Characterization of endogenous cereblon substrates with new chemical biology methods

Abstract

C-Terminal cyclic imides are posttranslational modifications on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. I therefore developed an antibody-like reagent, termed “cerebody,” for visualizing and enriching CRBN substrates. I describe my protein engineering campaign to identify cerebody and showcase cerebody’s utility in identifying CRBN substrates by Western blot and enrichment from whole cell and tissue lysates. Furthermore, CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation. In Chapter 1, I introduce the E3 ubiquitin ligase substrate adaptor CRBN and provide an overview of its clinical use, structure, substrates it recognizes, and recognition motifs. I then discuss methods to recognize CRBN substrates through the C-terminal cyclic imide modification, and criteria for improved methods for detection of CRBN substrates through this modification. In Chapter 2, I discuss the directed evolution of CRBN’s thalidomide binding domain through random mutagenesis phage display in pursuit of a tool protein for detection of CRBN substrates. In Chapter 3, I move from the thalidomide binding domain scaffold to CRBNmidi and describe the development of the tool protein called cerebody. I then detail its use for detection and enrichment of endogenous CRBN substrates. In Chapter 4, I describe the validation of UROD as a PCMT1-dependent CRBN substrate, a discovery which was enabled and accelerated by cerebody enrichment. In Chapter 5, I discuss the utility of cerebody beyond endogenous substrates, including its use for identifying CRBN neosubstrates and its potential alterations to also provide structural insight into the interface between CRBN and its substrates and neosubstrates. I also provide detailed protocols for the implementation of cerebody methods. I conclude by discussing the broad applicability for cerebody and derived methods to illuminate CRBN’s biology, and what happens when these pathways are altered.