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The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinβ, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinβ interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinβ represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinβ does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinβ. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.

International audience; Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.

International audience; Patients with the rare neurodevelopmental repair syndrome known as group A trichothiodystrophy (TTD-A) carry mutations in the gene encoding the p8 subunit of the transcription and DNA repair factor TFIIH. Here we describe the crystal structure of a minimal complex between Tfb5, the yeast ortholog of p8, and the C-terminal domain of Tfb2, the yeast p52 subunit of TFIIH. The structure revealed that these two polypeptides adopt the same fold, forming a compact pseudosymmetric heterodimer via a beta-strand addition and coiled coils interactions between terminal alpha-helices. Furthermore, Tfb5 protects a hydrophobic surface in Tfb2 from solvent, providing a rationale for the influence of p8 in the stabilization of p52 and explaining why mutations that weaken p8-p52 interactions lead to a reduced intracellular TFIIH concentration and a defect in nucleotide-excision repair, a common feature of TTD cells.

Trichothiodystrophy (TTD) is a rare hereditary multi-system disorder associated with defects in nucleotide excision repair (NER) and transcription as consequences of mutations in XPB, XPD and p8/TTD-A subunits of transcription factor IIH (TFIIH). Here, we report the solution structure of the p8/TTD-A protein, a small alpha/beta protein built around an antiparallel beta-sheet that forms a homodimer with an extended interface. In order to characterize the dimer interface, we have introduced a mutation at position 44, which destabilizes the dimeric form of the protein. We have shown that this mutation has no effect on the intrinsic ability of p8/TTD-A to stimulate NER in vitro, but affects the capacity of p8/TTD-A to restore TFIIH concentration in TTD-A fibroblasts. Point mutations found in TTD-A patients are discussed on the basis of the present structure.