A natural-product switch for a dynamic protein interface.

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces.

Proline primed helix length as a modulator of the nuclear receptor-coactivator interaction.

Nuclear receptor binding to coactivator proteins is an obligate first step in the regulation of gene transcription. Nuclear receptors preferentially bind to an LXXLL peptide motif which is highly conserved throughout the 300 or so natural coactivator proteins. This knowledge has shaped current understanding of this fundamental protein-protein interaction, and continues to inspire the search for new drug therapies. However, sequence specificity beyond the LXXLL motif and the molecular functioning of flanking residues still requires urgent addressing. Here, ribosome display has been used to reassess the estrogen receptor for new and enlarged peptide recognition motifs, leading to the discovery of a potent and highly evolved PXLXXLLXXP binding consensus. Molecular modeling and X-ray crystallography studies have provided the molecular insights on the role of the flanking prolines in priming the length of the α-helix and enabling optimal interactions of the α-helix dipole and its surrounding amino acids with the surface charge clamp and the receptor activation function 2. These findings represent new structural parameters for modulating the nuclear receptor-coactivator interaction based on linear sequences of proteinogenic amino acids and for the design of chemically modified inhibitors.

Author Correction: Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons.

In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.

CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations.

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population.

Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons.

In Huntington's disease (HD), whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model, an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins, each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover, healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether, our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD.

Design and evaluation of fragment-like estrogen receptor tetrahydroisoquinoline ligands from a scaffold-detection approach.

A library of small tetrahydroisoquinoline ligands, previously identified via structure- and chemistry-based hierarchical organization of library scaffolds in tree-like arrangements, has been generated as novel estrogen receptor agonistic fragments via traditional medicinal chemistry exploration. The approach described has allowed for the rapid evaluation of a structure-activity relationship of the ligands concerning estrogen receptor affinity and estrogen receptor ß subtype selectivity. The structural biological insights obtained from the fragments aid the understanding of larger analogues and constitute attractive starting points for further optimization.