Publications by authors named "Domenico Sanfelice"

Article Synopsis
  • - The Chemical Probes Portal is a free online resource that helps researchers evaluate and select high-quality small-molecule compounds, known as chemical probes, essential for studying protein functions in complex biological systems.
  • - This Portal aims to improve the reliability of biomedical research by addressing the issue of low-quality compounds, which can lead to incorrect conclusions.
  • - Recent updates to the Portal include an increased number of chemical probes and human protein targets, enhanced expert reviews, and new tools that support researchers in their studies.
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canSAR (https://cansar.ai) is the largest public cancer drug discovery and translational research knowledgebase. Now hosted in its new home at MD Anderson Cancer Center, canSAR integrates billions of experimental measurements from across molecular profiling, pharmacology, chemistry, structural and systems biology.

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We describe the Chemical Probes Portal (https://www.chemicalprobes.org/), an expert review-based public resource to empower chemical probe assessment, selection and use.

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Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074.

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Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS.

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Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study.

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Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation.

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Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation.

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What is the mechanism that determines the denaturation of proteins at low temperatures, which is, by now, recognized as a fundamental property of all proteins? We present experimental evidence that clarifies the role of specific interactions that favor the entrance of water into the hydrophobic core, a mechanism originally proposed by Privalov but never proved experimentally. By using a combination of molecular dynamics simulation, molecular biology, and biophysics, we identified a cluster of negatively charged residues that represents a preferential gate for the entrance of water molecules into the core. Even single-residue mutations in this cluster, from acidic to neutral residues, affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees.

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Protein stability is an important issue for the interpretation of a wide variety of biological problems but its assessment is at times difficult. The most common parameter employed to describe protein stability is the temperature of melting, at which the populations of folded and unfolded species are identical. This parameter may yield ambiguous results.

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We have exploited the capability of in-cell NMR to selectively observe flexible regions within folded proteins to carry out a comparative study of two members of the highly conserved frataxin family which are found both in prokaryotes and in eukaryotes. They all contain a globular domain which shares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing the mitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaY cannot be observed in the homologous E.

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As for a variety of other molecular recognition processes, conformational fluctuations play an important role in the cleavage of polyubiquitin chains by the Josephin domain of ataxin-3. The interaction between Josephin and ubiquitin appears to be mediated by the motions of α-helical hairpin that is unusual among deubiquitinating enzymes. Here, we characterized the conformational fluctuations of the helical hairpin by incorporating NMR measurements as replica-averaged restraints in molecular dynamics simulations, and by validating the results by small-angle x-ray scattering measurements.

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Frataxins are a family of metal binding proteins associated with the human Friedreich's ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength.

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The role of the denatured state in protein folding represents a key issue for the proper evaluation of folding kinetics and mechanisms. The yeast ortholog of the human frataxin, a mitochondrial protein essential for iron homeostasis and responsible for Friedreich's ataxia, has been shown to undergo cold denaturation above 0 °C, in the absence of chemical denaturants. This interesting property provides the unique opportunity to explore experimentally the molecular mechanism of both the hot and cold denaturation.

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The availability of solid state structures of opioid receptors has prompted us to reconsider a crucial question concerning bioactive peptides: can their conformation be studied without any knowledge of the structure of their receptors? The possibility of giving a meaningful answer to this query rests ultimately on the ease of dealing with the flexibility of bioactive peptides, and amongst them one of the most flexible bioactive peptides, enkephalin. All solution studies of enkephalin hint at an inextricable mixture of quasi isoenergetic conformers. In this study we refer to the only NMR work that yielded inter-residue NOEs, performed at very low temperature.

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Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence.

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Crowding and confinement can affect protein stability, favouring the more compact species amongst the folded and unfolded conformations. An unbiased assessment of the relative efficacy of crowded and confined environments has been hampered so far by the paucity of homogeneous comparisons on the same protein. This paper reports spectroscopic studies on yeast frataxin (Yfh1), a protein which provides an excellent model system for stability studies since it undergoes both cold and heat denaturation at measurable temperatures.

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Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions.

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Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I.

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An NMR study of the thermal stability of titin I28 in the temperature range from -16 to 65 degrees C showed that this protein can undergo cold denaturation at physiological conditions. This is the second case of a protein undergoing unbiased cold denaturation. Comparison of the stability curves in buffer and in crowded conditions shows that it is possible to measure thermodynamics parameters for unfolding even when proteins aggregate at high temperature.

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(1)H, (15)N and (13)C chemical shift assignments are presented for the N-terminal region of human La protein, in the apo and 5'-UUUU RNA-bound state. Secondary structure analysis shows conformational changes in the interdomain linker upon complex formation.

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The eukaryotic La protein recognizes the 3' poly(U) sequences of nascent RNA polymerase III transcripts to assist folding and maturation. The 3' ends of such RNAs are bound by the N-terminal domain of La (LaNTD). We have solved the crystal structures of four LaNTD:RNA complexes, each containing a different single-stranded RNA oligomer, and compared them to the structure of a previously published LaNTD:RNA complex containing partially duplex RNA.

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Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors.

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Eukaryotic initiation factor 5 (eIF5) plays multiple roles in translation initiation. Its N-terminal domain functions as a GTPase-activator protein (GAP) for GTP bound to eIF2, while its C-terminal region nucleates the interactions between multiple translation factors, including eIF1, which acts to inhibit GTP hydrolysis or P(i) release, and the beta subunit of eIF2. These proteins and the events in which they participate are critical for the accurate recognition of the correct start codon during translation initiation.

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