1,721,049 research outputs found
Intestinal nutrient transport in the cradle of the Mediterranean Diet: 23(rd) EITG meeting report
No full textDevelopment of a Non-Radioactive, No-Wash Biochemical Assay for High-Throughput Screening of Small Molecule Modulators of PHF20.
No full textPlant homeodomain finger protein 20 (PHF20) is a multidomain protein mainly involved in the activation of p53 and in the prevention of its ubiquitylation. Furthermore, it uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting that it plays a role in the cross-talk between lysine methylation and histone acetylation.
Writer and eraser proteins have been the main focus of therapeutic development but over the past few years a relatively underexplored group of proteins, the readers, have emerged as promising targets operating at the interface of translating histone marks. While therapeutic potential is evident, there’s need to establish specific biochemical assay for drug discovery.
We describe here the development of a high-throughput, nonradioactive bead-based assay that is suitable for screening applications to identify new PHF20 ligands.
The Tudor domain of the protein was expressed in E.Coli and purified by affinity chromatography using the GST tag. Biotinylated peptide H4K20me2 was incubated with the protein, afterwards the addition of biotinilated donor beads and anti-GST acceptor beads allowed us to measure the activity of the protein. The optimization of the assay was performed varying assay buffer, reaction time, substrate and protein concentration.
Overall, the results presented demonstrate that this novel homogenous and nonradioactive PHF20 assay could represent a powerful technology for measuring readers activity
Biomolecular and biophysical approaches to interrogate p300: a platform for drug discovery.
No full textLysine acetylation is a protein post-translational modification which effect the relaxing of the chromatin structure, making chromosomal DNA more accessible. Among the different enzymes responsible for this transformation (KATs), p300 is one of the most studied: the dysregulation of its activity leads to many human diseases.
Nevertheless, a limited number of p300 modulators have been described so far: one of the main problem is the absence of a gold standard screening technique for this enzyme because of the intrinsic limitation of each method. We decided to develop a robust and widely usable combined screening platform to identify small molecule modulators of p300, using different biophysical and biomolecular techniques to interrogate the target and to validate the outputs.
The multiple platform was applied to two different libraries of small molecule compounds, derived from the molecular pruning of anacardic acid and garcinol, natural inhibitors of p300.
This combined approach allowed us to identify and deeply characterize the activity of new chemical probes, very useful for the study of p300-mediated lysine acetylation and its implications in physiological and/or pathological processes
An AP1 regulatory element is involved in transcriptional regulation of Δ9-desaturase gene of Histoplasma capsulatum
No full textWe have characterized a region of the promoter of a cloned delta9-desaturase gene (Ole1) of Histoplasma capsulatum, a dimorphic pathogenic fungus of humans. The product of the delta9-desaturase gene is involved in regulating membrane fluid state in animal cells and microorganisms. To identify sequences critical for Ole1 expression in both the saprobic mycelial and parasitic yeast phases of this organism, we performed a deletion analysis. Evidence is presented that a 240 nt region of the proximal promoter is involved in a phase-specific binding in vitro. By sequence analysis we have identified one likely regulatory element that coincides with an AP1 binding site (TGACTAA) that is located at -740 nt of 5'-upstream from the ATG. Using gel mobility shift assays, we show that this cis-acting element binds nuclear proteins extracted from the yeast and mycelial phases of H. capsulatum that may participate in control of expression of the delta9-desaturase gene
Isolamento e caratterizzazione di geni regolati dal rame e/o dal ferro nell'intestino di ratto.
No full textIsolamento e caratterizzazione di geni regolati dal rame e/o dal ferro nell'intestino del ratto
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Editorial on: Genetic Determinants and Prediction of Antibiotic Resistance Phenotypes in Helicobacter pylori
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The influence of copper on secretion and biological activity of the gastrointestinal factor TFF1
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