Investigation of the C-terminal cyclic imide degron targeted by cereblon

Abstract

The E3 ubiquitin ligase substrate adapter cereblon (CRBN) has garnered widespread interest from the research lab to the clinic. Since its first association with neurological development and subsequent discovery as the primary target of thalidomide, CRBN has emerged as a promising E3 ligase for targeted protein degradation, an emerging drug modality that promotes degradation of challenging therapeutic targets. However, while there is a strong understanding of small molecule-induced substrates and activities of CRBN, the endogenous substrates and physiological functions of CRBN have not been fully elucidated. Here, I present the identification of the C-terminal cyclic imide modification as an endogenous mark on substrates, termed a degron, that are recognized and removed by CRBN, followed by the systematic characterization of two separate mechanisms of C-terminal cyclic imide formation in biological systems. In Chapter 1, I introduce the ubiquitin-proteasome system and outline the families and substrate recognition mechanisms of E3 ubiquitin ligases. I then detail the chemically induced activity of CRBN, focusing on the history of CRBN-engaging agents and their more recent application in targeted protein degradation. Finally, I review the ubiquitin-dependent and - independent interactors of CRBN and outline the association of CRBN with multiple biological pathways and diseases. In Chapter 2, I describe the efforts to identify C-terminal cyclic imides that occur by cyclization of asparagine and glutamine (denoted as cN and cQ, respectively) as a degron of CRBN. I recount the chemical biology and proteomic approaches used to uncover and validate the functional engagement of cN and cQ moieties by CRBN and the prevalence of these modifications in the proteome. I then briefly discuss the use of the C-terminal cyclic imide degron as versatile and selective ligands for targeted protein degradation. In Chapter 3, I investigate the C-terminal cyclic imides that stem from spontaneous intramolecular cleavage that constitutes a previously overlooked form of protein damage. The intrinsic and extrinsic factors that promote asparagine cleavage events to afford C-terminal cyclic imides in comparison to deamidation, a related form of protein damage, are discussed. Using glutathione synthetase (GSS) as a representative substrate, I demonstrate the regulation of protein damage that progresses through the C-terminal cyclic imide in the presence of CRBN and the association of unmitigated cleavage events with protein aggregation in the absence of CRBN. In Chapter 4, I report the discovery of an enzymatic mechanism of C-terminal cyclic imide formation. I delineate the efforts to characterize the enzyme-mediated formation of the aspartimide on multiple peptides and proteins ending with C-terminal asparagine and the recognition of these substrates by CRBN. Finally, I present glutamine synthetase (GLUL) as a substrate of CRBN that is recognized via the enzymatically generated aspartimide degron. I conclude with a discussion of the implications of this emerging pathway and future perspectives on the biology of CRBN and C-terminal cyclic imide modifications. Collectively, the degron formation mechanisms and endogenous substrates shed light on the role of CRBN in protein quality control, signaling, and metabolism.