Articoli di riviste sul tema "Ligand/substrate identification"
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1
Singh, Manvi, Priya Kempanna e Kavitha Bharatham. "Identification of Mtb GlmU Uridyltransferase Domain Inhibitors by Ligand-Based and Structure-Based Drug Design Approaches". Molecules 27, n. 9 (28 aprile 2022): 2805. http://dx.doi.org/10.3390/molecules27092805.
Abstract (sommario):
Targeting enzymes that play a role in the biosynthesis of the bacterial cell wall has long been a strategy for antibacterial discovery. In particular, the cell wall of Mycobacterium tuberculosis (Mtb) is a complex of three layers, one of which is Peptidoglycan, an essential component providing rigidity and strength. UDP-GlcNAc, a precursor for the synthesis of peptidoglycan, is formed by GlmU, a bi-functional enzyme. Inhibiting GlmU Uridyltransferase activity has been proven to be an effective anti-bacterial, but its similarity with human enzymes has been a deterrent to drug development. To develop Mtb selective hits, the Mtb GlmU substrate binding pocket was compared with structurally similar human enzymes to identify selectivity determining factors. Substrate binding pockets and conformational changes upon substrate binding were analyzed and MD simulations with substrates were performed to quantify crucial interactions to develop critical pharmacophore features. Thereafter, two strategies were applied to propose potent and selective bacterial GlmU Uridyltransferase domain inhibitors: (i) optimization of existing inhibitors, and (ii) identification by virtual screening. The binding modes of hits identified from virtual screening and ligand growing approaches were evaluated further for their ability to retain stable contacts within the pocket during 20 ns MD simulations. Hits that are predicted to be more potent than existing inhibitors and selective against human homologues could be of great interest for rejuvenating drug discovery efforts towards targeting the Mtb cell wall for antibacterial discovery.
2
Rothweiler, Elisabeth M., Paul E. Brennan e Kilian V. M. Huber. "Covalent fragment-based ligand screening approaches for identification of novel ubiquitin proteasome system modulators". Biological Chemistry 403, n. 4 (23 febbraio 2022): 391–402. http://dx.doi.org/10.1515/hsz-2021-0396.
Abstract (sommario):
Abstract Ubiquitination is a key regulatory mechanism vital for maintenance of cellular homeostasis. Protein degradation is induced by E3 ligases via attachment of ubiquitin chains to substrates. Pharmacological exploitation of this phenomenon via targeted protein degradation (TPD) can be achieved with molecular glues or bifunctional molecules facilitating the formation of ternary complexes between an E3 ligase and a given protein of interest (POI), resulting in ubiquitination of the substrate and subsequent proteolysis by the proteasome. Recently, the development of novel covalent fragment screening approaches has enabled the identification of first-in-class ligands for E3 ligases and deubiquitinases revealing so far unexplored binding sites which highlights the potential of these methods to uncover and expand druggable space for new target classes.
3
Fernández, Rico-Jiménez, Ortega, Daddaoua, García García, Martín-Mora, Torres, Tajuelo, Matilla e Krell. "Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins". International Journal of Molecular Sciences 20, n. 20 (17 ottobre 2019): 5156. http://dx.doi.org/10.3390/ijms20205156.
Abstract (sommario):
Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for γ-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.
4
Wang, Wenyuan, Junli Zhu, Qi Huang, Lei Zhu, Ding Wang, Weimin Li e Wenjie Yu. "DFT Exploration of Metal Ion–Ligand Binding: Toward Rational Design of Chelating Agent in Semiconductor Manufacturing". Molecules 29, n. 2 (8 gennaio 2024): 308. http://dx.doi.org/10.3390/molecules29020308.
Abstract (sommario):
Chelating agents are commonly employed in microelectronic processes to prevent metal ion contamination. The ligand fragments of a chelating agent largely determine its binding strength to metal ions. Identification of ligands with suitable characteristics will facilitate the design of chelating agents to enhance the capture and removal of metal ions from the substrate in microelectronic processes. This study employed quantum chemical calculations to simulate the binding process between eleven ligands and the hydrated forms of Ni2+, Cu2+, Al3+, and Fe3+ ions. The binding strength between the metal ions and ligands was quantified using binding energy and binding enthalpy. Additionally, we explored the binding interaction mechanisms and explained the differences in binding abilities of the eleven ligands using frontier molecular orbitals, nucleophilic indexes, electrostatic potentials, and energy decomposition calculations based on molecular force fields. Based on our computational results, promising chelating agent structures are proposed, aiming to guide the design of new chelating agents to address metal ion contamination issues in integrated circuit processes.
5
Weng, Z., S. M. Thomas, R. J. Rickles, J. A. Taylor, A. W. Brauer, C. Seidel-Dugan, W. M. Michael, G. Dreyfuss e J. S. Brugge. "Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains". Molecular and Cellular Biology 14, n. 7 (luglio 1994): 4509–21. http://dx.doi.org/10.1128/mcb.14.7.4509-4521.1994.
Abstract (sommario):
Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.
6
Weng, Z., S. M. Thomas, R. J. Rickles, J. A. Taylor, A. W. Brauer, C. Seidel-Dugan, W. M. Michael, G. Dreyfuss e J. S. Brugge. "Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains." Molecular and Cellular Biology 14, n. 7 (luglio 1994): 4509–21. http://dx.doi.org/10.1128/mcb.14.7.4509.
Abstract (sommario):
Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.
7
Evans, S. W., D. Rennick e W. L. Farrar. "Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity". Biochemical Journal 244, n. 3 (15 giugno 1987): 683–91. http://dx.doi.org/10.1042/bj2440683.
Abstract (sommario):
The haematopoietic growth factors multi-colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2 specifically control the production and proliferation of distinct leucocyte series. Each growth factor acts on a unique surface receptor associated with an appropriate signal-transduction apparatus. In this report we identify a 68 kDa substrate which is phosphorylated after stimulation of different cell types with multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2. The 68 kDa substrate is also phosphorylated in each cell line stimulated with synthetic diacylglycerol, a direct activator of protein kinase C. Interestingly, granulocyte/macrophage colony-stimulating factor does not induce phosphorylation of the 68 kDa molecule. The 68 kDa molecule that is phosphorylated after stimulation with each ligand yielded similar peptide maps after chymotryptic digestion; furthermore, the substrate was always phosphorylated on threonine residues. Phosphorylation of the same residues in the 68 kDa substrate suggests that activation of protein kinase C is one common signal-transduction event associated with the action of multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2.
8
Duarte Filho, Luiz Antonio Miranda de Souza, Cintia Emi Yanaguibashi Leal, Pierre-Edouard Bodet, Edilson Beserra de Alencar Filho, Jackson Roberto Guedes da Silva Almeida, Manon Porta Zapata, Oussama Achour et al. "The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy". Marine Drugs 22, n. 6 (27 maggio 2024): 244. http://dx.doi.org/10.3390/md22060244.
Abstract (sommario):
Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme’s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease’s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.
9
Drexler, Hannes C. A., Matthias Vockel, Christian Polaschegg, Maike Frye, Kevin Peters e Dietmar Vestweber. "Vascular Endothelial Receptor Tyrosine Phosphatase: Identification of Novel Substrates Related to Junctions and a Ternary Complex with EPHB4 and TIE2". Molecular & Cellular Proteomics 18, n. 10 (19 agosto 2019): 2058–77. http://dx.doi.org/10.1074/mcp.ra119.001716.
Abstract (sommario):
Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.
10
Lamaze, C., e S. L. Schmid. "Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase." Journal of Cell Biology 129, n. 1 (1 aprile 1995): 47–54. http://dx.doi.org/10.1083/jcb.129.1.47.
Abstract (sommario):
EGF-receptor (EGF-R) tyrosine kinase is required for the down-regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).
11
Mora Lagares, Liadys, Yunierkis Pérez-Castillo, Nikola Minovski e Marjana Novič. "Structure–Function Relationships in the Human P-Glycoprotein (ABCB1): Insights from Molecular Dynamics Simulations". International Journal of Molecular Sciences 23, n. 1 (29 dicembre 2021): 362. http://dx.doi.org/10.3390/ijms23010362.
Abstract (sommario):
P-Glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette superfamily of transporters, and it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping compounds out of cells. P-gp contributes to a reduction in toxicity, and has broad substrate specificity. It is involved in the failure of many cancer and antiviral chemotherapies due to the phenomenon of multidrug resistance (MDR), in which the membrane transporter removes chemotherapeutic drugs from target cells. Understanding the details of the ligand–P-gp interaction is therefore critical for the development of drugs that can overcome the MDR phenomenon, for the early identification of P-gp substrates that will help us to obtain a more effective prediction of toxicity, and for the subsequent outdesign of substrate properties if needed. In this work, a series of molecular dynamics (MD) simulations of human P-gp (hP-gp) in an explicit membrane-and-water environment were performed to investigate the effects of binding different compounds on the conformational dynamics of P-gp. The results revealed significant differences in the behaviour of P-gp in the presence of active and non-active compounds within the binding pocket, as different patterns of movement were identified that could be correlated with conformational changes leading to the activation of the translocation mechanism. The predicted ligand–P-gp interactions are in good agreement with the available experimental data, as well as the estimation of the binding-free energies of the studied complexes, demonstrating the validity of the results derived from the MD simulations.
12
Ruan, Ke-He. "High resolution nuclear magnetic resonance spectroscopy-guided mutagenesis for characterization of membrane-bound proteins: Experimental designs and applications". Spectroscopy 18, n. 1 (2004): 13–29. http://dx.doi.org/10.1155/2004/802728.
Abstract (sommario):
High resolution Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for determining the solution structures of peptides and small proteins, and their ligand binding functions. Molecular biology mutagenesis is a widely used and powerful approach for identification of the protein functions. We have developed a strategy integrating NMR experiments with mutagenesis studies to advance and extend the approaches used for structure/function relationship studies of proteins, especially for membrane-bound proteins, which play important roles in physiopathological processes. The procedures include the design of the functional protein domain, identification of the solution structure and intermolecular contacts between the protein segment and its ligand. These determinations are resolved by high-resolution 2D NMR spectroscopy, and followed by site-directed mutagenesis of the residues suggested from the NMR experiment for the membrane-bound proteins. The residues important to the protein functions, identified by the mutagenesis, were further used to re-assign the NMR spectra and finalize the docking of the protein with its ligand. A structural model of the protein/ligand interaction can be constructed at an atomic level based on the NMR spectroscopy and mutagenesis results. As an application, the strategy has enhanced our knowledge in the understanding of the structure/function relationship for a membrane-bound G protein coupling receptor, the thromboxane A2receptor (TP receptor), interacting with its ligand, and a microsomal P450, prostacyclin synthase (PGIS), docking with its substrate in the endoplasmic reticulum (ER) membrane. In this review, we have summarized the principles and applications for this newly developed technique.
13
Reynolds, A. B., J. Daniel, P. D. McCrea, M. J. Wheelock, J. Wu e Z. Zhang. "Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes". Molecular and Cellular Biology 14, n. 12 (dicembre 1994): 8333–42. http://dx.doi.org/10.1128/mcb.14.12.8333-8342.1994.
Abstract (sommario):
p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.
14
Reynolds, A. B., J. Daniel, P. D. McCrea, M. J. Wheelock, J. Wu e Z. Zhang. "Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes." Molecular and Cellular Biology 14, n. 12 (dicembre 1994): 8333–42. http://dx.doi.org/10.1128/mcb.14.12.8333.
Abstract (sommario):
p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.
15
Adams, James, Benjamin P. Thornton e Lydia Tabernero. "A New Paradigm for KIM-PTP Drug Discovery: Identification of Allosteric Sites with Potential for Selective Inhibition Using Virtual Screening and LEI Analysis". International Journal of Molecular Sciences 22, n. 22 (11 novembre 2021): 12206. http://dx.doi.org/10.3390/ijms222212206.
Abstract (sommario):
The kinase interaction motif protein tyrosine phosphatases (KIM-PTPs), HePTP, PTPSL and STEP, are involved in the negative regulation of mitogen-activated protein kinase (MAPK) signalling pathways and are important therapeutic targets for a number of diseases. We have used VSpipe, a virtual screening pipeline, to identify a ligand cluster distribution that is unique to this subfamily of PTPs. Several clusters map onto KIM-PTP specific sequence motifs in contrast to the cluster distribution obtained for PTP1B, a classic PTP that mapped to general PTP motifs. Importantly, the ligand clusters coincide with previously reported functional and substrate binding sites in KIM-PTPs. Assessment of the KIM-PTP specific clusters, using ligand efficiency index (LEI) plots generated by the VSpipe, ascertained that the binders in these clusters reside in a more drug-like chemical–biological space than those at the active site. LEI analysis showed differences between clusters across all KIM-PTPs, highlighting a distinct and specific profile for each phosphatase. The most druggable cluster sites are unexplored allosteric functional sites unique to each target. Exploiting these sites may facilitate the delivery of inhibitors with improved drug-like properties, with selectivity amongst the KIM-PTPs and over other classical PTPs.
16
Yadav, Rolly, Anwesh Pandey, Nidhi Awasthi e Anamika Shukla. "Molecular Docking Studies of Enzyme Binding Drugs on Family of Cytochrome P450". Advanced Science, Engineering and Medicine 12, n. 1 (1 gennaio 2020): 83–87. http://dx.doi.org/10.1166/asem.2020.2520.
Abstract (sommario):
The combination of experimental and computational strategies has been of great value in the identification and development of metabolism of drugs. Nowadays modern drug design, molecular docking methods are helpful in exploring the ligand conformations adopted within the binding sites of macro-molecular targets such as DNA, proteins, and enzymes, there by reducing cost, time and wayward efforts of chemist. Since the development of the algorithms in the 1980s, molecular docking became an important tool in drug discovery like investigation of crucial molecular events, including ligand binding modes and the corresponding intermolecular interactions that stabilize the ligand-receptor complex, can be conveniently performed. In present study we have tried to investigate the drug binding pocket of various cytochrome (CYP) enzymes found in humans. All structures of drugs are optimized at B3LYP/6-31** level of theory using Gaussian program suite. Docking of substrate-enzyme duo was done using AUTODOCK 4.0. Computational docking revealed that almost all drugs have same binding pocket with varied binding affinities due to change in interactions and interacting distance from heme prosthetic moiety with transition metal iron as chelating ion.
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BARNETT, Stanley F., Deborah DEFEO-JONES, Sheng FU, Paula J. HANCOCK, Kathleen M. HASKELL, Raymond E. JONES, Jason A. KAHANA et al. "Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors". Biochemical Journal 385, n. 2 (7 gennaio 2005): 399–408. http://dx.doi.org/10.1042/bj20041140.
Abstract (sommario):
We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 μM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 μM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 μM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
18
Wei, Min, Richard Wynn, Gregory Hollis, Boshan Liao, Alexander Margulis, Brian G. Reid, Ronald Klabe et al. "High-Throughput Determination of Mode of Inhibition in Lead Identification and Optimization". Journal of Biomolecular Screening 12, n. 2 (11 gennaio 2007): 220–28. http://dx.doi.org/10.1177/1087057106296679.
Abstract (sommario):
After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC50 or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.
19
Takeuchi, Hajime, Daniel J. Rigden, Bahram Ebrahimi, Philip C. Turner e Huw H. Rees. "Regulation of ecdysteroid signalling during Drosophila development: identification, characterization and modelling of ecdysone oxidase, an enzyme involved in control of ligand concentration". Biochemical Journal 389, n. 3 (26 luglio 2005): 637–45. http://dx.doi.org/10.1042/bj20050498.
Abstract (sommario):
The steroidal moulting hormones (ecdysteroids) mediate developmental transitions in insects, and their regulation is mainly controlled by the production and inactivation of these steroid hormones at the appropriate developmental times. One route of metabolism of ecdysteroids in insects involves EO (ecdysone oxidase)-catalysed conversion into 3-dehydroecdysteroid, which undergoes reduction to the corresponding 3-epiecdysteroid. By a twin-stranded bioinformatics approach, employing both phylogenomics and model structure-based analysis, we first predicted that DmEO (the EO of Drosophila melanogaster) corresponds to the protein product of gene CG9504. When CG9504 was expressed in COS7 cells, significant conversion of ecdysone into 3-dehydroecdysone was observed. Quantitative PCR and enzyme assay showed that DmEO was mainly expressed in the midgut during the late instars at a time corresponding to a hormone titre peak. DmEO shares only 27% amino acid sequence identity with Spodoptera littoralis (Lepidoptera) EO, yet key substrate-binding residues are well conserved. A model of DmEO is consistent with an inability to catalyse reaction of cholesterol derivatives. The significance of DmEO in ligand activation is discussed in relation to new evidence suggesting that 3-dehydro- and 3-epiecdysteroids may be functionally active as ligands in a novel, atypical ecdysteroid signalling pathway involving the Drosophila orphan nuclear receptor, DHR38, rather than being merely hormone inactivation products.
20
D’Arrigo, Giulia, Ida Autiero, Eleonora Gianquinto, Lydia Siragusa, Massimo Baroni, Gabriele Cruciani e Francesca Spyrakis. "Exploring Ligand Binding Domain Dynamics in the NRs Superfamily". International Journal of Molecular Sciences 23, n. 15 (5 agosto 2022): 8732. http://dx.doi.org/10.3390/ijms23158732.
Abstract (sommario):
Nuclear receptors (NRs) are transcription factors that play an important role in multiple diseases, such as cancer, inflammation, and metabolic disorders. They share a common structural organization composed of five domains, of which the ligand-binding domain (LBD) can adopt different conformations in response to substrate, agonist, and antagonist binding, leading to distinct transcription effects. A key feature of NRs is, indeed, their intrinsic dynamics that make them a challenging target in drug discovery. This work aims to provide a meaningful investigation of NR structural variability to outline a dynamic profile for each of them. To do that, we propose a methodology based on the computation and comparison of protein cavities among the crystallographic structures of NR LBDs. First, pockets were detected with the FLAPsite algorithm and then an “all against all” approach was applied by comparing each pair of pockets within the same sub-family on the basis of their similarity score. The analysis concerned all the detectable cavities in NRs, with particular attention paid to the active site pockets. This approach can guide the investigation of NR intrinsic dynamics, the selection of reference structures to be used in drug design and the easy identification of alternative binding sites.
21
Basu, Nirmalya, Rashna Bhandari, Vivek T. Natarajan e Sandhya S. Visweswariah. "Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis". Molecular and Cellular Biology 29, n. 19 (20 luglio 2009): 5277–89. http://dx.doi.org/10.1128/mcb.00001-09.
Abstract (sommario):
ABSTRACT Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.
22
Astegno, Alessandra, Alejandro Giorgetti, Alessandra Allegrini, Barbara Cellini e Paola Dominici. "Characterization of C-S Lyase fromC. diphtheriae: A Possible Target for New Antimicrobial Drugs". BioMed Research International 2013 (2013): 1–13. http://dx.doi.org/10.1155/2013/701536.
Abstract (sommario):
The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase fromCorynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein inE. coliand analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin fromTreponema denticolaindicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site ofC. diphtheriaeC-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics.
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Sanguinetti, Manuel, Lucianna Helene Silva Santos, Juliette Dourron, Catalina Alamón, Juan Idiarte, Sotiris Amillis, Sergio Pantano e Ana Ramón. "Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter". International Journal of Molecular Sciences 23, n. 24 (16 dicembre 2022): 16039. http://dx.doi.org/10.3390/ijms232416039.
Abstract (sommario):
Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.
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Xia, Daosong, Renwen Zheng, Jianhua Huang, Sihan Lu e Qingfeng Tang. "Identification and Functional Analysis of Glutathione S-Transferases from Sitophilus zeamais in Olfactory Organ". Insects 13, n. 3 (5 marzo 2022): 259. http://dx.doi.org/10.3390/insects13030259.
Abstract (sommario):
Odorant-degrading enzymes (ODEs) play an important role in rapidly degrading and inactivating odorant molecules that have completed information transmission, as well as in maintaining the stability and sensitivity of insect olfactory sensing systems. Glutathione S-transferases (GSTs), as a group of ODEs, supposedly bear the ability to catalyze the conjugation of glutathione (GSH) and xenobiotic odorant molecules in the degrading process. However, there are few reports regarding the role of the GST genes of Sitophilus zeamais in the degrading process. Thus, we characterized 13 full-length genes encoding GST sequences from S. zeamais, of which only SzeaGSTd1 contained a high abundance in the antennae. Ligand-binding assays implied that SzeaGSTd1 was able to catalyze the conjugation of GSH with 2, 4-dinitrochlorobenzene (CDNB). We investigated whether recombinant SzeaGSTd1 bears the ability to degrade the volatile molecules of the host; among the host volatiles, and found capryl alcohol to be a suitable substrate for SzeaGSTd1. These results strongly suggest that SzeaGSTd1 probably plays a role in auxiliary host location by degrading the host volatiles of capryl alcohol and exhibits a potential biological function in the olfactory sensing system of S. zeamais. Knowledge of the potential functions of SzeaGSTd1 will provide new ideas for biological control strategies for S. zeamais.
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Thakur, Priti, Jowad Atway, Patrick A. Limbach e Balasubrahmanyam Addepalli. "RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site". International Journal of Molecular Sciences 23, n. 13 (24 giugno 2022): 7021. http://dx.doi.org/10.3390/ijms23137021.
Abstract (sommario):
Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5–1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this in silico approach for the identification of key interactions. Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.
26
Milot, Marie-Michelle, e Adrian Hofmann. "Shikimate kinase-like 1 plays an integral role in chloroplast biogenesis". Acta Crystallographica Section A Foundations and Advances 70, a1 (5 agosto 2014): C452. http://dx.doi.org/10.1107/s2053273314095473.
Abstract (sommario):
The shikimate pathway is found in plants, fungi, and microbes, but not mammals which makes it an attractive target for antimicrobials and herbicide development. In plants, the shikimate pathway produces precursors of compounds that are crucial for growth, defense and, survival. We focused on the enzyme shikimate kinase (SK) which catalyzes the phosphorylation of shikimate; the fifth reaction of the pathway. Plant SK underwent a gene duplication event yielding shikimate kinase-like genes (SKL1 and SKL2) roughly 400 MYA. Despite the overall similarity in sequence between SK and SKL1, a previous in vitro analysis showed that SKL1 does not catalyze a phosphorylation reaction in the presence of shikimate. When a three-dimensional structural model of SKL1 was compared with the microbial crystal structure of SK, we identified several highly conserved functional domains crucial for the shikimate kinase reaction: the shikimate binding domain (SBD) critical for substrate recognition, the LID domain integral for the transfer of phosphate to bound shikimate, and the walker A and B domains important for ATP binding. Of these domains, only the walker A and B domains are conserved in SKL1. Together with in vitro analysis, it is evident that SKL1 does not interact with shikimate, but the function is still unknown. Sequence alignment and modeled structure also revealed the presence of a novel phosphoglycerate mutase-like (PGML) domain within SKL1.This further suggests neofunctionalization of SKL1 compared to its parent gene SK. Current studies in our lab focus on determining the crystal structure of SKL1. This structure will be used for ligand docking experiments to identify potential substrates of the enzyme. As an additional means of substrate identification, we will also be performing crystal soaking experiments using plant extracts.
27
Venkatraman, Janani, Jyothi Bhat, Suresh M. Solapure, Jatheendranath Sandesh, Debasmita Sarkar, Sundaram Aishwarya, Kakoli Mukherjee et al. "Screening, Identification, and Characterization of Mechanistically Diverse Inhibitors of the Mycobacterium Tuberculosis Enzyme, Pantothenate Kinase (CoaA)". Journal of Biomolecular Screening 17, n. 3 (15 novembre 2011): 293–302. http://dx.doi.org/10.1177/1087057111423069.
Abstract (sommario):
The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis ( Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift–based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated Kie and Kies for representative compounds and through nuclear magnetic resonance–based ligand displacement assays.
28
N'Diaye, Elsa-Noah, Aylin C. Hanyaloglu, Kimberly K. Kajihara, Manojkumar A. Puthenveedu, Ping Wu, Mark von Zastrow e Eric J. Brown. "The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis". Molecular Biology of the Cell 19, n. 3 (marzo 2008): 1252–60. http://dx.doi.org/10.1091/mbc.e07-08-0775.
Abstract (sommario):
The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics.
29
Pallen, C. J., D. S. Y. Lai, H. P. Chia, I. Boulet e P. H. Tong. "Purification and characterization of a higher-molecular-mass form of protein phosphotyrosine phosphatase (PTP 1B) from placental membranes". Biochemical Journal 276, n. 2 (1 giugno 1991): 315–23. http://dx.doi.org/10.1042/bj2760315.
Abstract (sommario):
Purification of a major placental membrane protein phosphotyrosine phosphatase (PTP-I) through the use of a nonhydrolysable phosphotyrosine analogue affinity ligand has enabled identification of the enzyme as a single polypeptide of at least 46 kDa. This phosphatase specifically dephosphorylates phosphotyrosine-containing substrates, including the src peptide, the epidermal-growth-factor receptor tyrosine kinase and the non-receptor tyrosine kinase p56lck. The p56lck can be dephosphorylated by PTP-I at two tyrosine residues (Tyr-394 and Tyr-505), which are differentially phosphorylated in vitro and in vivo and have been suggested to modulate kinase activity. The activity of PTP-I towards these substrates indicates a possible function of regulation of cellular tyrosine phosphorylation pathways at the level of growth-factor receptor and/or oncogene/proto-oncogene tyrosine kinases. Kinetic analyses show that PTP-I exhibits a Km value of about 2 microM with either src peptide or reduced, carboxyamidomethylated and maleylated (RCM)-lysozyme as substrate, and is inhibited in a mixed competitive manner by the polyanions heparin and poly(Glu4,Tyr1). Sequencing of PTP-I peptides reveals almost complete identity with sequences within the N-terminal half of the 37 kDa non-receptor tyrosine phosphatase 1B. However, the size and amino acid composition of PTP-I are similar to that of a higher-molecular-mass form of PTP 1B predicted from cDNA cloning. These results suggest that the 37 kDa PTP 1B is a proteolysed form of PTP-I, and provide evidence that a larger form of PTP 1B exists in vivo, at least in association with placental membranes.
30
Powell, David W., Madhavi J. Rane, Brian A. Joughin, Ralitsa Kalmukova, Jeong-Ho Hong, Bruce Tidor, William L. Dean et al. "Proteomic Identification of 14-3-3ζ as a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Substrate: Role in Dimer Formation and Ligand Binding". Molecular and Cellular Biology 23, n. 15 (1 agosto 2003): 5376–87. http://dx.doi.org/10.1128/mcb.23.15.5376-5387.2003.
Abstract (sommario):
ABSTRACT Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3ζ. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3ζ in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3ζ at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3ζ to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3ζ dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3ζ functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.
31
Dryuchko, Oleksandr, Natalia Bunyakina, Bogdan Korobko, Oleksandr Shefer, Kateryna Kytaihora e Iryna Ivanytska. "SEARCH FOR WAYS OF CONTROLLED MODIFICATION OF CHARACTERISTICS OF FUNCTIONAL UNITS OF ADAPTIVE AIR PURIFICATION SYSTEMS". Bulletin of the National Technical University "KhPI". Series: Chemistry, Chemical Technology and Ecology, n. 2(6) (23 dicembre 2021): 34–51. http://dx.doi.org/10.20998/2079-0821.2021.02.06.
Abstract (sommario):
Information on alkaline coordination nitrates of rare-earth elements of the cerium subgroup - precursors of promising modern multifunctional materials - on the conditions of their formation and existence, the nature of the chemical bond, the composition, structure, shape of the Ln coordination polyhedra, the type of ligand coordination, and the existence of isotypic series in stoichiometry are generalized. composition, structure, characteristic properties. The data obtained (as primary information) is the basis for the detection, identification, and control of the phase state of processing objects in the preparatory stages, the choice of criteria for the compatibility of the components in the formation of single-layer and layered nanostructured oxide composite systems of lanthanides and transition elements for general purposes, with catalytic and photocatalytic activity, coatings self-cleaning with hydrophilic properties; development of various combined methods for their activation and identification of technological functional dependencies; controlled modification of the properties of the obtained target products. To increase the photocatalytic activity of coating samples based on highly dispersed anatase TiO2, a methodology for chemical modification of oxidation centers in their surface layer with heat treatment in contact with thermolysis products of alkaline coordination lanthanide nitrate melts is proposed. An effective test photocatalytic destruction of organic substrates vapors has been discovered using ethanol as an example.Effective activation of the functioning of functional units in the composition of self-adjusting air purification complexes using new photocatalytically active three-layer perovskite-like oxide materials M2Ln2Ti3O10 (M - Li, Na, K; Ln - La, Nd) has been proven. The variability of such methods for creating and modifying the characteristics of polyfunctional coatings is determined by the number and individual properties of representatives of the natural series of lanthanides, alkali metals of the periodic system, the peculiarities of their cooperative behavior in the preparatory technological stages, conditions and methods of activation of formation processes, the nature of the substrate, and other factors.
32
Aytenfisu, Asaminew H., Daniel Deredge, Erik H. Klontz, Jonathan Du, Eric J. Sundberg e Alexander D. MacKerell. "Insights into substrate recognition and specificity for IgG by Endoglycosidase S2". PLOS Computational Biology 17, n. 7 (26 luglio 2021): e1009103. http://dx.doi.org/10.1371/journal.pcbi.1009103.
Abstract (sommario):
Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins’ catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.
33
Byrne, Dominic P., Yong Li, Pawin Ngamlert, Krithika Ramakrishnan, Claire E. Eyers, Carrow Wells, David H. Drewry et al. "New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors". Biochemical Journal 475, n. 15 (14 agosto 2018): 2435–55. http://dx.doi.org/10.1042/bcj20180266.
Abstract (sommario):
Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.
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Lobell, Robert B., Joseph P. Davide, Nancy E. Kohl, H. Donald Burns, Wai-Si Eng e Raymond E. Gibson. "A Cell-Based Radioligand Binding Assay for Farnesyl: Protein Transferase Inhibitors". Journal of Biomolecular Screening 8, n. 4 (agosto 2003): 430–38. http://dx.doi.org/10.1177/1087057103256153.
Abstract (sommario):
Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [3H]-labeled or [125I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.
35
Vargas-Pérez, María de los Ángeles, Damien Paul Devos e Guillermo López-Lluch. "An AlphaFold Structure Analysis of COQ2 as Key a Component of the Coenzyme Q Synthesis Complex". Antioxidants 13, n. 4 (21 aprile 2024): 496. http://dx.doi.org/10.3390/antiox13040496.
Abstract (sommario):
Coenzyme Q (CoQ) is a lipidic compound that is widely distributed in nature, with crucial functions in metabolism, protection against oxidative damage and ferroptosis and other processes. CoQ biosynthesis is a conserved and complex pathway involving several proteins. COQ2 is a member of the UbiA family of transmembrane prenyltransferases that catalyzes the condensation of the head and tail precursors of CoQ, which is a key step in the process, because its product is the first intermediate that will be modified in the head by the next components of the synthesis process. Mutations in this protein have been linked to primary CoQ deficiency in humans, a rare disease predominantly affecting organs with a high energy demand. The reaction catalyzed by COQ2 and its mechanism are still unknown. Here, we aimed at clarifying the COQ2 reaction by exploring possible substrate binding sites using a strategy based on homology, comprising the identification of available ligand-bound homologs with solved structures in the Protein Data Bank (PDB) and their subsequent structural superposition in the AlphaFold predicted model for COQ2. The results highlight some residues located on the central cavity or the matrix loops that may be involved in substrate interaction, some of which are mutated in primary CoQ deficiency patients. Furthermore, we analyze the structural modifications introduced by the pathogenic mutations found in humans. These findings shed new light on the understanding of COQ2’s function and, thus, CoQ’s biosynthesis and the pathogenicity of primary CoQ deficiency.
36
Ma, Peilin, Raghuveer Mali, Li-Fan Zeng, Holly Martin, Baskar Ramdas, Yantao He, Emily Sims et al. "KIT Induced Myeloproliferative Disease Is Dependent on PI3Kinase and SHP2 Phosphatase: Identification of SHP2 As a Druggable Target for Treating MPD and AML". Blood 118, n. 21 (18 novembre 2011): 868. http://dx.doi.org/10.1182/blood.v118.21.868.868.
Abstract (sommario):
Abstract Abstract 868 Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). An activating KIT receptor mutation of aspartic acid to valine at codon 814 in mice (KITD814V) or codon 816 in humans (KITD816V) results in altered substrate recognition and constitutive tyrosine autophosphorylation leading to promiscuous signaling. Consequently, cells bearing oncogenic form of KIT (KITD814V) demonstrate ligand independent proliferation in vitro and MPD in vivo. However, the intracellular signals that contribute to KITD814V induced MPD are not known. Here, we show the constitutive phosphorylation of SHP2 in cells bearing KITD814V, but not WT KIT, which was inhibited by treatment with a novel SHP2 inhibitor, II-B08 (*p < 0.05). In addition, treatment with II-B08 suppressed the growth of cells bearing KITD814V, but not WT KIT (*p < 0.05), Human mast cell line HMC1.2 (*p < 0.05), and Human CD34+ cells bearing KITD816V (*p < 0.05). Likewise, deficiency of SHP2 in primary bone marrow cells resulted in a significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). To determine the mechanism behind the repression in ligand independent growth of cells bearing KITD814V by II-B08, we examined the role of SHP2 in cell survival. We observed a dose dependent increase in apoptosis of cells bearing KITD814V compared to WT KIT in the presence of II-B08 (*p < 0.05). Similarly, deficiency of SHP2 resulted in increased apoptosis of cells bearing KITD814V (*p < 0.05). In an effort to identify the mechanism behind reduced growth and increased apoptosis of cells bearing KITD814V in the presence of II-B08, we examined whether SHP2 regulates the activation of AKT in KITD814V bearing cells. We found constitutive activation of AKT in cells bearing KITD814V, but not WT KIT, which was significantly inhibited upon II-B08 treatment suggesting that SHP2 regulates ligand independent growth and survival of KITD814V bearing cells in part by regulating the activation of AKT. To further determine the signaling molecules that co-operate with SHP2 in KITD814V induced MPD, we examined proteins that potentially interact with SHP2 in KITD814V bearing cells and whether inhibition of SHP2 activity by II-B08 suppresses these co-operating protein interactions. SHP2 constitutively bound to p85α and Gab2 in cells bearing KITD814V, but not in WT KIT bearing cells, which was inhibited upon II-B08 treatment. To further assess whether the binding of SHP2 to p85α, Gab2 and KITD814V is sufficient to induce MPD, we generated a KIT mutant receptor, KITD814V-F7, in which seven tyrosine residues in KITD814V (known to bind SH2 containing proteins at tyrosine 567, 569, 702, 719, 728, 745, and 934) were converted to phenylalanine, and KITD814V-Y719, in which only tyrosine residue 719 (binding site for p85α) was added back to the KITD814V-F7 receptor. We observed constitutive binding of SHP2 and Gab2 to p85α in cells bearing KITD814V and KITD814V-Y719, but not in WT KIT or KITD814V-F7 bearing cells. In addition, conversion of all the seven intracellular tyrosine residues in KITD814V to phenylalanine (KITD814V-F7) resulted in complete loss of ligand independent growth in vitro (*p < 0.05) and significantly delayed the progression of MPD in vivo (*p < 0.05). Importantly, restoration of tyrosine residue at position 719 (KITD814V-Y719) was sufficient to induce ligand independent growth in vitro and MPD in vivo to KITD814V levels. Furthermore, deficiency of Gab2 resulted in significant repression in constitutive growth of cells bearing KITD814V (*p < 0.05). These results demonstrate that p85α recruits SHP2 and Gab2 to KITD814V at Y719, which might contribute to KITD814V induced MPD. Moreover, II-B08 enhances the efficacy of PI3Kinase inhibitor LY294002 in suppressing KITD814V induced ligand independent growth in vitro (*p < 0.05) and MPD in vivo (*p < 0.05). Taken together our results demonstrate that SHP2 is a druggable target which cooperates with PI3Kinase in inducing MPD. Disclosures: No relevant conflicts of interest to declare.
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Wang, Xingdong, Jie Pei, Pengjia Bao, Chunnian Liang, Min Chu, Shaoke Guo, Ping Yan e Xian Guo. "Identification of Yak’s TLR4 Alternative Spliceosomes and Bioinformatic Analysis of TLR4 Protein Structure and Function". Animals 11, n. 1 (26 dicembre 2020): 32. http://dx.doi.org/10.3390/ani11010032.
Abstract (sommario):
In this study, the yak’s TLR4 gene alternative spliceosomes were investigated using PCR amplification and cloning to improve disease-resistance in yak and promote efficient utilization of yak’s resources. qRT-PCR was used to determine the expression levels of two alternatively spliced transcripts of the TLR4 gene in seven distinct tissues. To predict the function of proteins expressed by each TLR4 spliceosome, bioinformatic analysis of yak’s TLR4 protein structure and function was performed, which led to the identification of two alternative spliceosomes of yak’s TLR4 gene. The TLR4-X1 sequence length was 2526 bp, and it encoded full-length TLR4 protein (841 amino acids). The sequence length of the exon-2 deleted TLR4-X2 sequence was 1926 bp, and it encoded truncated TLR4 protein (641 amino acids). TLR4-X2 sequence was consistent with the predicted sequence of the TLR4 gene in GenBank. Each tissue showed significantly different expression levels of these two alternative spliceosomes. As per the bioinformatic analysis of the structure and function of TLR4 protein, deletion of exon-2 in the TLR4 gene resulted in frameshift mutations of the reading frame in the corresponding protein, which altered its ligand-binding and active sites. Besides, biological property such as substrate specificity of truncated TLR4 protein was also altered, leading to altered protein function. This study has laid a theoretical foundation for exploring the role of two variants of the TLR4 gene in yak’s disease resistance. Besides, this study’s data could be analyzed further to explore the molecular mechanism associated with disease-resistance in the yak.
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Perduca, Massimiliano, Michele Bovi, Mattia Bertinelli, Edoardo Bertini, Laura Destefanis, Maria E. Carrizo, Stefano Capaldi e Hugo L. Monaco. "High-resolution structures of mutants of residues that affect access to the ligand-binding cavity of human lipocalin-type prostaglandin D synthase". Acta Crystallographica Section D Biological Crystallography 70, n. 8 (25 luglio 2014): 2125–38. http://dx.doi.org/10.1107/s1399004714012462.
Abstract (sommario):
Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2(prostaglandin H2) to produce PGD2(prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as β-trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 Å resolution and of the double mutant C65A/K59A at 1.60 Å resolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance–exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin.
39
ZECHEL, David L., Shouming HE, Claude DUPONT e Stephen G. WITHERS. "Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB". Biochemical Journal 336, n. 1 (15 novembre 1998): 139–45. http://dx.doi.org/10.1042/bj3360139.
Abstract (sommario):
Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 Å resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid–base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032–16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2´,4´-dinitrophenyl 2-deoxy-2-fluoro-β-d-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact = 0.29±0.02 min-1 and Kinact = 0.72±0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki = 9±1 mM), a behaviour that was also observed with the substrate 2´,4´-dinitrophenyl β-d-cellobioside (kcat = 40±1 s-1, Km = 0.35±0.03 mM, Ki = 24±4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O = 0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact = 0.024 min-1, Kreact = 54 mM). Digestion of the labelled enzyme by pepsin followed by LC–ESI–tandem MS (MS–MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI–MS–MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid–base catalyst Glu-203 are conserved in all known family-12 sequences.
40
Cooper, William C., Yi Jin e Trevor M. Penning. "Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate". Journal of Biological Chemistry 282, n. 46 (11 settembre 2007): 33484–93. http://dx.doi.org/10.1074/jbc.m703414200.
Abstract (sommario):
Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3α-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP+ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP+ controlled kcat, whereas the chemical event also contributed to this term. kcat was insensitive to [2H]NADPH, whereas Dkcat/Km and the Dklim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated kcat, whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall Keq favored reduction. The kinetic Haldane and free energy diagram confirmed that Keq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
41
Purushotham, K. R., Y. Nakagawa, M. G. Humphreys-Beher, N. Maeda e C. A. Schneyer. "Rat Parotid Gland Acinar Cell Proliferation: Signal Transduction at the Plasma Membrane". Critical Reviews in Oral Biology & Medicine 4, n. 3 (aprile 1993): 537–43. http://dx.doi.org/10.1177/10454411930040034001.
Abstract (sommario):
Galactosyltransferase (Gal Tase) is involved in a "receptor-ligand-type" interaction at the cell surface that mediates signal transduction following isoproterenol (ISO) treatment leading to acinar cell proliferation. Evidence is presented herein for the identification of the cell-surface glycoprotein signaling component. Using intact cells or isolated plasma membranes, the EGF-receptor (EGF-R) was specifically radiolabeled with [14C]-Galactose following ISO treatment. Injection of a polyclonal antibody monospecific for rat EGF-R also inhibited proliferation in a dose-dependent manner. The immunoaffinity purified receptor demonstrated altered lectin binding and increased in vitro Gal Tase substrate capacity following β-agonist treatment when compared with EGF-R isolated from control animals. When acinar cells were incubated in the presence of EGF, plasma membranes from control and ISO-treated animals showed autophosphorylation of EGF-R tyrosine moieties, transient increases in membrane associated phospholipase Cy, and increased cellular levels of cAMP. These properties of the tyrosine phosphate signaling pathway could be duplicated by the exogenous addition of bovine Gal Tase to ISO-treated cells but not control cells. The results suggest that cell surface Gal Tase interacts with a form of the EGF-R, having altered carbohydrate moieties to promote intracellular signaling for acinar cell proliferation.
42
Diaz-Bárcena, Alba, Luis Fernandez-Pacios e Patricia Giraldo. "Structural Characterization and Molecular Dynamics Study of the REPI Fusion Protein from Papaver somniferum L." Biomolecules 14, n. 1 (19 dicembre 2023): 2. http://dx.doi.org/10.3390/biom14010002.
Abstract (sommario):
REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.
43
Gómez-Melero, Sara, Fé Isabel García-Maceira, Tania García-Maceira, Verónica Luna-Guerrero, Gracia Montero-Peñalvo, Javier Caballero-Villarraso, Isaac Túnez e Elier Paz-Rojas. "Development of a High-Throughput Calcium Mobilization Assay for CCR6 Receptor Coupled to Hydrolase Activity Readout". Biomedicines 10, n. 2 (10 febbraio 2022): 422. http://dx.doi.org/10.3390/biomedicines10020422.
Abstract (sommario):
CCR6 is a chemokine receptor highly implicated in inflammatory diseases and could be a potential therapeutic target; however, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Development of a high-throughput screening assay for CCR6 should facilitate the identification of novel compounds against CCR6. To develop a cell-based assay, RBL-2H3 cells were transfected with plasmids encoding β-hexosaminidase and CCR6. Intracellular calcium mobilization of transfected cells was measured with a fluorescent substrate using the activity of released hexosaminidase as readout of the assay. This stable, transfected cell showed a specific signal to the background ratio of 19.1 with low variability of the signal along the time. The assay was validated and optimized for high-throughput screening. The cell-based calcium mobilization assay responded to the specific CCR6 ligand, CCL20, in a dose-dependent manner with an EC50 value of 10.72 nM. Furthermore, the assay was deemed robust and reproducible with a Z’ factor of 0.63 and a signal window of 7.75. We have established a cell-based high-throughput calcium mobilization assay for CCR6 receptor. This assay monitors calcium mobilization, due to CCR6h activation by CCL20, using hexosaminidase activity as readout. This assay was proved to be robust, easy to automate and could be used as method for screening of CCR6 modulators.
44
Chao, Nan, Wen-Ting Jiang, Xue-Chun Wang, Xiang-Ning Jiang e Ying Gai. "Novel motif is capable of determining CCR and CCR-like proteins based on the divergence of CCRs in plants". Tree Physiology 39, n. 12 (21 novembre 2019): 2019–26. http://dx.doi.org/10.1093/treephys/tpz098.
Abstract (sommario):
Abstract Cinnamoyl-coenzyme A reductases (CCRs) have been reported as key enzymes involved in monolignol biosynthesis. In this study, a motif-aware workflow based on a new signature motif effectively distinguished CCRs from CCR-like proteins. The divergence of CCRs and CCR-like sequences in Populus tomentosa Carr, Panicum virgatum L, Oryza sativa L and Selaginella moellendorffii Hieron suggests that NWYCY is not efficient for CCR recognition. The novel motif H202(X)2K205 (CCR-SBM or CCR substrate binding motif) was introduced to distinguish between CCRs and CCR-like proteins. The site-directed mutant R205K in Os(I)CCR-like and H202 in PtoCCR7 resulted in the rescue and loss of activity, respectively, further validating the fact that CCR-SBM is critical for maintaining CCR activity. The molecular docking using feruloyl-cinnamoyl-coenzyme A (CoA) as the ligand and binary PhCCR-NADP structures as receptors indicated an interaction between H202 and K205 with CoA moiety. The genuine CCRs and CCR-like proteins from several angiosperms and gymnosperms were screened using a motif-aware workflow and were validated using a biochemical assay. Our results suggest that the motif-aware workflow is efficient and effective for the identification of CCRs and CCR-like proteins in land plants and can be used as a more accurate way of identifying genuine CCRs among land plants.
45
Zhao, Yi, Eliud Morales Morales Dávila, Xue Li, Beiyu Tang, Ariana I. Rabinowitsch, Jose Manuel Perez-Aguilar e Carl P. Blobel. "Identification of Molecular Determinants in iRhoms1 and 2 That Contribute to the Substrate Selectivity of Stimulated ADAM17". International Journal of Molecular Sciences 23, n. 21 (24 ottobre 2022): 12796. http://dx.doi.org/10.3390/ijms232112796.
Abstract (sommario):
The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated shedding of most ADAM17 substrates tested to date can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of very few ADAM17 substrates, such as TGFα. The first transmembrane domain (TMD1) of iRhom2 and the sole TMD of ADAM17 are important for the stimulated shedding of ADAM17 substrates by iRhom2. However, little is currently known about how the iRhoms interact with different substrates to control their stimulated shedding by ADAM17. To provide new insights into this topic, we tested how various chimeras between iRhom1 and iRhom2 affect the stimulated processing of the EGFR-ligands TGFα (iRhom1- or 2-dependent) and EREG (iRhom2-selective) by ADAM17. This uncovered an important role for the TMD7 of the iRhoms in determining their substrate selectivity. Computational methods utilized to characterize the iRhom1/2/substrate interactions suggest that the substrate selectivity is determined, at least in part, by a distinct accessibility of the substrate cleavage site to stimulated ADAM17. These studies not only provide new insights into why the substrate selectivity of stimulated iRhom2/ADAM17 differs from that of iRhom1/ADAM17, but also suggest new approaches for targeting the release of specific ADAM17 substrates.
46
Alves, Krisnna M. A., Fábio José Bonfim Cardoso, Kathia M. Honorio e Fábio A. de Molfetta. "Design of Inhibitors for Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Enzyme of Leishmania mexicana". Medicinal Chemistry 16, n. 6 (7 settembre 2020): 784–95. http://dx.doi.org/10.2174/1573406415666190712111139.
Abstract (sommario):
Background:: Leishmaniosis is a neglected tropical disease and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is a key enzyme in the design of new drugs to fight this disease. Objective:: The present study aimed to evaluate potential inhibitors of GAPDH enzyme found in Leishmania mexicana (L. mexicana). Methods: A search for novel antileishmanial molecules was carried out based on similarities from the pharmacophoric point of view related to the binding site of the crystallographic enzyme using the ZINCPharmer server. The molecules selected in this screening were subjected to molecular docking and molecular dynamics simulations. Results:: Consensual analysis of the docking energy values was performed, resulting in the selection of ten compounds. These ligand-receptor complexes were visually inspected in order to analyze the main interactions and subjected to toxicophoric evaluation, culminating in the selection of three compounds, which were subsequently submitted to molecular dynamics simulations. The docking results showed that the selected compounds interacted with GAPDH from L. mexicana, especially by hydrogen bonds with Cys166, Arg249, His194, Thr167, and Thr226. From the results obtained from molecular dynamics, it was observed that one of the loop regions, corresponding to the residues 195-222, can be related to the fitting of the substrate at the binding site, assisting in the positioning and the molecular recognition via residues responsible for the catalytic activity. Conclusion:: he use of molecular modeling techniques enabled the identification of promising compounds as inhibitors of the GAPDH enzyme from L. mexicana, and the results obtained here can serve as a starting point to design new and more effective compounds than those currently available.
47
Gao, Dan, Mohamed Madi, Cherlyn Ding, Matthew Fok, Thomas Steele, Christopher Ford, Leif Hunter e Chen Bing. "Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes". American Journal of Physiology-Endocrinology and Metabolism 307, n. 3 (1 agosto 2014): E289—E304. http://dx.doi.org/10.1152/ajpendo.00430.2013.
Abstract (sommario):
Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue.
48
Takacs, Gregory, Yujia Zhou, David J. King, Amy Meacham, Loguinov Alex, Abderrahmane Tagmount, Amin Sobh, Max Russo, Chris D. Vulpe e Christopher R. Cogle. "CRISPR Dropout Screens Identify DHODH,PIK3C3, and Crkl as Potential Therapeutic Targets in Acute Myeloid Leukemia". Blood 134, Supplement_1 (13 novembre 2019): 1452. http://dx.doi.org/10.1182/blood-2019-128180.
Abstract (sommario):
Relapsed disease is still a major challenge in AML despite recently approved targeted therapies. New therapeutic targets are needed. To identify vulnerabilities in AML, screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library (DepMap) was analyzed by examining gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ). 438 gene KOs were identified as essential to AML cell survival in 14/15 AML cell lines. 247/438 (56%) genes had no active compound or approved drug interacting with its gene-products (DGIdb, canSAR Black) but were tractable targets based on 3D modeling or ligand-based druggability scoring (canSAR Black). This refined list of targets made up a new screening platform termed disKO. For clinical relevance, a primary AML specimen was treated with a custom-made lentiviral-based pooled CRISPR-Cas9 library including disKO targets using guide RNA's constructed against a parsed version of the druggable genome. Following transduction, PCR and oligo-sequencing were performed on Days 0, 7, and 14. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) was used to identify gene KOs associating with AML dropout over time. Four gene KOs from disKO were identified as essential in primary AML: NUP93, DHODH, CRKL, and PIK3C3. NUP93 loss of function mutations have been associated with steroid-resistant nephrotic syndrome in patients, suggesting inhibition may be toxic. DHODH inhibitors are currently being tested in AML. PIK3C3 codes for lipid kinases involved in autophagocytosis, and PIK3C3 inhibitors are in preclinical stage of development. CRKL is a proto-oncogene encoding src homology domains (SH2, SH3), which activate RAS and JNK signaling pathways. Although CRKL is a substrate of BCR-ABL CML, its essentiality in our study was found in AML cell lines and primary AML lacking the BCR-ABL fusion. Differential gene expression analysis showed CRKL to have lower expression in AML when compared to normal hematopoietic cells. Its druggability score based on six 3D structures and 1.3 ligand z-score indicates promising pharmacologic inhibition. In conclusion, a new CRISPR-Cas9 screening method termed disKO led to the identification of three viable targets for AML: PIK3C3, DHODH, and CRKL. Current efforts are focused on target validation followed by compound screening to find inhibitors with therapeutic potential. Disclosures No relevant conflicts of interest to declare.
49
Rieger, Leonie, Kilian Irlinger, Franziska Fuechsl, Nicolas Barbian, Marlene Tietje, Melanie Faber, Tobias Schulze et al. "Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma". Blood 142, Supplement 1 (28 novembre 2023): 3310. http://dx.doi.org/10.1182/blood-2023-187349.
Abstract (sommario):
Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM. Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138 + cells were obtained from bone marrow aspirations followed by MACS ® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified. Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138 + MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co-cultures with MM cells and primary CD138 + MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated. Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.
50
Mou, Peipei, Zhao Zeng, Lijie Ren, Qiang Li, Kenneth M. Wannemacher, Xi Mo, Lawrence F. Brass e Li Zhu. "Identification of a Region within the Cytoplasmic Domain of Sema4D That Binds Calmodulin and Regulates Shedding of the Sema4D Exodomain in Platelets",. Blood 118, n. 21 (18 novembre 2011): 3249. http://dx.doi.org/10.1182/blood.v118.21.3249.3249.
Abstract (sommario):
Abstract Abstract 3249 We have previously shown that the 150 kDa semaphorin family member, Sema4D (or CD100), is expressed on the surface of human and mouse platelets, where it is able to selectively reinforce collagen-initiated platelet activation by engaging receptors in trans on adjoining platelets in a contact-dependent manner. Key to this effect is the Sema4D extracellular domain, which in addition to being a ligand for Sema4D receptors, is a substrate for the metalloprotease, ADAM17 in platelets. Our previous studies suggest that ADAM17 cleaves Sema4D and other platelet surface proteins close to the platelet plasma membrane gradually producing, in the case of Sema4D, a single large (≈120 kDa) exodomain fragment and a smaller (≈28 kDa) fragment that includes the transmembrane domain and the cytoplasmic domain and remains associated with the platelet (Zhu, et al., PNAS 2007). Exodomain shedding in platelets can be triggered by the phorbol ester, PMA, and by physiologic agonists such as thrombin that raise the cytoplasmic Ca++ concentration, but the mechanisms that regulate the shedding of Sema4D have not been defined. Here we have studied the potential role of an interaction between calmodulin and the Sema4D cytoplasmic domain. Using a public resource (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html), we identified a potential calmodulin-binding sequence (GYLPRQCLKFRSALLIGKKKPKS-COOH, Gly758–Ser780) within the membrane-proximal region of the Sema4D cytoplasmic domain. To test whether this region binds calmodulin, a 23 amino acid peptide corresponding to the predicted Sema4D calmodulin binding sequence (SCBP) was synthesized, as was a scrambled control peptide (RLIKACRQPKPKYKLLGFGSSKL or scrambled SCBP), which is not predicted to bind calmodulin. The results show that SCBP, but not scrambled SCBP, was able to bind to calmodulin-agarose and retrieve calmodulin from platelet lysates. As constitutive association of calmodulin with glycoprotein (GP) Ib has been shown prevent ADAM17-dependent GPIb alpha shedding in platelets, we incubated human platelets with the calmodulin inhibitor, W7. The inhibitor induced gradual Sema4D shedding that was detectable after 5 min and reached a maximum at 60 min, kinetics that are similar to those we have observed with platelet agonists. However, in contrast to platelet agonists, W7-induced Sema4D shedding generated a smaller retained fragment (≈24 kDa Vs. 28 kDa) suggesting that there is either a second or different site of cleavage. Despite their polybasic sequences, flow cytometry and confocal microscopy showed that FITC-conjugated SCBP and scrambled-SCBP are able to cross the plasma membrane. Addition of SCBP, but not scrambled-SCBP, to platelet caused cleavage of Sema4D, producing the same 28 kDa fragment observed with thrombin and PMA. In all cases cleavage of Sema4D was blocked by the metalloprotease inhibitor, TAPI-2. Combined with our earlier observations, these results suggest that 1) Sema4D is a calmodulin binding protein with a site of interaction in the membrane-proximal cytoplasmic domain and a site of cleavage by ADAM17 in the membrane-proximal exodomain, 2) the detachment of calmodulin from Sema4D may be the trigger for Sema4D cleavage in response to platelet agonists, and 3) in contrast to W7, decoying calmodulin from binding sites on Sema4D and other metalloprotease substrates on the platelet surface, as we have done here with a Sema4D cytoplasmic domain peptide, may trigger the same events seen in activated platelets and provide a tool to understand the underlying mechanisms. Disclosures: No relevant conflicts of interest to declare.