Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results

Histone Deacetylase SIRT1 Targets Plk2 to Regulate Centriole Duplication

Summary: The protein deacetylase SIRT1 (Sirtuin 1) regulates many cellular processes, including cell-cycle progression, DNA damage response, and metabolism. Although the centrosome is a key regulator of cell-cycle progression and genome stability, little is known concerning SIRT1 controlled centrosome-associated events. Here we report that the centrosome protein Plk2 is acetylated and undergoes deacetylation by SIRT1. Acetylation protects Plk2 from ubiquitination, and SIRT1-mediated deacetylation promotes ubiquitin-dependent degradation of Plk2. SIRT1 controls centriole duplication by temporally modulating centrosomal Plk2 levels. AURKA phosphorylates SIRT1 and promotes the SIRT1-Plk2 interaction in mitosis. In early-mid G1, phosphorylated SIRT1 deacetylates and promotes Plk2 degradation. In late G1, SIRT1 is hypophosphorylated and its affinity to Plk2 is decreased, resulting in a rapid accumulation of centrosomal Plk2, which contributes to the timely initiation of centriole duplication. Collectively, our findings uncover a critical role of SIRT1 in centriole duplication and provide a mechanistic insight into SIRT1-mediated centrosome-associated functions. : Ling et al. demonstrate that SIRT1 deacetylates and thereby destabilizes Plk2, which in turn results in the suppression of centriole duplication