTULIN by systematically screening up to 20,000 proteins. We identified many potential interacting proteins of LUBAC and OTULIN, which may function as regulators or substrates of linear ubiquitination. Interestingly, our results also hint that linear ubiquitination may have broad functions in diverse pathways. In addition, we recognized lymphocyte activation gene-3 (LAG3, CD223), a transmembrane receptor that negatively regulates lymphocyte functions as a novel substrate of linear ubiquitination in the adaptive immunity pathway. In conclusion, our results provide searchable, accessible data for the interacting proteins of LUBAC and OTULIN, which broaden our understanding of linear ubiquitination.
Highlights
Ubiquitination is a reversible posttranslational modification and plays crucial roles in the regulation of various cellular pathways, such as the cell cycle, DNA damage repair, immune signaling, and diverse signal transduction (Komander and Rape, 2012; Swatek and Komander, 2016)
OTULIN expresses as a soluble protein in Escherichia coli, but the purification of HOIP is troublesome in prokaryotic expression systems
Using HEK 293T cells as the protein expression system, HIS-tagged HOIP and the linear ubiquitin chain assembly complex (LUBAC) subunit HOIL-1 were successfully purified from the soluble cell lysate
Summary
Ubiquitination is a reversible posttranslational modification and plays crucial roles in the regulation of various cellular pathways, such as the cell cycle, DNA damage repair, immune signaling, and diverse signal transduction (Komander and Rape, 2012; Swatek and Komander, 2016). The substrates can be modified by monoubiquitination or poly-ubiquitination at lysine or non-lysine residues, such as serine, threonine, and cysteine (Cadwell, 2005; Shimizu et al, 2010; Swatek and Komander, 2016; Wang et al, 2017; Pao et al, 2018). Poly-ubiquitination occurs by diverse ubiquitin chain linkage via the formation of isopeptide bonds at the seven lysine sites of proximal ubiquitin. The first methionine (M1) of ubiquitin can be modified by linking to another ubiquitin molecule via a peptide bond named linear ubiquitination or M1 ubiquitination (Kirisako et al, 2006; Spit et al, 2019). Linear ubiquitination is a distinct linkage type of poly-ubiquitination, as the formation and erasure are catalyzed by unique enzymes named linear ubiquitin chain assembly complex (LUBAC)
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