scholarly journals Interactions of ATP, oestradiol, genistein and the anti-oestrogens, faslodex (ICI 182780) and tamoxifen, with the human erythrocyte glucose transporter, GLUT1

2002 ◽  
Vol 365 (3) ◽  
pp. 707-719 ◽  
Author(s):  
Iram AFZAL ◽  
Philip CUNNINGHAM ◽  
Richard. J. NAFTALIN
Keyword(s):  
Binding Site ◽  
Oestrogen Receptor ◽  
Glucose Transporter ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Atp Binding ◽  
Binding Motif ◽  
Transmembrane Helices ◽  
Selective Oestrogen Receptor Modulators ◽  
Dependent Inhibition

17β-Oestradiol (ED when subscript to K) and the phytoestrogen isoflavone genistein (GEN) inhibit glucose transport in human erythrocytes and erythrocyte ghosts. The selective oestrogen receptor modulators or anti-oestrogens, faslodex (ICI 182780) (FAS) and tamoxifen (TAM), competitively antagonize oestradiol inhibition of glucose exit from erythrocytes (Ki(ED/FAS) = 2.84±0.16μM and Ki(ED/TAM) = 100±2nM). Faslodex has no significant inhibitory effect on glucose exit, but tamoxifen alone inhibits glucose exit (Ki(TAM) = 300±100nM). In ghosts, ATP (1–4mM) competitively antagonizes oestradiol, genistein and cytochalasin B (CB)-dependent inhibitions of glucose exit, (Ki(ATP/ED) = 2.5±0.23mM, Ki(ATP/GEN) = 0.99±0.17mM and Ki(ATP/CB) = 0.76±0.08mM). Tamoxifen and faslodex reverse oestradiol-dependent inhibition of glucose exit with ATP>1mM (Ki(ED/TAM) = 130±5nM and Ki(ED/FAS) = 2.7±0.9μM). The cytoplasmic surface of the glucose transporter (GLUT)1 contains four sequences with close homologies to sequences in the ligand-binding domain of human oestrogen receptor β (hesr-2). One homology is adjacent to the Walker ATP-binding motif II (GLUT1, residues 225–229) in the large cytoplasmic segment linking transmembrane helices 6 and 7; another GLUT (residues 421–423) contains the Walker ATP-binding motif III. Mapping of these regions on to a three-dimensional template of GLUT indicates that a possible oestrogen-binding site lies between His337, Arg349 and Glu249 at the cytoplasmic entrance to the hydrophilic pore spanning GLUT, which have a similar topology to His475, Glu305 and Arg346 in hesr-2 that anchor the head and tail hydroxy groups of oestradiol and genistein, and thus are suitably placed to provide an ATP-sensitive oestrogen binding site that could modulate glucose export.

scholarly journals The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis

2004 ◽  
Vol 378 (3) ◽  
pp. 793-799 ◽  
Author(s):  
Xueji WU ◽  
Mihiro YANO ◽  
Hiroyo WASHIDA ◽  
Hiroshi KIDO
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Site ◽  
Metal Binding ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Binding Motif ◽  
Metal Binding Site

The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP–ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403–414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.

Calcium control of macrophage cytoplasmic gelation: evidence for the involvement of the 70,000 Mr actin-bundling protein

1987 ◽  
Vol 88 (1) ◽  
pp. 81-94
Author(s):  
M. Pacaud ◽  
M.C. Harricane
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Supernatant Fraction ◽  
Actin Networks ◽  
Dependent Inhibition ◽  
Section Electron ◽  
Filament Bundles ◽  
Gel Network

Under appropriate conditions macrophage cytosolic extracts can form a three-dimensional gel network of cross-linked actin filaments. These cytoplasmic gels are mainly composed of actin, filamin, alpha-actinin, and two new proteins of about 70,000 and 55,000 Mr (70 and 55 K). The behaviour of 70 K protein was found to be remarkably affected by Ca2+. Ca2+ treatment of isolated cytoplasmic gels led to the selective solubilization of the 70 K protein along with a 17 K polypeptide. Half-maximal recovery in the supernatant fraction was obtained from about 0.15 microM free Ca2+. The cytoplasmic gel constituents solubilized in high ionic strength buffer were able to re-assemble into an insoluble actin network when returned to near physiological ionic conditions. However, the inclusion of micromolar Ca2+ prevented the re-association of 70 K protein with actin in these complexes. As compared to the 70 K protein, alpha-actinin was fully resistant to any variations in Ca2+ concentrations. On the other hand, purified 70 K protein displayed the property of assembling actin filaments into bundles at low Ca2+ concentrations (less than 0.15 microM). However, the bundling activity decreased progressively at higher Ca2+, as detected by co-sedimentation and electron microscopy of the 70 K protein-actin mixtures. Half-maximal inhibition was observed at about 0.3 microM free Ca2+. Re-assembly of actin filaments into bundles occurred after chelation of Ca2+ by EGTA, indicating that the inhibitory effect of Ca2+ was reversible. Severing of actin filaments by 70 K protein was not observed in any of the solution conditions used. The Ca2+-dependent inhibition of the ability of 70 K protein to interact with actin networks resulted in a marked distortion of the overall organization of actin filaments, as revealed by thin-section electron microscopy of cytoplasmic gels formed in the presence and absence of Ca2+. Large zones of oriented bundles of filaments were replaced by a looser mesh. When the actin gel constituents were re-assembled in the presence of Ca2+ and exogenous gelsolin, it was also observed that the filament bundles (essentially generated by alpha-actinin) collapsed into dense aggregates. Furthermore, gelsolin did not significantly affect the ability of actin to re-combine with other proteins. The data presented here and previously led us to suspect that the Ca2+ control of the functional state of 70 K protein might be one of the prime factors in the triggering of rapid assembly and disassembly of microfilaments within macrophages.

Hybrid rotors in F1Fo ATP synthases: subunit composition, distribution, and physiological significance

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1031-1042 ◽  
Author(s):  
Karsten Brandt ◽  
Volker Müller
Keyword(s):  
Binding Site ◽  
Amino Acid Level ◽  
Ion Binding ◽  
Binding Motif ◽  
Transmembrane Helices ◽  
Acetobacterium Woodii ◽  
Composition Distribution ◽  
Gene Encoding ◽  
Acetogenic Bacterium ◽  
Encoding Genes

Abstract The c ring of the Na+ F1Fo ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE1, atpE2 and atpE3. Subunit c1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c2 and c3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid FoVo c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F1Fo ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one Fo- and one Vo-type c subunit in one operon. Their c subunits have the conserved binding motif for Na+. The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed.

The three-dimensional structure of MAP kinase p38β: different features of the ATP-binding site in p38β compared with p38α

2009 ◽  
Vol 65 (8) ◽  
pp. 777-785 ◽  
Author(s):  
Sangita B. Patel ◽  
Patricia M. Cameron ◽  
Stephen J. O'Keefe ◽  
Betsy Frantz-Wattley ◽  
Jed Thompson ◽  
...  
Keyword(s):  
Map Kinase ◽  
Binding Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Atp Binding ◽  
Three Dimensional Structure ◽  
Atp Binding Site

scholarly journals Novel Biphenyl Amines Inhibit Oestrogen Receptor (ER)-α in ER-Positive Mammary Carcinoma Cells

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 783
Author(s):  
Basappa Basappa ◽  
Baburajeev Chumadathil Pookunoth ◽  
Mamatha Shinduvalli Kempasiddegowda ◽  
Rangappa Knchugarakoppal Subbegowda ◽  
Peter E. Lobie ◽  
...  
Keyword(s):  
Cell Viability ◽  
Mammary Carcinoma ◽  
Oestrogen Receptor ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Structural Basis ◽  
Dependent Manner ◽  
Luciferase Reporter Construct ◽  
Ic50 Value

Herein, the activity of adamantanyl-tethered-biphenyl amines (ATBAs) as oestrogen receptor alpha (ERα) modulating ligands is reported. Using an ERα competitor assay it was demonstrated that ATBA compound 3-(adamantan-1-yl)-4-methoxy-N-(4-(trifluoromethyl) phenyl) aniline (AMTA) exhibited an inhibitory concentration 50% (IC50) value of 62.84 nM and demonstrated better binding affinity compared to tamoxifen (IC50 = 79.48 nM). Treatment of ERα positive (ER+) mammary carcinoma (MC) cells (Michigan Cancer Foundation-7 (MCF7)) with AMTA significantly decreased cell viability at an IC50 value of 6.4 μM. AMTA treatment of MC cell-generated three-dimensional (3D) spheroids resulted in significantly decreased cell viability. AMTA demonstrated a superior inhibitory effect compared to tamoxifen-treated MC cell spheroids. Subsequently, by use of an oestrogen response element (ERE) luciferase reporter construct, it was demonstrated that AMTA treatment significantly deceased ERE transcriptional activity in MC cells. Concordantly, AMTA treatment of MC cells also significantly decreased protein levels of oestrogen-regulated CCND1 in a dose-dependent manner. In silico molecular docking analysis suggested that AMTA compounds interact with the ligand-binding domain of ERα compared to the co-crystal ligand, 5-(4-hydroxyphenoxy)-6-(3-hydroxyphenyl)-7- methylnaphthalen-2-ol. Therefore, an analogue of AMTA may provide a structural basis to develop a newer class of ERα partial agonists.

Inhibition of the Activities of α2-Plasmin inhibitor (PI) and CI inhibitor (CI-Inh) by the Synthetic Fibrinolytic Agent, 3-Hydroxypropyl Flufenamide (Flu-HPA)

1979 ◽  
Author(s):  
L Miles ◽  
J Burnier ◽  
M Verlander ◽  
M Goodman ◽  
A Kleiss ◽  
...  
Keyword(s):  
Plasminogen Activators ◽  
Inhibitory Effect ◽  
Mechanisms Of Action ◽  
Flufenamic Acid ◽  
Clot Lysis ◽  
Factor Xiia ◽  
Dependent Inhibition ◽  
Normal Human ◽  
Plasmin Inhibitor

Flu-HPA is one of a series of flufenamic acid derivations that enhances plasminogen-dependent clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The influence of Flu-HPA on the inhibition of purified plasmin by purified PI was studied. PI activity was assessed by its inhibition of the clevage of the tripeptide S-2251 (H-D-Val-Leu-Lys-pNA) by plasmin. Flu-HPA was dissolved in DMF or in methonol and preincubated with PI before addition of plasmin. At Flu-HPA concentrations greater than 1mM and up to 60mM, the inhibitory activity of PI was totally lost. The inhibitory effect of normal human plasma on plasmin was also completely abolished at concentrations of Flu-HPA between 2.5 and 40mM. The effect of Flu-HPA on the inhibition of purified plasma kallikrein by purified CI-Inh was also studied. CI-Inh activity was measured by its inhibition of cleavage of the tripeptide Bz-Pro-Phe-Arg-pNA by kallikrein. When Flu-HPA, dissolved in DMF or in methonol, was preincubated with CI-Inh, a concentration dependent inhibition of CI-Inh activity was observed. CI-Inh activity was abolished by concentrations of Flu-HPA greater than 1mM. Flu-HPA also inhibited the activity of CI-Inh on purified Factor XIIa. These observations suggest that this flufenamic acid derivative may enhance fibrinolysis not only by inhibiting PI activity but also by decreasing the inactivation of plasminogen activators by CI-Inh.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

2020 ◽  
Vol 21 (2) ◽  
pp. 117-130 ◽  
Author(s):  
Mohammad J. Hosen ◽  
Mahmudul Hasan ◽  
Sourav Chakraborty ◽  
Ruhshan A. Abir ◽  
Abdullah Zubaer ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Binding Site ◽  
Glucose Transporter ◽  
Substrate Binding ◽  
Mutation Screening ◽  
Homology Model ◽  
Connective Tissue Disorder ◽  
Crystallographic Structure ◽  
Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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