The Ubiquitin Proteasome System (UPS) is involved in virtually any cellular process and frequently implicated in human pathologies. Ubiquitin, through the action of a three-enzyme cascade (E1, E2 and E3), becomes attached to substrate proteins. The posttranslational modification with ubiquitin can serve a multitude of functions depending on the type and length of the ubiquitin chain attached to the substrate, including the control of protein expression. The human genome encodes for more than 600 E3 ligases, which confer specificity in the ubiquitin signaling cascade. While the process of ubiquitin transfer is well understood, the biological function and molecular mechanisms of the majority of ubiquitin ligases remain obscure.

We combine structural biology, cell biology and biochemical reconstitutions to address the molecular workings of these multi-component ubiquitin ligases. In particular, we are interested in protein complexes and pathways that contribute to the control of gene expression and are frequently associated with human disease and cancer. Intimate understanding of the structure allows us to dissect the complex mechanisms that underlie function and regulation of such molecules and to probe their biology in a cellular context. We seek to leverage our molecular understanding to propose and test new avenues of therapeutic intervention.

Work on the efficacy target of the widely used anti-cancer drugs thalidomide, lenalidomide, pomalidomide, the CRL4CRBN ubiquitin ligase, illustrates the general approach. We successfully determined the structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure established CRBN as the CRL4CRBN substrate receptor, which enantioselectively binds thalidomide and its analogues, lenalidomide and pomalidomide. Utilizing mass spectrometry and protein microarrays, we performed unbiased proteome-wide screens to identify CRL4CRBN ligase substrates. Using biochemistry, chemical biology and cell biology methods, we confirmed that thalidomide prevents MEIS2 binding to CRBN thus inhibiting MEIS2 ubiquitination and turnover. At the same time, we could provide a structural rationale for the action of thalidomide analogues in promoting CRL4CRBN mediated degradation of Ikaros/Aiolos transcription factors as well as Ck1. Our findings thus, provide a molecular mechanism for thalidomide action that involves inhibition of CRL4CRBN-mediated ubiquitination of endogenous substrates, while simultaneously acting as agonist on neo-substrates such as Ikaros transcription factors.