Cereblon influences the timing of muscle differentiation in Ciona tadpoles.

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.

Profiling CELMoD-Mediated Degradation of Cereblon Neosubstrates.

Targeted protein degradation is garnering increased attention as a therapeutic modality due in part to its promise of modulating targets previously considered undruggable. Cereblon E3 Ligase Modulating Drugs (CELMoDs) are one of the most well-characterized therapeutics employing this modality. CELMoDs hijack Cereblon E3 ligase activity causing neosubstrates to be ubiquitinated and degraded in the proteasome. Here, we describe a suite of assays-cellular substrate degradation, confirmation of CELMoD mechanism of action, in vitro ubiquitination, and Cereblon binding-that can be used to characterize CELMoD-mediated degradation of Cereblon neosubstrates. While the assays presented herein can be run independently, when combined they provide a strong platform to support the discovery and optimization of CELMoDs and fuel validation of targets degraded by this drug modality.

Cereblon Modulators Target ZBTB16 and Its Oncogenic Fusion Partners for Degradation via Distinct Structural Degrons.

There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate. We also report two new Cereblon modulators, CC-3060 and CC-647, that promote ZBTB16 degradation. Unexpectedly, CC-3060 and CC-647 target ZBTB16 for degradation by primarily engaging distinct structural degrons on different zinc finger domains. The reciprocal fusion proteins, ZBTB16-RARα and RARα-ZBTB16, which cause a rare acute promyelocytic leukemia, contain these same structural degrons and can be targeted for proteasomal degradation with Cereblon modulator treatment. Thus, a targeted protein degradation approach via Cereblon modulators may represent a novel therapeutic strategy in acute promyelocytic leukemia where ZBTB16/RARA rearrangements are critical disease drivers.

Quantitative Lys-ϵ-Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin-mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 ligase HUWE1.

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.

Hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5) communicates cellular iron and oxygen availability by distinct mechanisms.

Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.

Protein degradation and iron homeostasis.

Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals.

Structural and molecular characterization of iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).

Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.

An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.

Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.