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  • br Significance Our understanding of the

    2020-09-21


    Significance Our understanding of the ubiquitin biology has been rapidly expanding. The role of the ubiquitin system in the pathogenesis of numerous disease states has increased the interest in finding new strategies to pharmacologically interfere with the enzymes responsible of the ubiquitination process. However, the development of molecules targeting the ubiquitin cascade, especially the E2 conjugating pepstatin australia enzymes and E3 ligases, has not being extensively sustained by the availability of robust and affordable technologies for extensive primary screening of pepstatin australia libraries. Performing high-throughput screening in the ubiquitin field remains challenging and it usually requires engineered proteins or the synthesis of chemical probes. Here we show that the MALDI-TOF E2-E3 assay is a robust, scalable, label-free assay that can be employed for primary screening of compound libraries against E2 conjugating enzymes and E3 ligases belonging to different families and representative of all the currently known ubiquitylation mechanisms. The MALDI-TOF E2/E3 assay is a readily accessible addition to the drug discovery toolbox with the potential to accelerate drug discovery efforts in the ubiquitin pathway.
    STAR★Methods
    Acknowledgments We would like to thank the DNA cloning, Protein Production, DNA sequencing facility, and mass spectrometry teams of the MRC Protein Phosphorylation and Ubiquitylation Unit for their support. We would like to thank Prof. Dario Alessi, Prof. Ronald Hay, Prof. Philip Cohen, Prof. Katrin Rittinger, Dr. Satpal Virdee, Dr. Sarah Buhrlage, Dr. Natalia Shpiro, Dr. Siddharth Bakshi, Dr. Andrea Testa, and Dr. Francesca Morreale for tools and helpful discussions; Bruker Daltonics, particularly Meike Hamester, Rainer Paape, and Anja Resemann for their technical support. We thank Dr. Anthony Hope, Alex Cookson, and Lorna Campbell for providing the FDA-approved compound library and support with the liquid handling. This work was funded by Medical Research Council UK (MC_UU_12016/5), and the pharmaceutical companies supporting the Division of Signal Transduction Therapy (DSTT) (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA).
    Main Text Cullin-RING E3 ubiquitin ligases (CRLs) are major regulators of eukaryotic cell biology, controlling the half-lives, activities, assemblies, and localizations of thousands of proteins. This depends on hundreds of distinct CRLs in humans—and even more in plants and other organisms—assembling from structurally related modules, whereby a cullin-RING complex is a scaffold bridging a variable cullin-binding substrate receptor module with a RING-binding E2 or E3 enzyme that delivers ubiquitin to the receptor-bound substrate (Lydeard et al., 2013). The first discovered and prototypical CRLs comprise the SCF subfamily, in which the scaffold is CUL1-RBX1, and substrate receptor module consists of SKP1 and an associated F-box protein (FBP). The sheer number of FBPs (69 in humans), combined with a ∼4-fold higher concentration of SKP1:CUL1 in human cells, poses an interesting question: how does the limited pool of CUL1 select among a sea of FBPs while, at the same time, managing to not exclude others when needed for expedient substrate ubiquitylation? If affinity toward CUL1 were the sole determinant of SCF assembly, tightest binding SKP1-FBP pairs would dominate. Yet different signaling pathways, cell types, and developmental programs depend on altering the expression patterns of FBPs to cope with ever-changing needs to ubiquitylate different substrates (Reitsma et al., 2017). What then determines how a given SKP1-FBP module is assembled into active an SCF E3 ligase? In this issue, Liu et al. (2018) report the development of a mathematical model that provides new insights and ways to investigate the rules governing the repertoire of SCFs assembled as demanded for cellular regulation. A key premise of the new work is that SCF assembly is coordinated with continuous cycles of CUL1 conjugation to/deconjugation from the ubiquitin-like protein NEDD8 (“neddylation,” Figure 1, reviewed in Lydeard et al., 2013). In the absence of NEDD8, different SKP1-FBP modules continuously sample CUL1 due to CAND1 or CAND2 (referred to here collectively as “CAND”) expelling SKP1-FBPs from unmodified CUL1-RBX1 and vice-versa (Pierce et al., 2013, Wu et al., 2013, Zemla et al., 2013). CAND binding to CUL1 is structurally incompatible with neddylation (Goldenberg et al., 2004). Although SKP1-FBP-bound/CAND-free CUL1 is readily neddylated, NEDD8 is rapidly deconjugated from CUL1 by the COP9 signalosome (CSN). The deneddylated SCF can then undergo additional cycles of CAND-catalyzed SKP1-FBP exchange. Substrates are capable of putting an abrupt halt to this cycling by sterically blocking CSN-dependent deneddylation. By preventing deneddylation, an FBP-bound substrate indirectly preserves NEDD8 on its associated SCF, thereby preventing disassembly and maintaining ubiquitylation activity (Bornstein et al., 2006). While this attractive model explains some aspects of SCF assembly, it remains unclear to what extent substrate ubiquitylation relies on CAND-driven FBP exchange, or why cells developed such a seemingly complex regulatory system. The vast number of players competing for SCF assembly and disassembly, and their ever changing levels as cells respond to various cues (Reitsma et al., 2017), has presented a significant challenge for predicting system-wide changes upon perturbation.