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  • 98 synthesis br Results and Discussion br Conclusions Unders

    2020-04-02


    Results and Discussion
    Conclusions Understanding the connections between function and fitness is a primary goal of many biological disciplines including systems biology and molecular evolution. While sound approaches have been developed to understand the connections between function and fitness for proteins that perform a single function [19], [24], [31], [32], [33], investigating potential interdependencies in multi-functional proteins had posed daunting technical challenges. This study demonstrates that systematic analyses of the effects of mutations on biochemical function and growth rates provide a powerful approach to investigate how edge-rich protein interaction networks contribute to overall biological function.
    Materials and Methods Libraries of ubiquitin point mutants were displayed on the surface of yeast as C-terminal fusions with Aga2-HA similar to previous descriptions [73], [74]. Pools of yeast-displayed mutants were reacted with E1, labeled with fluorescent 98 synthesis directed to either E1 or the HA tag. FACS was used to isolate E1-reactive cells (E1 and HA positive) and/or HA-displaying (HA positive) cells. Deep sequencing [38] was used to determine the enrichment or depletion of each mutation in E1-reactive cells compared to HA-displaying cells. The relative E1 reactivity of a panel of individual ubiquitin variants was independently determined relative to wild-type ubiquitin using purified proteins. The accumulation pattern of His6-ubiquitin variants in yeast harboring untagged wild-type ubiquitin was monitored by inducing expression of the epitope-tagged variant followed by Western blotting.
    Acknowledgements This work was aided by discussions with R. Hietpas, P. Mishra, L. Jiang, C. R. Matthews, R. Gilmore, and P. Pryciak. We are grateful for assistance from the University of Massachusetts Medical School Core Flow Cytometry Laboratory and R. Konz with cell sorting experiments. This work was supported by the National Institutes of Health (grant number R01-GM083038 to D.N.B.).
    Main Text Post-translational modifications by ubiquitin (Ub) and ubiquitin-like proteins (Ubl) regulate a diverse array of cellular and physiological processes including protein degradation, localization, and activation. While ubiquitination has primary responsibility for targeting substrates for proteasomal degradation, Ubls like SUMO, Nedd8, and ISG15 often regulate substrate activity by serving as docking sites for protein-protein interactions. Ub/Ubls are covalently linked to their target proteins through a three-enzyme cascade. An E1 activating enzyme forms a thioester conjugate with Ub/Ubl through ATP hydrolysis, which is then transferred to an E2 conjugating enzyme, and finally an E3 ligase transfers Ub/Ubl to its target protein. Different Ub/Ubl proteins have their own cognate E1, E2, and E3 enzyme machinery for protein conjugation (Kerscher et al., 2006). Over the last two decades, Ub- and Ubl-dependent pathways have emerged as attractive targets for inhibitor development. Small molecules targeting E1-activating enzymes and E3 ligases have been developed for specific systems of Ubls. These compounds have shown promise in regulating “undruggable” targets such as transcription factors in therapeutic contexts (Wertz and Wang, 2019). Notably, inhibitors of the E1s for Ub (TAK-243) (Hyer et al., 2018), Nedd8 (MLN4924) (Brownell et al., 2010), and SUMO (ML-792) (He et al., 2017) have been developed and shown promise in the clinic (Shah et al., 2016). These inhibitors are mechanistically similar and form covalent adducts with Ub/Ubl, catalyzed by their respective E1s. This adduct mimics the Ub/Ubl∼adenylate intermediate of the E1 activation cycle, which then binds to E1 and inhibits further Ubl-dependent activation. In this issue of Cell Chemical Biology, Chen and colleagues (Li et al., 2019) expand the scope of E1 inhibition by developing an allosteric inhibitor for small ubiquitin-like modifiers (SUMO) activating enzyme E1. This novel compound covalently binds in a buried site of the enzyme away from the active site, in contrast to previously characterized E1 inhibitors (Li et al., 2019; Figure 1).