The many enzymes comprising the ubiquitin-proteasome pathway are necessary partners in the intricacy of the ubiquitin code. The combined activities of E1, E2, and E3 enzymes lead to the conjugation of ubiquitin to protein substrates, while deubiquitylases cleave polyubiquitin chains and remove ubiquitin from substrates. Thus, these enzymes participate in the “writing” and “erasing” ubiquitin modifications onto substrate proteins, dictating the biological fates of ubiquitylated substrates. While cellular mixtures contain many conjugating enzymes, ligases, and DUBs, it is possible to reconstitute ubiquitylation and deubiquitylation reactions in a purified in vitro mixture. Of course, enzymes with high purity and activity are essential for producing a robust ubiquitylation reaction.
The diversity of topology and length in ubiquitin chains enables ubiquitylation to function as a complex signaling code that regulates many essential cellular processes. The interactions between E1 and E2 enzymes, and the subsequent pairing of E2 and E3 enzymes, play an integral role in substrate selection and chain type. LifeSensors provides a wide selection of ELISA-based assays to study the activity and selectivity of these enzymes. These assays allow researchers to determine the compatibility of various pairs of enzymes in vitro, enabling hypothesis-driven research into the resulting biological roles of ubiquitin pathway enzymes.