Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions

2020 ◽  
Vol 14 (2) ◽  
pp. 88-99
Author(s):  
Mateen A. Khan
Keyword(s):  
Ionic Strength ◽  
Binding Affinity ◽  
Electrostatic Interactions ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Scatchard Analysis ◽  
Ph Effects ◽  
Protein Fluorescence ◽  
Iron Regulatory Proteins ◽  
Tryptophan Residues

Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. e168-e179 ◽  
Author(s):  
Mayka Sanchez ◽  
Bruno Galy ◽  
Bjoern Schwanhaeusser ◽  
Jonathon Blake ◽  
Tomi Bähr-Ivacevic ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Rna Binding ◽  
Isotope Labeling ◽  
Rna Binding Proteins ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Ribonucleoprotein Complexes ◽  
Cellular Iron ◽  
Irp1 And Irp2

Abstract Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

scholarly journals Electrostatic interactions affecting the active site of class Sigma glutathione S-transferase

2000 ◽  
Vol 347 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Julie M. STEVENS ◽  
Richard N. ARMSTRONG ◽  
Heini W. DIRR
Keyword(s):  
Ionic Strength ◽  
Conformational Changes ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Size Exclusion ◽  
Tryptophan Residue ◽  
Glutathione S Transferase ◽  
Protein Fluorescence ◽  
Subunit Interface ◽  
Α Helix

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the α-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.

scholarly journals Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis

2004 ◽  
Vol 23 (2) ◽  
pp. 386-395 ◽  
Author(s):  
Esther G Meyron-Holtz ◽  
Manik C Ghosh ◽  
Kazuhiro Iwai ◽  
Timothy LaVaute ◽  
Xavier Brazzolotto ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Iron Regulatory Protein ◽  
Iron Regulatory Proteins

scholarly journals Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization

2021 ◽  
Author(s):  
Yingying Xing ◽  
Ning Xu ◽  
Deepak D Bhandari ◽  
Dmitry Lapin ◽  
Xinhua Sun ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Iron Uptake ◽  
Iron Acquisition ◽  
Iron Accumulation ◽  
Regulatory Proteins ◽  
Mineral Nutrient ◽  
Effector Protein ◽  
Iron Regulatory Proteins ◽  
Protein Levels ◽  
Pathogen Colonization

Abstract Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle

1980 ◽  
Vol 186 (1) ◽  
pp. 89-98 ◽  
Author(s):  
T P Walsh ◽  
D J Winzor ◽  
F M Clarke ◽  
C J Masters ◽  
D J Morton
Keyword(s):  
Skeletal Muscle ◽  
Binding Constant ◽  
Moving Boundary ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Affinity Constant ◽  
Quantitative Studies ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Troponin Complex

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

scholarly journals Expression of Iron Regulatory Proteins in Healthy and Osteoarthritic Chondrocytes

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Brandon J Rose ◽  
Nathan F Behrens ◽  
Shelby T Harris ◽  
Sarah A Maloy ◽  
Tanner K Nelson ◽  
...  
Keyword(s):  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Osteoarthritic Chondrocytes
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