scholarly journals Deciphering the TLT-1 Fibrinogen Ligand Interaction

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4207-4207
Author(s):  
Siobhan Laken Branfield ◽  
Xariana D Valez ◽  
José A González Feliciano ◽  
Pearl Akamine ◽  
Laura Campbell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Complex Formation ◽  
Molecular Interaction ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Clot Formation ◽  
Ligand Interaction ◽  
Platelet Receptor ◽  
Competitive Kinetics ◽  
Kd Values

Abstract Background: Platelets, derived from megakaryocytes primarily play a central role in thrombosis and hemostasis, however, they extend beyond this role as immune cells that initiate and accelerate various vascular inflammatory conditions. Upon activation, platelets release TREM-Like Transcript-1 (TLT-1) from their a-granules onto their surface. Early studies by amino link columns preloaded with soluble TLT-1 followed by mass spectrometry and immunoblotting identified fibrinogen as a ligand for TLT-1. Fibrinogen is a plasma protein that is essential for clot formation, during inflammation and hypercoagulable states tissue deposition and plasma concentration of fibrinogen are increased, demonstrating a role of fibrinogen in both thrombosis and inflammation. TLT-1 binding fibrinogen was a surprising discovery since αIIbβ3, the most abundant platelet receptor, also binds fibrinogen and facilitates platelet aggregation. It is difficult to understand why there are two platelet specific receptors that have the same ligand, drawing us to question what the difference in function between the two is? Our studies suggest that although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory setting like αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Very little is known about the TLT-1-Fibrinogen interaction, further studies would set the stage for a better understanding as to why two fibrinogen ligands exist on platelets and potentially outline a novel platelet therapeutic target during hypercoagulable and/or hyperinflammatory states. We set out to determine the binding affinity and localize the binding sites for the TLT-1 fibrinogen molecular interaction. Aims: Delineate the TLT-1 fibrinogen molecular interaction and elucidate the mechanism by which this interaction drives inflammation and thrombosis-hemostasis. Methods: To confirm the TLT-1 fibrinogen ligand interaction we carried out a kinetics assay using an Octet Qk e Bio-layer Interferometry (BLI) that measures biomolecular complex formation in real time. The TLT-1 Chimera was captured onto an Anti-Human Fc Capture (AHC) Biosensor, washed in kinetics buffer to limit nonspecific binding and submerged in a 96 well plate containing varying concentrations of Fibrinogen. To localize the exact binding sites for this molecular interaction, we digested fibrinogen using trypsin and carried out an immunoprecipitation (IP) followed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). Results: The curve (Figure 1) shows that the TLT-1 fibrinogen interaction has increasing bimolecular complex formation with increases in concentration of Fibrinogen (15.625nM - 250nM), with a concentration of 250nM showing the best bicomplex formation. In the control well with HIV01 4E10 capture, reference and sensor well, no bicomplex formation is shown, highlighting the specificity of the TLT-1 fibrinogen interaction. The curve illustrates a strong association with no dissociation, suggesting a strong interaction between the proteins. We isolated and identified four potential peptides (Alpha chain: GGSTSYGTGSETESPR, GSESGIFTNTK, Beta chain: QDGSVDFGR , QGFGNVATNTDGK) that bind TLT-1. We are currently performing BLI Competitive kinetics assays using biotinylated constructs of the peptides isolated from the Immunoprecipitation/ LC-MS/MS. The BLI competitive assays using the four peptides are suggestive of an interaction between TLT-1 and the four peptides as illustrated by increasing bimolecular complex formation with increasing concentration of soluble TLT-1 for all four peptides(data not shown). Conclusions: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 fibrinogen interaction, suggesting a high affinity interaction between TLT-1 and fibrinogen. In our preliminary results from the BLI Competitive kinetics assays we obtained KD values within the nanomolar concentration range and are currently conducting experiments to optimize conditions to obtain our final bicomplex binding curve and KD values. We are currently assessing the identified peptides for potential of mediating the molecular interaction between TLT-1 and fibrinogen. Our poster will report the current state of these studies . Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Four SR Proteins Play Opposing Roles in the Regulated Biosynthesis of Tissue Factor in Human Monocytic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2128-2128
Author(s):  
Sajiv Chandradas ◽  
Gintaras Deikus ◽  
Jonathan G. Tardos ◽  
David H Bechhofer ◽  
Vladimir Y Bogdanov
Keyword(s):  
Complex Formation ◽  
Tissue Factor ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Sr Proteins ◽  
Human Monocytes ◽  
Wild Type ◽  
Binding Motifs ◽  
Rna Probes

Abstract Abstract 2128 Poster Board II-105 Background. Circulating Tissue Factor (TF) is a major contributor to the etiology of thrombotic disorders. Blood monocytes are the primary source of circulating TF, which they express in two forms: full length TF (flTF), a transmembrane surface protein, and alternatively spliced TF (asTF), a secreted soluble protein. The presence or absence of the internal cassette exon 5 in the TF mRNA determines whether the encoded protein is flTF or asTF, respectively. While the procoagulant potential of flTF vastly exceeds that of asTF when assessed using conventional static assays, asTF exhibits unique angiogenic properties distinct from flTF. Investigation of the molecular mechanisms governing TF exon 5 processing is of much interest as it may afford novel approaches to modulate monocyte-mediated blood thrombogenicity as well as monocyte-induced angiogenesis. Using a splicing reporter system developed to study the processing of TF pre-mRNA, we previously determined that SR proteins ASF/SF2 and SRp55 are vital for TF exon 5 inclusion in human monocytes (Tardos et al, J Thromb Haemost 6:877-884, 2008). In the course of more recent experiments, we found that in contrast to ASF/SF2 and SRp55, two other SR proteins – SC35 and SRp40 – appear to promote exclusion of this variable exon: weakening of the binding motifs for SC35 (sites 33 and 81) and SRp40 (site 44), whose positions in exon 5 overlap with the binding sites for ASF/SF2 and SRp55, results in the decrease of the splicing event unique to asTF. The competition of these four SR proteins for binding to their overlapping binding sites may thus play a role in the maintenance of the asTF / flTF ratio; however, a physical association of SC35 and SRp40 with their putative sites in exon 5 was not demonstrated. Objective. To determine whether SC35 and/or SRp40 physically associate with their putative binding sites. Results. To evaluate interaction of the SR proteins SC35 and SRp40 with their putative binding sites in TF exon 5, we employed RNA mobility shift assay methodology using freshly prepared nuclear extracts of THP-1 cells, a monocytic cell line, and in vitro transcribed, uniformly labeled RNA probes comprising the two regions of interest – one spanning SC35 site 33 and SRp40 site 44, and the other spanning the SC35 site 81. As expected, these RNA probes yielded reproducible band shift products, confirming RNA-protein complex formation. We then developed three counterpart mutant RNA probes in which the SC35 and SRp40 binding motifs were selectively weakened by targeted site-directed mutagenesis, and performed RNA gel shift assays alongside the corresponding wild-type probes. Each mutant probe exhibited a significantly weaker interaction with the SR proteins compared to its wild-type counterpart: the SC35 site 33 mutant probe produced a 31.1% reduction in complex formation relative to wild-type (p = 0.011), the SRp40 site 44 mutant probe also produced a 31.1% reduction in complex formation relative to wild-type (p = 0.0001), and the SC35 site 81 mutant probe produced a 33.0% reduction in complex formation relative to wild-type (p = 0.0151). Conclusions. We show for the first time that SR proteins SC35 and SRp40 physically associate with functional binding sites within TF exon 5. The SC35 and SRp40 binding sites overlap with the binding sites for ASF/SF2 and SRp55, the SR proteins that promote exon 5 inclusion. The opposing effects of distinct SR proteins on TF exon 5 processing reveal a heretofore unknown mechanism governing regulated TF biosynthesis. Further studies of SR protein-mediated effects on the TF profile of human monocytes are likely to aid in the development of novel therapeutic strategies aimed at selective targeting of biologically distinct TF forms. Disclosures: No relevant conflicts of interest to declare.

Inverse Effects of EDTA on αIIbβ3-Mediated Adhesion to Immobilized Fibrinogen Versus the D98 Plasmin Fragment of Fibrinogen

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1036-1036
Author(s):  
Hina Zafar ◽  
Jihong Li ◽  
George A David ◽  
Barry S. Coller
Keyword(s):  
Binding Sites ◽  
Metal Ion ◽  
Conflicts Of Interest ◽  
Divalent Cations ◽  
Hek293 Cells ◽  
Clot Retraction ◽  
Ligand Interaction ◽  
Fibrinogen Binding ◽  
A Site ◽  
Adhesion Of Platelets

Abstract αIIbβ3 is known to mediate adhesion of platelets to immobilized fibrinogen through its interaction with the C-terminal γ chain dodecapeptide (γ12) and EDTA inhibits the adhesion by binding divalent cations required for ligand interaction with the β3 metal ion-dependent adhesion site (MIDAS) cation. Studies by several groups, however, suggest that αIIβb3 can also interact with other sites on fibrin(ogen). To identify potential additional sites of interaction between fibrinogen and αIIbβ3, we studied the adhesion of HEK293 cells expressing αIIbβ3 (αIIbβ3-HEK) to the D98 plasmin fragment of fibrinogen, which lacks the γ12 peptide. The D98 fragment did not contain the γ12 peptide as judged by both immunoblotting with mAb 7E9 (anti-γ12) and mass spectroscopy. αIIbβ3-HEK did not bind to immobilized D98 (10 µg/ml coating concentration) in the presence of 2 mM Ca2+/1 mM Mg2+, but they did bind to immobilized intact fibrinogen (10 µg/ml coating concentration) and the adhesion was inhibited by mAbs 10E5 (anti-αIIbβ3), 7E3 (anti-αIIbβ3 + αVβ3), and 7E9. Adhesion of αIIbβ3-HEK to fibrinogen was nearly eliminated by 10 mM EDTA [13,007 ± 3,676 vs 304 ± 331 arbitrary fluorescence intensity units (AFU); n=9; p<0.001]. Unexpectedly, and in dramatic contrast, 10 mM EDTA increased adhesion of αIIbβ3-HEK to D98 nearly 25-fold, from 458 ± 601 to 10,718 ± 3,106 AFU (n=9; p=0.001). The adhesion to D98 in the presence of EDTA was not inhibited by mAb 7E9 or mAb LM609 (anti-αVβ3), and was inhibited by mAb 7E3 by less than 10%. EDTA-dependent adhesion was, however, inhibited by mAb 10E5, which binds to the αIIb cap domain and inhibits fibrinogen binding to αIIbβ3, by 85% ± 4% (n=7; p=0.003). Dose-response experiments demonstrated that 3 mM EDTA was sufficient to block adhesion to fibrinogen and 3-4 mM EDTA was required to enhance adhesion to D98. Adding the β3 D119 mutation to αIIbβ3-HEK (αIIbβ3-D119-HEK), which disrupts the MIDAS, eliminated adhesion to fibrinogen (αIIbβ3-HEK: 17,342 ± 6,148 vs. αIIbβ3-D119-HEK: 0 ± 241 AFU; n=3; p=0.006), but had little or no effect on the binding to D98 in the presence of EDTA (αIIbβ3-HEK: 11,363 ± 3,700 vs. αIIbβ3-D119-HEK: 9,026 ± 3,252 AFU; n=3; p=0.054). However, unlike EDTA-dependent adhesion of αIIbβ3-HEK to D98, the adhesion of αIIbβ3-D119-HEK was inhibited by mAb 10E5 by only 20% ± 19% (n=3; p=0.247). We conclude that EDTA exposes one or more sites on αIIbβ3 that bind(s) to a site(s) on immobilized D98 that is either not accessible or not expressed on intact fibrinogen. These data are consistent with the known effect of EDTA in altering the conformation of αIIbβ3 as judged by its exposing "ligand-induced" binding sites recognized by mAbs such as AP5 and PMI-1, even in the absence of ligand, and the ability of αIIbβ3 to mediate interactions with fibrin to support clot retraction even in the presence of EDTA. EDTA-treated αIIbβ3 may, therefore, provide insights into potential ancillary sites of interaction between αIIbβ3 and fibrin(ogen). Disclosures No relevant conflicts of interest to declare.

Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

2019 ◽  
Author(s):  
Michael Olp ◽  
Daniel Sprague ◽  
Stefan Kathman ◽  
Ziyang Xu ◽  
Alexandar Statsyuk ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Site ◽  
Binding Sites ◽  
Computational Prediction ◽  
Chemical Probes ◽  
Computational Docking ◽  
Fragment Screening ◽  
Covalent Inhibitors ◽  
Bet Protein ◽  
Proof Of Principle

<p>Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the <i>C</i>-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.</p>

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Mitochondrion ◽  
2013 ◽  
Vol 13 (6) ◽  
pp. 823-830 ◽  
Author(s):  
Carla Rossini Crepaldi ◽  
Phelipe Augusto Mariano Vitale ◽  
Andrea Cristina Tesch ◽  
Hélen Julie Laure ◽  
José César Rosa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Protein Complexes ◽  
Type I ◽  
Sds Page

Mapping the protein-binding sites for iridium(iii)-based CO-releasing molecules

2016 ◽  
Vol 45 (30) ◽  
pp. 12206-12214 ◽  
Author(s):  
Marco Caterino ◽  
Ariel A. Petruk ◽  
Alessandro Vergara ◽  
Giarita Ferraro ◽  
Daniela Marasco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Binding ◽  
Binding Sites ◽  
Raman Microspectroscopy ◽  
Rnase A ◽  
Protein Binding Sites ◽  
X Ray ◽  
X Ray Crystallography ◽  
Model Protein ◽  
Circular Dichroïsm

Mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography have been used to investigate the reaction of CO-releasing molecule Cs2IrCl5CO with the model protein RNase A.

scholarly journals Elucidating Binding Sites and Affinities of ERα Agonist and Antagonist to Human Alpha-Fetoprotein by in silico Modeling and Point Mutagenesis

2019 ◽  
Author(s):  
Nurbubu T. Moldogazieva ◽  
Daria S. Ostroverkhova ◽  
Nikolai N. Kuzmich ◽  
Vladimir V. Kadochnikov ◽  
Alexander A. Terentiev ◽  
...  
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Disulfide Bonds ◽  
Electrostatic Interactions ◽  
Molecular Mechanisms ◽  
3D Structure ◽  
Scoring Function ◽  
Alpha Fetoprotein ◽  
Ligand Interaction ◽  
Ligand Interactions

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting variety of hydrophobic ligands including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth and this can be attributed to its estrogen-binding ability. Despite AFP has long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP-ligand interaction remain obscure. In our study we constructed homology-based 3D model of human AFP (HAFP) with the purpose to perform docking of ER&alpha; ligands, three agonists (17&beta;-estradiol, estrone and diethylstilbestrol) and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on ligand docked scoring function, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity sites were located in (i) a tunnel formed within HAFP subdomains IB and IIA and (ii) opposite side of the molecule in a groove originating from cavity formed between domains I and III, while (iii) the third low-affinity site was found at the bottom of the cavity. 100 ns MD simulation allowed studying their geometries and showed that HAFP-estrogen interactions occur due to van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP-antiestrogen binding. MM/GBSA rescoring method estimated binding free energies (&Delta;Gbind) and showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP-ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues along two disulfide bonds, Cys224-Cys270 and Cys269-Cys277 have key roles in both HAFP-estrogen and HAFP-antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein-ligand interactions and anti-cancer therapy strategies based on ER-binding ligands.

scholarly journals Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5056-5056 ◽  
Author(s):  
Julia Fremerey ◽  
Pavel Morozov ◽  
Cindy Meyer ◽  
Aitor Garzia ◽  
Marianna Teplova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Binding Properties ◽  
Expression Levels ◽  
Rna Targets ◽  
Polymerase I

Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

scholarly journals The structural chemistry of zinc(ii) and nickel(ii) dithiocarbamate complexes

2021 ◽  
Vol 19 (1) ◽  
pp. 974-986
Author(s):  
Tanzimjahan A. Saiyed ◽  
Jerry O. Adeyemi ◽  
Damian C. Onwudiwe
Keyword(s):  
Complex Formation ◽  
Metal Ions ◽  
Structural Properties ◽  
Binding Sites ◽  
Biological Systems ◽  
Structural Chemistry ◽  
Metal Coordination ◽  
Nickel Ions ◽  
Coordination Modes ◽  
Biomedical Fields

Abstract Dithiocarbamate complexes are of immense interest due to their diverse structural properties and extensive application in various areas. They possess two sulfur atoms that often act as the binding sites for metal coordination in a monodentate, bidentate, or anisodentate fashion. These different coordination modes enhance the possibility for complex formation and make them useful in different areas especially in biomedical fields. A synergy exists in the metal ions and dithiocarbamate moieties, which tends to exert better properties than the respective individual components of the complex. These improved properties have also been attributed to the presence of the C–S bonds. Zinc and nickel ions have been majorly found to bind to the dithiocarbamate in bidentate modes, and consequently different geometries have resulted from this interaction. The aim of this review is to present some studies on the synthesis, structural chemistry, and the relevance of zinc and nickel dithiocarbamates complexes especially in biological systems.

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