Substrate recognition and specificity modulation of Cullin4-RING E3 ubiquitin ligase substrate receptors

• Applicant: Dr. David Schwefel
• Subject Area: Biochemistry
• Term: from 2015 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 280022178

Cullin4-RING E3 ubiquitin ligases evolved a modular architecture for the targeted modification of cellular proteins with ubiquitin, in order to mark substrates for proteasomal degradation or to induce other regulatory processes, with implications for DNA repair, DNA replication and cell cycle control. Substrate specificity is achieved by exchangeable substrate receptors (DCAFs); these are linked to the catalytic Cullin4-RING core via an adaptor protein (DDB1). Knowledge of the molecular determinants for receptor binding to the catalytic core and for the recognition of specific substrate sequences by the receptor are critical to understand the mechanism of targeted ubiquitin ligation and for the exploration of the cellular consequences. In the first part of the proposed project, insights into the endogenous function of the DCAF1 substrate receptor will be gained. DCAF1 is essential for the regulation of DNA replication and methyltransferase-dependent gene transcription. A combination of in vitro and cell-based methods is proposed to identify individual functional domains of DCAF1 and to understand their DDB1 and substrate interactions. This approach will clarify how DCAF1 is integrated in the E3 ligase complex, how recognition of methylated and other substrate sequences induces substrate degradation, and what the cellular consequences are.In virus-infected cells, the Cullin4-RING system is also modified by viral factors. The so-called viral accessory proteins bind to DCAF1 and modify its molecular surface to redirect the specificity of the system towards cellular antiviral factors. In this way, better conditions for viral replications are achieved. In the second part of the proposed project, the interaction of DCAF1 with the accessory protein Vpr from immunodeficiency viruses will be studied, in order to understand the molecular mechanism of Vpr-induced DCAF1 specificity modulation and the resulting degradation of antiviral factors. This approach will not only provide mechanistic insights into viral modification of the Cullin4-RING system, but also contribute to further understanding of retroviral replication.Apart from DCAF1, DCAF2 and DDB2, the substrate spectrum of other DCAF receptors is presently under-determined. Consequently, in the third part of the proposed project, an expression library of 13 further human DCAF proteins will be cloned and their DDB1 interactions will be verified. Pull-down experiments in vitro and in vivo together with mass spectrometry methods will be conducted to identify specific DCAF substrate candidates. These candidates will be verified in further biochemical, structural and functional assays to gain new insights into Cullin4 receptor specificity and hence into the biology of the Cullin4-RING system.

• DFG Programme: Independent Junior Research Groups