scholarly journals Binding Site Analysis of c-Myb: Screening of Potential Binding Sites by Using the Mutation Matrix Derived from Systematic Binding Affinity Measurements

1996 ◽  
Vol 24 (4) ◽  
pp. 766-774 ◽  
Author(s):  
Q.-L. Deng ◽  
S. Ishii ◽  
A. Sarai
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Site Analysis ◽  
Mutation Matrix ◽  
Potential Binding

scholarly journals DNA Binding Site Selection of Dimeric and Tetrameric Stat5 Proteins Reveals a Large Repertoire of Divergent Tetrameric Stat5a Binding Sites

2000 ◽  
Vol 20 (1) ◽  
pp. 389-401 ◽  
Author(s):  
Elisabetta Soldaini ◽  
Susan John ◽  
Stefano Moro ◽  
Julie Bollenbacher ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Site Selection ◽  
Binding Sites ◽  
Tetramer Binding ◽  
Dna Binding Site ◽  
Potential Binding ◽  
Wide Range ◽  
Large Repertoire ◽  
Selection Of

ABSTRACT We have defined the optimal binding sites for Stat5a and Stat5b homodimers and found that they share similar core TTC(T/C)N(G/A)GAA interferon gamma-activated sequence (GAS) motifs. Stat5a tetramers can bind to tandemly linked GAS motifs, but the binding site selection revealed that tetrameric binding also can be seen with a wide range of nonconsensus motifs, which in many cases did not allow Stat5a binding as a dimer. This indicates a greater degree of flexibility in the DNA sequences that allow binding of Stat5a tetramers than dimers. Indeed, in an oligonucleotide that could bind both dimers and tetramers, it was possible to design mutants that affected dimer binding without affecting tetramer binding. A spacing of 6 bp between the GAS sites was most frequently selected, demonstrating that this distance is favorable for Stat5a tetramer binding. These data provide insights into tetramer formation by Stat5a and indicate that the repertoire of potential binding sites for this transcription factor is broader than expected.

Abstract P622: Characterization of 125-I-Angiotensin (1-7) Binding to Rat Kidney

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Filipe F Conti ◽  
Andrea Linares ◽  
Leena E Couling ◽  
Mariana Morris ◽  
Katia De Angelis ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Binding ◽  
Rat Kidney ◽  
Albino Rats ◽  
Angiotensin Peptides ◽  
Putative Receptor ◽  
Wet Weight

Despite the plethora of data indicating beneficial effects of angiotensin (1-7) (Ang 1-7) on the cardiovascular system, its putative receptor, Mas, has not been characterized in tissue membrane preparations other than single concentration demonstrations of the localization of 125 I-Ang 1-7 binding sites in rat kidney. This does not indicate the specificity of 125 I-Ang 1-7 binding nor does it indicate the actual densities of the binding sites, i.e., B max (fmoles/mg tissue), or dissociation constant (K D ) to indicate binding affinity of 125 I-Ang 1-7 for its putative receptor. To characterize 125 I-Ang 1-7 binding in the kidney we prepared a low specific activity, monoradioiodinated Ang 1-7 using a 1:19 mix of 125 iodine : 127 iodine which allows for assessment of the B max and K D with concentrations of radioligand up to 100 nM. Frozen kidneys from adult male albino rats were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:MgCl 2 (50:1) pH 7.2 and incubated with 12 concentrations of 125/127 I-Ang 1-7 ranging from ~3-100 nM for 30 min at 22 C, after which bound 125/127 I-Ang 1-7 was resolved from unbound 125/127 I-Ang 1-7 by filtration and measured with a gamma counter. Specific binding (defined as 100 μM Ang 1-7 displaceable binding) of 125/127 I-Ang 1-7 showed a moderate binding affinity (K D = 14.7 ± 1.8 nM) and binding site density (B max = 24.5 ± 9.9 fmoles/mg initial wet weight). The B max value tended to be lower than that in the liver (B max = 62.3 ± 20.1 fmoles/mg initial wet weight) and the K D value was significantly greater (lower affinity) than that in the liver ( K D = 5.7 ± 0.6 nM, p = 0.0085). Of note, competition for 125/127 I-Ang 1-7 binding Ang 1-7 indicated that the IC 50 for Ang 1-7 competition for 125/127 I-Ang 1-7 binding was 42.5 μM. Moreover, the ability of a variety of angiotensin peptides to inhibit 125/127 I-Ang 1-7 binding at 100 μM, Ang 1-7 was less potent that the other angiotensin peptides: Ang III > Ang II > Ang I ~ Ang IV > Ang 2-7 > Ang 1-7 ~ Ang 3-7. These studies suggest that the binding site for 125/127 I-Ang 1-7 is not specific for the putative Ang 1-7 receptor mas, and may represent a low affinity binding to the AT 1 or AT 2 receptor

Mir-590 Is a Novel STAT5 Regulated Oncogenic miRNA and Targets FasL In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3811-3811 ◽  
Author(s):  
Amanda J Favreau ◽  
Fariba Shaffiey ◽  
Erin Cross ◽  
Pradeep Sathyanarayana
Keyword(s):  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Functional Role ◽  
Luciferase Assay ◽  
Potential Binding ◽  
Flt3 Mutations ◽  
Acute Myeloid

Abstract The recent discovery of new molecular lesions with prognostic significance in acute myeloid leukemia (AML) is enhancing our understanding of leukemia biology and our ability to identify new therapeutic targets. Previously, using the unique leukemic myeloid progenitor line AML-193, we profiled IL-3-, GM-CSF-, and G-CSF-regulated miRNA signatures. 301 miRNAs were commonly regulated by these three cytokines, and the most highly induced miRNA was miR-590-5p. Herein, we have attempted to define the functional role and clinical relevance of miR-590 in AML. We first examined the relative miR-590 expression in steady state hematopoiesis and showed it was highest at CD34+ and declined its expression through myeloid lineage differentiation (ANOVA, p<0.0001). To functionally determine the role of increased miR-590 expression, we generated a gain-of-function model in human CD34+ hematopoietic stem cells (HSC) via lentivirus transduction. Increased expression of miR-590 in CD34+ cells resulted in significant increases in CFU-GM colonies, strongly suggesting that dysregulation of miR-590 expression may be myeloproliferative. In AML (n=33) and control (n=9) bone marrow samples, miR-590 expression was determined via RT-qPCR. miR-590-5p expression was highly upregulated in 22 of the samples (67%) compared to control subjects. In silico analysis of the miR-590-5p promoter revealed three potential binding sites for STAT5 (-249, -749, -1499). To functionally determine whether STAT5 directly regulates miR-590-5p expression, we performed a ChIP assay, which showed that STAT5 binds to the -749 region of miR-590-5p promoter. To conclusively determine the STAT5 binding sites, we cloned the miR-590 promoter in a luciferase vector and performed site directed mutagenesis for each potential binding site. This assay confirmed that the -749 binding site was the major STAT5 regulatory site for miR-590 (p<0.002). Importantly, constitutive activation of STAT5 is a hallmark of AML associated with FLT3 mutations, therefore, we set out to determine if specific STAT5 and FLT3 inhibitors could decrease miR-590 expression. We pretreated MV4-11 cells, which harbors the FLT-ITD mutation and has increased STAT5 activation, with 100uM STAT5 inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) for 90 minutes or 100nM FLT3 inhibitor (EMD Millipore, 343020) for 12 hours, both of which resulted in significant inhibition of miR-590-5p expression (p<0.05). To evaluate whether the AML samples with high miR-590 expression also possess elevated phospho-STAT5 or phospho-FLT3 levels, we performed immunohistochemistry analysis on a custom-made tissue microarray. In AML samples with high miR-590 levels, increased activation of FLT3 and STAT5 was observed compared to controls. Since FLT3 mutations result in decreased survival and poorer prognosis in AML, it may be that miR-590-5p plays an important role in the pathology of AML associated with dysregulated FLT3 and STAT5. To understand the complete functional role of miR-590 in AML, the predicted targets need to be identified and validated for their roles in leukemogenesis. Upon molecular screening of several predicted targets, FasL was experimentally found to be a conserved target of miR-590. More specifically, 3’UTR analysis of FasL revealed three potential seed sequences for miR-590 which have been verified experimentally via luciferase assay. Furthermore, significantly increased levels of FasL protein and transcript expression was detected in the MV4-11 cells stably expressing anti-miR-590 compared to control cells. Additionally, we identified the levels of Fas/CD95 (FasL receptor) on AML-193 and MV4-11 cell lines and found these cells had high Fas/CD95 expression on the cell surface as analyzed via flow cytometry. In order to determine the physiological significance of Fas/FasL, these cells were treated with soluble FasL (100ng) for 24 hours and apoptosis was analyzed via Annexin V staining. FasL treatment induced increased apoptosis compared to the untreated cells. Taken together, we have identified miR-590 as a candidate oncomiR that is regulated via the STAT5 pathway and targets FasL to promote cell survival. Thus, our data suggests that further understanding of miR-590’s role in AML may lead to development of novel anti-miR-590 therapeutic strategies in AML associated with dysregulated STAT5. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interaction of unsaturated fatty acids with anti-oestrogen-binding sites

1987 ◽  
Vol 243 (2) ◽  
pp. 359-364 ◽  
Author(s):  
P L H Hwang
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Unsaturated Fatty Acids ◽  
Biological Significance ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Endogenous Ligands

Specific high-affinity binding sites for non-steroidal anti-oestrogens such as tamoxifen have been identified in many animal and human tissues. The function of these binding sites and the nature of their endogenous ligands are currently unknown. Our laboratory has previously reported that unsaturated fatty acids at micromolar concentrations inhibited [3H]tamoxifen binding to the anti-oestrogen-binding sites in rat liver, raising the possibility that fatty acids might represent endogenous ligands for these sites. These studies have now been extended to examine the mechanism by which fatty acids inhibit [3H]tamoxifen binding to the anti-oestrogen-binding site. Saturation analysis revealed that increasing concentrations of oleic acid progressively decreased the apparent binding affinity of these sites for [3H]tamoxifen without decreasing the total number of binding sites; however, the apparent dissociation constant did not vary linearly with the prevailing oleic acid concentration, suggesting that the inhibition of [3H]tamoxifen binding by fatty acid was not competitive in nature. Kinetic studies of [3H]tamoxifen binding showed that oleic acid did not affect the rate of association, but increased the rate of dissociation of [3H]tamoxifen from the anti-oestrogen-binding site; the latter finding would not be expected if oleic acid acted as a competitive inhibitor. Furthermore, incubation of a rat microsomal fraction with [3H]oleic acid in the absence and presence of excess non-radioactively labelled tamoxifen also failed to demonstrate direct competition between oleic acid and tamoxifen for the same binding site. It is concluded that oleic acid, and presumably other unsaturated fatty acids, do not compete for the anti-oestrogen-binding site and probably reduce its tamoxifen-binding affinity by some other mechanism, such as perturbation of the lipid environment of the binding site. The biological significance of this interaction of unsaturated fatty acids with the anti-oestrogen-binding site remains to be elucidated.

Protein–carbohydrate complexes: Binding site analysis, prediction, binding affinity and molecular dynamics simulations

2020 ◽  
pp. 299-332 ◽  
Author(s):  
K. Veluraja ◽  
N. R. Siva Shanmugam ◽  
J. Jino Blessy ◽  
R. A. Jeyaram ◽  
B. Lalithamaheswari ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Binding Site ◽  
Binding Affinity ◽  
Site Analysis ◽  
Dynamics Simulations ◽  
Analysis Prediction

scholarly journals In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 453
Author(s):  
Jiayi Yuan ◽  
Chen Jiang ◽  
Junmei Wang ◽  
Chih-Jung Chen ◽  
Yixuan Hao ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Cannabinoid Receptors ◽  
Md Simulations ◽  
Docking Studies ◽  
Allosteric Modulators ◽  
3D Structures ◽  
Potential Binding ◽  
Allosteric Sites ◽  
Allosteric Binding Sites

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Abstract 46: Differential Gene and Transcription Factor Binding Site Expression Between the Intima and Media Drives Intimal Hyperplasia

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Kenneth DeSart ◽  
Zhihua Jiang ◽  
Ming Tao ◽  
Kerri O'Malley ◽  
Scott Berceli
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Cell Mass ◽  
Active Phase ◽  
Time Frame ◽  
Site Analysis ◽  
Vein Grafts ◽  
Whole Genome Microarray ◽  
And Migration ◽  
Cycle Regulation

Background: Vein grafts (VG) fail due to rapid intimal expansion in the weeks to months following implantation. Histologically, this results in a metabolically active intima juxtaposed to a quiescent medial layer. In this study, we leverage this dichotomy to identify the key genes that control intimal growth and the critical transcription factors (TF) that drive their activation. Methods: Microsurgical techniques were used to separate the intima and media of rabbit carotid VGs harvested at 1 (n=4), 3 (n=6), and 6 (n=4) months (m) following implantation. Each layer was independently analyzed for differential mRNA expression using a whole-genome microarray. rVista and TRANSFAC were used to identify TF binding sites within 5000 bp of differentially expressed genes. Results: Early growth of the intima was driven by an enhanced proliferative response and an accumulation of cell mass (Fig). During this early hyperplastic phase, substantial differences in intimal and medial gene expression were observed (197 genes at 1m and 346 at 3m, p<0.001) that resolved by 6m (8 genes). Genes involved in cell cycle regulation and cellular movement dominated the 1 and 3m differences (Fig). TF binding site analysis of up-regulated intimal genes at 1 month revealed enrichment of ETS and HOX, while CREB and bHLH-ZIP were the most over-represented among up-regulated 3m intimal genes. In the medial layer, RXR and zinc finger TF binding sites were most prominent among up-regulated genes at 1 and 3m, respectively. Conclusion: Corresponding to the most active phase of intimal growth, smooth muscle cells demonstrated marked up-regulation of an array of genes that modulate cell proliferation and migration, with these genes under the control of a discrete set of TFs. While previous attempts at modifying TF binding (via an E2F decoy) in the initial days following VG implantation have been unsuccessful, manipulation of a broader set of TFs in the time frame of weeks to months may prove a more promising strategy.

Modulation of Fox-Regulated Alternative Splicing Events during Erythropoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 142-142
Author(s):  
Thomas L. Gallagher ◽  
Sherry L. Gee ◽  
Christina L. Schluepen ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Dependent Manner ◽  
Enhancer Activity ◽  
Tissue Specific ◽  
High Affinity ◽  
Splicing Efficiency ◽  
Protein 4.1R

Abstract A switch in the alternative splicing pattern in protein 4.1R pre-mRNA, involving activation of exon 16 splicing, occurs in late erythropoiesis. Because the inclusion of exon 16 leads to synthesis of protein isoforms with high affinity for spectrin and actin, and mechanical strengthening of the membrane, this switch is critical for normal erythyroid differentiation. Previous studies have shown that Fox-2 protein is a splicing regulator that binds to intronic enhancer sequences downstream of exon 16, and that this binding stimulates the inclusion of exon 16. Fox binding sites are also located downstream of other tissue-specific alternative exons, some of which exhibit variable splicing efficiencies, leading us to investigate whether splicing switch strength is influenced by variations in the sequence of the Fox binding-site motif (UGCAUG) and by the number of these protein binding sites. A series of minigenes was created in which protein 4.1R E16 splicing efficiency was measured in transfection studies. Mutation of the first U residue of the Fox binding site resulted in a significantly weaker, but still Fox dependent activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. Insertion of two wild-type UGCAUG elements enhanced splicing substantially in a Fox protein concentration-dependent manner, and four elements gave even stronger inclusion. To test whether the reduced splicing efficiency observed for minigenes containing mutated enhancer elements was due to a reduction in Fox binding, surface plasmon resonance (SPR) was employed. Our binding studies indicate that full-length mouse Fox-2a bound to the UGCAUG sequence with high affinity (KD ∼100nM). Furthermore, in agreement with our functional splicing assays, Fox binding affinity for the hexameric sequence was reduced with mutations in position one, and almost eliminated with mutations in position six. There was an excellent correlation between binding affinity of Fox protein for the enhancer motif, and strength of enhancer activity measured in functional splicing assays. Additional studies demonstrated that Fox enhancer activity can be reduced by the presence of a closely linked binding site for hnRNP A1, a known splicing inhibitor. Taken together, these studies demonstrate that the composition and number of binding sites for Fox can significantly affect the efficiency of splicing for E16. It is likely that through these combinatorial mechanisms, Fox splicing factors are able to modulate the efficiency with which tissue specific exons in the erythroid alternative splicing program can be spliced into mature mRNA.

VEGF Mediated Regulation of KCl Cotransporters Gene Expression in Erythroid Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3163-3163
Author(s):  
Caryn S Gonsalves ◽  
Scott C Crable ◽  
Sharat Chandra ◽  
Clinton H. Joiner
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Promoter Activity ◽  
K562 Cells ◽  
Vegf Receptor ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Potential Binding ◽  
In Erythroid Cells

Abstract Abstract 3163 The KCl co-transporter (KCC) family of proteins catalyzes the electroneutral, coupled movement of K+ and Cl− ions across the plasma membrane, thereby mediating transepithelial ion transport and regulating cell volume. These proteins play an important role in disease states such as cancer, numerous neurological conditions as well as sickle cell disease (SCD). KCC activity is increased in sickle red blood cells and contributes to their dehydration, which potentiates sickling. The mechanisms of increased KCC activity and its abnormal regulation are not understood. Of the four mammalian KCC isoforms, KCC 1, 3 and 4 are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Hiki et. al. showed that the angiogenic factor vascular endothelial growth factor (VEGF) increased KCC 3a expression in HUVEC cells (J.B.C. 274, 10661–10667, 1999). As levels of VEGF and related family member, placenta growth factor (PlGF) are elevated in sickle cell patients, we hypothesized that VEGF and PlGF may influence KCC expression in erythroid cells. RT-PCR revealed that erythroid K562 cells expressed the VEGF receptor-1 (VEGF-R1, or Flt-1) but not VEGF receptor-2, (VEGF-R2 or Flk-1). Additionally, flow cytometric analysis of WT C57Bl6 mouse bone marrow showed the presence of the Flt-1 receptor, but not Flk-1 or Flk-3 in erythroid progenitors and expression decreased with maturation. VEGF treatment (50 ng/ml) of K562 cells increased KCC 1, 3a, 3b and 4 mRNA levels; PlGF treatment increased KCC 1, 3a and 4 mRNA levels but not KCC 3b. The VEGF receptor inhibitor, SU5416, ablated the effect of VEGF. VEGF-stimulated KCC 4 expression was blocked by pharmacological inhibitors that implicated PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), as with other VEGF effects. Analysis of the KCC 4 promoter showed that the −875 and −90 bp promoter luciferase constructs exhibited similar levels of activity as the −1200 bp promoter construct, when compared to the promoterless reporter plasmid. Deleted constructs corresponding to −65 bp from transcription start site showed ∼90% reduced promoter activity. In silico analysis of the −90 bp region of the KCC 4 promoter showed potential binding sites for transcription factor SP-1 and HIF-1α. Binding sites for transcription factor SP-1 at positions −35 to −44 bp and −56 to −64 bp were shown to be active by site directed mutagenesis. Mutation of the HIF-1α binding site at −73 to −76 bp significantly inhibited promoter activity, whereas mutation of the HIF-1α binding site at position −21 bp to −18 bp did not have any effect on activity. Similar analysis of the KCC 3a promoter indicate potential binding sites for SP-1 at positions −8 to −4 bp and a HIF-1α binding site at position −23 to −20 bp, and the KCC 3b promoter has binding sites for HIF-1α at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at position −13 to −10 bp. Luciferase assays with KCC 3b promoter constructs indicated that the −190 bp promoter region containing HIF-1α sites at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at −13 to −10 bp contained minimal promoter required for transcription activity. Mutations within both HIF-1α binding sites attenuated promoter activity indicating a role for HIF-1α in regulating KCC 3b activity, as well. EMSA and ChIP assays with the KCC 4 promoter demonstrated that VEGF treatment of K562 cells increased HIF-1α binding to the HIF-1α sites, which was abrogated by mutating these sites. Similar results were obtained for the KCC 3a and 3b promoters.These results suggest that activation of VEGF-R1 by VEGF, and presumably its other ligand, PlGF, leads to non-hypoxic activation of HIF-1α and SP-1-mediated up-regulation of KCC3a, 3b and 4 expressions in erythroid K562 cells via its canonical signaling pathways. Variation in KCC gene expression and its modulation by cytokines and growth factors may be a source of inter-individual variation in SS RBC volume regulation and thus of phenotypic variability of SCD. Identifying the factors that modulate transcriptional control of KCC expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC. Disclosures: No relevant conflicts of interest to declare.

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