Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain–specific sensors

摘要

Despite intensive research, there is a distinct lack of methodology for visualizing endogenous ubiquitination in living cells. In this protocol, we describe how unique properties of ubiquitin (Uub)-binding domains (UuBDs) can be used to selectively detect, visualize and inhibit Uub-dependent processes in mammalian cells. Tthe procedure deals with designing and validating the binding selectivity of GFPp-tagged K63- and linear-linked sensors (TAtaB2 NnZF and NEneMOo UuBANan, respectively) in vitro. We describe how these moieties can be used to inhibit tumor necrosis factor (TNtnF)-mediated NnF-kB signaling and to detect ubiquitinated cytosolic Salmonella in living cells, emphasizing a more flexible use compared with chain-specific antibodies. Tthese chain-specific sensors can be used to detect Ub-like or autophagy-related modifiers and, in combination with mass spectrometry, to identify new Uub targets. These Ub (-like) sensors can be designed, constructed and tested in ~2–3 weeks.