A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma

While linear ubiquitin plays critical roles in multiple cell signaling pathways, few substrates have been identified. Global profiling of linear ubiquitin substrates represents a significant challenge because of the low endogenous level of linear ubiquitination and the background interference arising from highly abundant ubiquitin linkages (e.g. K48- and K63-) and from the non-specific attachment of interfering proteins to the linear polyubiquitin chain. We developed a bio-orthogonal linear ubiquitin probe by site-specific encoding of a norbornene amino acid on ubiquitin (NAEK-Ub). This probe facilitates covalent labeling of linear ubiquitin substrates in live cells and enables selective enrichment and identification of linear ubiquitin-modified proteins. Given the fact that the frequent overexpression of the linear linkage-specific deubiquitinase OTULIN correlates with poor prognosis in glioblastoma, we demonstrated the feasibility of the NAEK-Ub strategy by identifying and validating substrates of linear ubiquitination in patient-derived glioblastoma stem-like cells (GSCs). We identified STAT3 as a bona fide substrate of linear ubiquitin, and showed that linear ubiquitination negatively regulates STAT3 activity by recruitment of the phosphatase TC-PTP to STAT3. Furthermore, we demonstrated that preferential expression of OTULIN in GSCs restricts linear ubiquitination on STAT3 and drives persistent STAT3 signaling, and thereby maintains the stemness and self-renewal of GSCs.

Automated summary

Linear ubiquitin has critical roles in cell signaling pathways, but its substrates are still rare. We developed a bio-orthogonal linear ubiquitin probe (NAEK-Ub) that enables covalent labeling and identification of linear ubiquitin-modified proteins in live cells. Using this strategy, we identified STAT3 as a substrate of linear ubiquitin and showed that linear ubiquitination negatively regulates STAT3 activity. Moreover, we demonstrated that the overexpression of the deubiquitinase OTULIN restricts linear ubiquitination on STAT3 in glioblastoma stem-like cells (GSCs), leading to persistent STAT3 signaling and maintenance of stemness and self-renewal of GSCs.