Valérie Vivat

A canonical structure for the ligand-binding domain of nuclear receptors

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXRα and the liganded RARγ LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specif ic signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXRα and glucocorticoid receptor ligand-binding sites according to the RARγ holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RARγ holo- and inactive RXRα apo-LBD structures.

The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways.

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR‐interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2–NR interaction and stimulation, in transfected cells, of the ligand‐induced activation function‐2 (AF‐2) present in the ligand‐binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo‐LBDs and stimulation of AF‐2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo‐LBDs and AF‐2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand‐induced AF‐2 activity of several NRs, indicating that NR AF‐2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo‐LBDs.

Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

Using a yeast two‐hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand‐ and AF‐2 activating domain (AF‐2 AD)‐dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha‐helix core of the AF‐2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF‐2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF‐2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF‐2 of nuclear receptors through different mechanisms.

Dimerization with retinoid X receptors and phosphorylation modulate the retinoic acid-induced degradation of retinoic acid receptors alpha and gamma through the ubiquitin-proteasome pathway.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor γ2 (RARγ2), RARα1, and retinoid X receptor α1 (RXRα1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RARγ2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXRα1. In contrast, RARα1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXRα1 degradation is also modulated by heterodimerization. Thus, each partner of RARγ/RXRα and RARα/RXRα heterodimers modulates the degradation of the other. We conclude that the ligand-dependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.

Discovery of Specific Inhibitors of Human USP7/HAUSP Deubiquitinating Enzyme

The human USP7 deubiquitinating enzyme was shown to regulate many proteins involved in the cell cycle, as well as tumor suppressors and oncogenes. Thus, USP7 offers a promising, strategic target for cancer therapy. Using biochemical assays and activity-based protein profiling in living systems, we identified small-molecule antagonists of USP7 and demonstrated USP7 inhibitor occupancy and selectivity in cancer cell lines. These compounds bind USP7 in the active site through a covalent mechanism. In cancer cells, these active-site-targeting inhibitors were shown to regulate the level of several USP7 substrates and thus recapitulated the USP7 knockdown phenotype that leads to G1 arrest in colon cancer cells. The data presented in this report provide proof of principle that USP7 inhibitors may be a valuable therapeutic for cancer. In addition, the discovery of such molecules offers interesting tools for studying deubiquitination.

A mutation mimicking ligand-induced conformational change yields a constitutive RXR that senses allosteric effects in heterodimers.

Mutations of a single residue in the retinoid X receptor α (RXRα) ligand‐binding pocket (LBP) generate constitutive, ligand‐binding‐competent mutants with structural and functional characteristics similar to those of agonist‐bound wild‐type RXR. Modelling of the mouse RXRαF318A LBP suggests that, like agonist binding, the mutation disrupts a cluster of van der Waals interactions that maintains helix H11 in the apo‐receptor location, thereby shifting the thermodynamic equilibrium to the holo form. Heterodimerization with some apo‐receptors (retinoic acid, thyroid hormone and vitamin D3 receptors) results in ‘silencing’ of RXRαF318A constitutive activity, which, on the other hand, efficiently contributes to synergistic transactivation within NGFI‐B‐RXR heterodimers. RAR mutants disabled for corepressor binding and/or lacking a functional AF‐2 activation domain, do not relieve RXR ‘silencing’. Not only RAR agonists, but also the RAR antagonist BMS614 induce conformational changes allowing RXR to exert constitutive (RXRαF318A) or agonist‐induced (wild‐type RXR) activity in heterodimers. Interestingly, the RXRαF318A constitutive activity generated within heterodimers in the presence of BMS614 requires the integrity of both RXR and RAR AF‐2 domains. These observations suggest that, within RXR‐RAR heterodimers, RAR can adopt a structure distinct from that of the active holo‐RAR, thus allowing RXR to become transcriptionally responsive to agonists.

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism.

Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.

STRUCTURAL BASIS FOR ENGINEERING OF RETINOIC ACID RECEPTOR ISOTYPE-SELECTIVE AGONISTS AND ANTAGONISTS

BACKGROUND Many synthetic retinoids have been generated that exhibit a distinct pattern of agonist/antagonist activities with the three retinoic acid receptors (RARalpha, RARbeta and RARgamma). Because these retinoids are selective tools with which to dissect the pleiotropic functions of the natural pan-agonist, retinoic acid, and might constitute new therapeutic drugs, we have determined the structural basis of their receptor specificity and compared their activities in animal and yeast cells. RESULTS There are only three divergent amino acid residues in the ligand binding pockets (LBPs) of RARalpha, RARbeta and RARgamma. We demonstrate here that the ability of monospecific (class I) retinoid agonists and antagonists to bind to and induce or inhibit transactivation by a given isotype is directly linked to the nature of these residues. The agonist/antagonist potential of class II retinoids, which bind to all three RARs but depending on the RAR isotype have the potential to act as agonists or antagonists, was also largely determined by the three divergent LBP residues. These mutational studies were complemented by modelling, on the basis of the three-dimensional structures of the RAR ligand-binding domains, and a comparison of the retinoid agonist/antagonist activities in animal and yeast cells. CONCLUSIONS Our results reveal the rational basis of RAR isotype selectivity, explain the existence of class I and II retinoids, and provide a structural concept of ligand-mediated antagonism. Interestingly, the agonist/antagonist characteristics of retinoids are not conserved in yeast cells, suggesting that yeast co-regulators interact with RARs in a different way than the animal cell homologues do.

Biophysical and structural characterization of mono/di-arylated lactosamine derivatives interaction with human galectin-3

Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases.

An Unexpected Mode Of Binding Defines BMS948 as A Full Retinoic Acid Receptor β (RARβ, NR1B2) Selective Agonist

Retinoic acid is an important regulator of cell differentiation which plays major roles in embryonic development and tissue remodeling. The biological action of retinoic acid is mediated by three nuclear receptors denoted RARα, β and γ. Multiple studies support that RARβ possesses functional characteristics of a tumor suppressor and indeed, its expression is frequently lost in neoplastic tissues. However, it has been recently reported that RARβ could also play a role in mammary gland tumorigenesis, thus demonstrating the important but yet incompletely understood function of this receptor in cancer development. As a consequence, there is a great need for RARβ-selective agonists and antagonists as tools to facilitate the pharmacological analysis of this protein in vitro and in vivo as well as for potential therapeutic interventions. Here we provide experimental evidences that the novel synthetic retinoid BMS948 is an RARβ-selective ligand exhibiting a full transcriptional agonistic activity and activating RARβ as efficiently as the reference agonist TTNPB. In addition, we solved the crystal structures of the RARβ ligand-binding domain in complex with BMS948 and two related compounds, BMS641 and BMS411. These structures provided a rationale to explain how a single retinoid can be at the same time an RARα antagonist and an RARβ full agonist, and revealed the structural basis of partial agonism. Finally, in addition to revealing that a flip by 180° of the amide linker, that usually confers RARα selectivity, accounts for the RARβ selectivity of BMS948, the structural analysis uncovers guidelines for the rational design of RARβ-selective antagonists.