scholarly journals Bile Acids Gate Dopamine Transporter Mediated Currents

2021 ◽  
Vol 9 ◽  
Author(s):  
Tiziana Romanazzi ◽  
Daniele Zanella ◽  
Mary Hongying Cheng ◽  
Behrgen Smith ◽  
Angela M. Carter ◽  
...  
Keyword(s):  
Binding Site ◽  
Bile Acids ◽  
Monoamine Transporters ◽  
Salt Bridges ◽  
Inward Current ◽  
Docking Simulations ◽  
Potential Binding ◽  
Direct Binding ◽  
Dietary Fat Absorption ◽  
New Evidence

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764–25773; Cheng and Bahar, Structure, 2015, 23, 2171–2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

scholarly journals Bile acids gate dopamine transporter mediated currents

2021 ◽  
Author(s):  
Tiziana Romanazzi ◽  
Daniele Zanella ◽  
Mary Hongying Cheng ◽  
Behrgen Smith ◽  
Angela M Carter ◽  
...  
Keyword(s):  
Binding Site ◽  
Bile Acids ◽  
Xenopus Laevis Oocytes ◽  
Monoamine Transporters ◽  
Salt Bridges ◽  
Inward Current ◽  
Docking Simulations ◽  
Direct Binding ◽  
Functional Relevance ◽  
Dietary Fat Absorption

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Interestingly, sterols such as cholesterol interact with monoamine transporters (MAT), including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward circuitries in the brain. The present study explores interactions of the BA, obeticholic acid (OCA), with DAT and mechanistically defines the regulation of DAT activity via both electrophysiology and molecular modeling. We express murine DAT (mDAT) in Xenopus laevis oocytes and confirm that DA induces an inward current that reaches a steady-state at a negative membrane voltage. Next, we show that OCA triggers an inward current through DAT that is Na+ dependent and not regulated by intracellular calcium. OCA also inhibits the DAT-mediated Li+ leak current, a feature that parallels DA action and indicates direct binding to the transporter. Interestingly, OCA does not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, in regard to DA. The current induced by OCA is transient in nature, returning to baseline in the continued presence of the BA. To understand the molecular mechanism of how OCA affects DAT electrical activity, we performed docking simulations. These simulations revealed two potential binding sites that provide important insights into the potential functional relevance of the OCA-DAT interaction. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., 2014;Cheng and Bahar, 2015). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is facilitated through the residues DAT R445 and D436. This binding may stabilize the inward-facing open (IFo) state by preventing the re-formation of the IF gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

2008 ◽  
Vol 19 (6) ◽  
pp. 2661-2672 ◽  
Author(s):  
Soomin Shim ◽  
Samuel A. Merrill ◽  
Phyllis I. Hanson
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Essential Role ◽  
Multivesicular Body ◽  
The Other ◽  
Aaa Atpase ◽  
Direct Binding ◽  
Novel Interactions

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

scholarly journals Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 259-268 ◽  
Author(s):  
Birgit Baumgarth ◽  
Frank Wilco Bartels ◽  
Dario Anselmetti ◽  
Anke Becker ◽  
Robert Ros
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Sinorhizobium Meliloti ◽  
Specific Binding ◽  
Dna Interaction ◽  
Dynamic Force ◽  
Sequence Motifs ◽  
Promoter Regions ◽  
Dissociation Kinetics ◽  
Direct Binding

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG–DNA interaction.

P0078INVESTIGATION THE VARIANTS AT THE BINDING SITE OF INFLAMMATORY TRANSCRIPTION FACTOR IN PATIENTS WITH ESRD

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
SUI-LUNG SU
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Disease Process ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Healthy Controls ◽  
Chip Assay ◽  
Potential Binding ◽  
Stage Renal Disease ◽  
End Stage

Abstract Background and Aims Inflammation is an important factor for enhancing the disease process from chronic kidney disease (CKD) to end-stage renal disease (ESRD). Nuclear factor-kappa B (NF-κB) is a transcription factor that regulates the expression of genes involved in inflammation. We investigated the potential association with the gene polymorphism of transcription factor binding site of NF-κB in ESRD patients. Method We used the Taiwan Biobank database, University of California, Santa Cruz, reference genome, chromatin immunoprecipitation sequencing database to find the SNPs at potential binding sites of NF-κB. In addition, we performed a case–control study and genotyped 847 patients with ESRD and 846 healthy controls at Tri-Service-General-Hospital from 2015 to 2016. Further we used ChIP-assay and Luciferase reporter assay to identify the binding activity at different genotype. Results Results of biometrics screening in the databases revealed 15 SNPs with the potential binding site of NF-κB. Genotype distributions of rs9395890 were significantly different in ESRD cases and healthy controls (P = 0.032). In the Dominant model, rs9395890 with T allele had a higher risk of ESRD (P = 0.032; odds ratio [OR] = 1.32, 95% confidence interval [CI] = 0.99–1.76). The ChIP assay reveals that around 1.49 times enrichment of NF-κB of the variant type TT when compared to that of the wild type GG in the rs9395890 (P<0.027; TT=3.20±0.16, GT=2.81±0.20, GG=1.71±0.18,) and the luciferase activity curve showed T allele was higher than G allele. Conclusion In conclusion, we demonstrate that rs9395890 may be associated with ESRD in the Taiwanese population.

scholarly journals On the prediction of DNA-binding preferences of C2H2-ZF domains using structural models: application on human CTCF

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Alberto Meseguer ◽  
Filip Årman ◽  
Oriol Fornes ◽  
Ruben Molina-Fernández ◽  
Jaume Bonet ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Computational Method ◽  
Chromatin Binding ◽  
Input Structure ◽  
Binding Factor ◽  
Potential Binding

Abstract Cis2-His2 zinc finger (C2H2-ZF) proteins are the largest family of transcription factors in human and higher metazoans. To date, the DNA-binding preferences of many members of this family remain unknown. We have developed a computational method to predict their DNA-binding preferences. We have computed theoretical position weight matrices (PWMs) of proteins composed by C2H2-ZF domains, with the only requirement of an input structure. We have predicted more than two-third of a single zinc-finger domain binding site for about 70% variants of Zif268, a classical member of this family. We have successfully matched between 60 and 90% of the binding-site motif of examples of proteins composed by three C2H2-ZF domains in JASPAR, a standard database of PWMs. The tests are used as a proof of the capacity to scan a DNA fragment and find the potential binding sites of transcription-factors formed by C2H2-ZF domains. As an example, we have tested the approach to predict the DNA-binding preferences of the human chromatin binding factor CTCF. We offer a server to model the structure of a zinc-finger protein and predict its PWM.

scholarly journals Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor

2020 ◽  
Vol 17 (16) ◽  
pp. 5664 ◽  
Author(s):  
Chao Zhang ◽  
Tiziana Schilirò ◽  
Marta Gea ◽  
Silvia Bianchi ◽  
Angelo Spinello ◽  
...  
Keyword(s):  
Competitive Inhibition ◽  
Estrogenic Activity ◽  
Endocrine Disrupting Chemicals ◽  
Binding Mode ◽  
Inhibition Mechanism ◽  
Aromatase Activity ◽  
Docking Simulations ◽  
Potential Binding ◽  
Endocrine Disrupting ◽  
17Β Estradiol

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17β-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 × 10−10 and 3.7 × 10−9, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling.

scholarly journals MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif

2020 ◽  
Vol 295 (52) ◽  
pp. 18266-18275
Author(s):  
Sebastian Kiehstaller ◽  
Christian Ottmann ◽  
Sven Hennig
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Viral Infections ◽  
Binding Mode ◽  
Aminopeptidase N ◽  
Binding Motif ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Phosphorylated Peptides ◽  
Direct Binding

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Sign in / Sign up

Export Citation Format

Share Document