Despite intense efforts over decades, many proteins implicated in human diseases have eluded pharmacological intervention. A novel type of drug targeting methodology has recently emerged that takes advantage of the ubiquitin transferase systems. Cullin-RING E3 ubiquitin ligases (CRLs) are a large family of regulatory protein complexes, responsible for ~20% of intracellular protein degradation in humans. CRLs are divided into six families based on their Cullin constituent (either CUL1, CUL2, CUL3, CUL4A/B or CUL5). Each Cullin binds a RING-domain containing protein (RBX1 or RBX2) and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 240 distinct CRLs. The structures of CRLs and their substrates vary enormously. All ~240 CRL family members are controlled by the COP9 signalosome complex (CSN). CSN removes NEDD8, an activator protein covalently attached to the Cullin C-terminal domain, and thereby inactivates the ligase. Prior to this work it had been unclear what enables a single complex, CSN, to be a master regulator of all CRLs. We studied how ubiquitin ligases can deal with activating signals ranging from DNA damage to modified proteins and how small-molecule degrader drugs exert their therapeutic benefit via the ubiquitin system.Overall objectives: The aim of the ERC grant is to understand the architecture, working and regulation of Cullin-RING ligases by the Cop9 signalosome. Given recent developments in the field of small molecule degraders, we extended our focus to questions of how small molecule drugs can reprogram CRLs and how these drugs interplay with CSN and other CRL regulators.Key learnings enabled by the ERC project: We were able to solve the first sub-nanometer resolution structure of CSN bound to a CRL substrate. By combining single particle electron microscopy with functional studies, we demonstrated that bulky substrates, irrespective of their chemical nature and protein sequence, are able to sterically displace CSN from their cognate CRL E3 ligase, providing a first universal mechanism for the regulation of diverse CRLs by diverse substrates (Cavadini et al., Nature 2016). The CRL ubiquitin Ligase System and its CSN master regulator, thus respond not to a specific protein epitope, but instead to the bulk of the protein. We found that this mechanism is similarly exploited by small molecules recruiting neo-substrates, for which we determined structures and provided the mode of action (Petzold et al., Nature 2016; Sievers et al., Science 2018; Słabicki et al., Nature 2020). If the proteins recruited by these small molecules have sufficient bulk and size, they are being recognized as true biological substrates, and as a consequence, the ligase remains intact and operational for the degradation of these neo-substrate targets. These principles of CRL action will guide future drug development efforts for novel therapeutics.Importance of our finding for human health: Protein degradation through the ubiquitin proteasome system is involved in virtually all biological processes. Accordingly, mutation of the ubiquitin ligases and their regulators are frequently found in cancer cells, as well as in other diseases ranging from neurodegeneration to developmental defects. Understanding these diseases and coming up with novel therapeutic solutions requires atomic-level understanding of the proteins involved.