The D816V activating mutation of the c-Kit kinase domain often causes human mastocytosis. Although inhibitors of wild-type c-Kit are known (e.g. STI-571), they are at least 10 times less active against the c-Kit mutant. Several derivatives of ellipticine (5,11-dimethyl-6H-pyrido[3,4-b]carbazole), substituted at positions 1, 2, 9, and 11, were found to inhibit purified D816V and wild-type c-Kit kinase domains with comparable potencies by competing with ATP binding. We investigated the difference between these inhibitors by modeling the D816V mutation in crystal structures of inactive and active c-Kit. Molecular dynamics simulations strongly suggested that the D816V point mutation shifts the conformational equilibrium of c-Kit kinase domain toward the active conformation. All ellipticine compounds were subsequently docked to the D816V mutant c-Kit model. The model provides possible explanations for the structure-activity relationships observed among ellipticine compounds, resulting in new insights into D816V c-Kit mutant inhibition.
Mass spectrometry analysis of the post-transcriptional modifications of histones H3 and H4 that were co-purified with histone methyltransferases Suv39h1 and G9a shows that, in HeLa cells, histone methyltransferases can be physically associated with acetylated histones, which normally mark transcriptionally active chromatin.
AbstractSpecific combinations of post-translational modifications of histones alter chromatin structure, facilitating gene transcription or silencing. Here we have investigated the 'histone code' associated with the histone methyltransferases Suv39h1 and G9a by combining double immunopurification and mass spectrometry. Our results confirm the previously reported histone modifications associated with Suv39h1 and G9a. Moreover, this method allowed us to demonstrate for the first time an association of acetylated histones with the repressor proteins Suv39h1 and G9a.
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