Covalent conjugation to ubiquitin (Ub) is one of the major post-translational modifications that regulates protein stability, function, and localization. Ubiquitination is the concerted action of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3). The specificity and efficiency of ubiquitination are largely determined by the E3 enzyme, which directs the last step of the Ub-conjugating cascade by binding to both an E2～Ub conjugate and a substrate protein. This step ensures the transfer of Ub from E2～Ub to the substrate, leading to its mono- or poly-ubiquitination.

The SMAD ubiquitination regulatory factor 1 (SMURF1) is a HECT-type E3 Ub ligase that regulates TGF-β/BMP pathways via ubiquitination of key signal transducers (SMAD1, SMAD2, or SMAD5), or TGF-β receptor I. SMURFs play a critical role in cell-type specification, tissue and organ development by regulating planar cell polarity signaling and convergent extension. SMURFs can also accelerate tumor progression, invasion, and metastasis as they regulate ubiquitination and subsequent proteasomal degradation of tumor-suppressing proteins including p53 as well as various cell signaling proteins. That is why SMURF1 and especially its Ub ligase activity is an attractive potential drug target in cancer immunotherapy. Like most E3 ligases, SMURF1 ubiquitinates itself.

The SMURF1 intrachain TR-FRET Assay Kit is a sensitive high-throughput screening (HTS) TR-FRET Assay Kit, designed to measure SMURF1 auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes a Europium cryptate-labeled Ub (donor) as well as Cy5-labeled Ub (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on SMURF1, this FRET-based assay requires no time-consuming washing steps, making it especially suitable for HTS applications as well as real-time kinetics analyses of polyubiquitination.

Figure 1. SMURF1 intrachain TR-FRET Assay Kit schematic