The CULT domain of cereblon: a pharmacological target and teratogenicity gateway

Half a century ago thalidomide caused one of the biggest pharmaceutical tragedies known by now. It was widely prescribed to pregnant women as a sedative, but displayed teratogenic properties, causing limb malformations and other developmental defects in more than 10,000 babies worldwide. Nevertheless, thalidomide and its derivatives are used nowadays in treatment of leprosy and multiple myeloma. The protein cereblon was identified as a primary target of thalidomide in the cell. As a substrate receptor, cereblon (CRBN) is linked via the damaged DNA binding protein 1 (DDB1) and cullin 4A to the E3 ubiquitin ligase machinery. The drug binds to the C-terminal region of cereblon, also referred to as CULT domain (cereblon domain of unknown activity, binding cellular ligands and thalidomide). This domain represents the most conserved part of cereblon and is also found solely in single-domain proteins in bacteria and, as a secreted form, in eukaryotes. Based on its ligand binding properties, its high degree of conservation and its intracellular as well as extracellular localization, a common interest arose in understanding the functional role of the CULT domain in vivo. The CULT domain carries a number of highly conserved cysteine and tryptophan residues within its amino acid sequence. In the solved crystal structure of the bacterial CULT domain, four conserved cysteines stabilize the protein fold by coordinating a zinc ion. Three invariant tryptophan residues build an aromatic cage, to which the ligand binds. Considering the structural similarity of uridine and thalidomide, we tested uridine binding to the hydrophobic pocket and could show an identical mode of binding. So far, uridine represents the only natural ligand, for which an interaction with the CULT domain has been shown. Further studies demonstrated that parts of the CULT domain fold upon ligand binding, thus stabilizing the protein. The pocket is highly similar to the aromatic cages found in histone readers that recognize methylated lysine or arginine residues in chromatin. This structural similarity suggests analogous ligands for cereblon, which include a distinct type of post-translational modifications. We developed a FRET-based in vitro assay for testing and characterizing ligand binding to cereblon. The determination and comparison of the substrate affinities for three CULT domains, from Homo sapiens, Magnetospirillum gryphiswaldense, and the secreted Caenorhabditis elegans, revealed similar values for the same ligands on a relative scale. This study convincingly confirmed the bacterial protein as a robust model system for both: (i) testing the specificity of the CULT domain to its ligands; and (ii) deciphering the function of cereblon proteins. Using the FRET-assay, we showed that various therapeutically relevant pharmaceuticals display affinities to the CULT domain with binding constants in the micromolar range. These off-target effects were further validated by applying an in vivo assay in zebrafish embryos to test the teratogenic potential of these compounds mediated via their interaction with cereblon. Searching for proteins interacting with cereblon in vivo, we identified transcription termination factor Rho to bind to the bacterial CULT domain. The assumption of a possible role of cereblon in transcriptional regulation is further supported by the fact that hCRBN interacts with LSD1, a demethylase which is essential for transcriptional regulation in multicellular organisms. Taken together, our data imply a potential function of cereblon in transcriptional repression and/or activation, particularly during limb formation

Chemical and biophysical methods to explore dynamic mechanisms of chromatin silencing

Chromatin, the nucleoprotein complex organizing the genome, is central in regulating gene expression and genome organization. Chromatin conformational dynamics, controlled by histone post-translational modifications (PTM) and effector proteins, play a key role in this regulatory function. Recent developments in chemical biology, cell biology, and biophysics sparked important new studies, which probe direct causal connections between histone PTMs, chromatin effector proteins that write or read these modifications, and the involved functional chromatin states. In particular, the mechanisms of heterochromatin silencing have been explored in great detail in recent years. These studies revealed the highly dynamic nature of this chromatin state, its conformational heterogeneity, and different mechanisms of its formation. Here, we review how chemical biology and biophysics shaped our current understanding of the dynamic processes observed in heterochromatin and discuss the emerging technologies to detect chromatin organization directly in the cellular environment

Bacillus thuringiensis DB27 Produces Two Novel Protoxins, Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Bacillus thuringiensis has been widely used as a biopesticide, primarily for the control of insect pests, but some B. thuringiensis strains specifically target nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using Caenorhabditis elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in Escherichia coli and fed to C. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins on C. elegans are synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC(50)s) for Cry21Fa1 and Cry21Ha1 to be 13.6 μg/ml and 23.9 μg/ml, respectively, which are comparable to the LC(50) of nematicidal Cry5B. Finally, we found that signaling pathways which protect C. elegans against Cry5B toxin are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins

A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage

The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play a key role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair

A FRET-Based Assay for the Identification and Characterization of Cereblon Ligands

Cereblon serves as an ubiquitin ligase substrate receptor that can be tuned toward different target proteins by various cereblon-binding agents. This offers one of the most promising avenues for targeted protein degradation in cancer therapy, but cereblon binding can also mediate teratogenic effects. We present an effective assay that is suited for high-throughput screening of compound libraries for off-target cereblon interactions but also can guide lead optimization and rational design of novel cereblon effector molecules

Structural dynamics of the cereblon ligand binding domain.

Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution

Thalidomide mimics uridine binding to an aromatic cage in cereblon

AbstractThalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity

Chemical Ligand Space of Cereblon

The protein cereblon serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degradation is exploited in different clinical settings and has sparked the development of a growing number of thalidomide derivatives. Here, we probe the chemical space of cereblon binding beyond such derivatives and work out a simple set of chemical requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures for a diverse set of compounds, including commonly used pharmaceuticals, but also find that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. Our results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors and provide a framework for the circumvention of unintended cereblon binding by negative design for future pharmaceuticals