Nitrogen Monoxide Activates Iron Regulatory Protein 1 RNA-Binding Activity by Two Possible Mechanisms:  Effect on the [4Fe-4S] Cluster and Iron Mobilization from Cells†

Biochemistry ◽  
2000 ◽  
Vol 39 (10) ◽  
pp. 2748-2758 ◽  
Author(s):  
S. L. Wardrop ◽  
R. N. Watts ◽  
D. R. Richardson
Keyword(s):  
Rna Binding ◽  
Regulatory Protein ◽  
Nitrogen Monoxide ◽  
Binding Activity ◽  
Iron Regulatory Protein ◽  
Iron Mobilization ◽  
Iron Regulatory Protein 1

The Iron-Sulfur Cluster of Iron Regulatory Protein 1 Modulates the Accessibility of RNA Binding and Phosphorylation Sites†

Biochemistry ◽  
1997 ◽  
Vol 36 (13) ◽  
pp. 3950-3958 ◽  
Author(s):  
Kevin L. Schalinske ◽  
Sheila A. Anderson ◽  
Polygena T. Tuazon ◽  
Opal S. Chen ◽  
M. Claire Kennedy ◽  
...  
Keyword(s):  
Rna Binding ◽  
Regulatory Protein ◽  
Phosphorylation Sites ◽  
Iron Regulatory Protein ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Regulatory Protein 1

Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a non-IRE-binding state

2010 ◽  
Vol 430 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Zvezdana Popovic ◽  
Douglas M. Templeton
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Capacity ◽  
Regulatory Protein ◽  
Product Formation ◽  
Binding Activity ◽  
Hill Coefficient ◽  
Iron Regulatory Protein ◽  
Aconitase Activity ◽  
Iron Regulatory Protein 1

In its aconitase-inactive form, IRP-1 (iron regulatory protein-1)/cytosolic aconitase binds to the IRE (iron-responsive element) of several mRNAs to effect post-transcriptional regulation. We have shown previously that IRP-1 has ATPase activity and that binding of ATP suppresses the IRP-1/IRE interaction. In the present study, we characterize the binding activity further. Binding is observed with both [α-32P]ATP and [α-32P]ADP, but not with [γ-32P]ATP. Recombinant IRP-1 binds approximately two molecules of ATP, and positive co-operativity is observed with a Hill coefficient of 1.67±0.36 (EC50=44 μM) commencing at 1 μM ATP. Similar characteristics are observed with both apoprotein and the aconitase form. On binding, ATP is hydrolysed to ADP, and similar binding parameters and co-operativity are seen with ADP, suggesting that ATP hydrolysis is not rate limiting in product formation. The non-hydrolysable analogue AMP-PNP (adenosine 5′-[β,γ-imido]triphosphate) does not induce co-operativity. Upon incubation of IRP-1 with increasing concentrations of ATP or ADP, the protein migrates more slowly on agarose gel electrophoresis, and there is a shift in the CD spectrum. In this new state, adenosine nucleotide binding is competed for by other nucleotides (CTP, GTP and AMP-PNP), although ATP and ADP, but not the other nucleotides, partially stabilize the protein against spontaneous loss of aconitase activity when incubated at 37 °C. A mutant IRP-1(C437S) lacking aconitase activity shows only one ATP-binding site and lacks co-operativity. It has increased IRE-binding capacity and lower ATPase activity (Km=75±17 nmol/min per mg of protein) compared with the wild-type protein (Km=147±48 nmol/min per mg of protein). Under normal cellular conditions, it is predicted that ATP/ADP will maintain IRP-1 in a non-IRE-binding state.

scholarly journals Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2

2009 ◽  
Vol 29 (8) ◽  
pp. 2219-2229 ◽  
Author(s):  
Kimberly B. Zumbrennen ◽  
Michelle L. Wallander ◽  
S. Joshua Romney ◽  
Elizabeth A. Leibold
Keyword(s):  
Oxidative Stress ◽  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Structural Basis ◽  
Iron Regulatory Protein ◽  
Transferrin Receptor 1 ◽  
Binding Cleft

ABSTRACT Iron regulatory protein 2 (IRP2) is an RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor 1. IRP2 RNA-binding activity is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that IRP2 RNA binding is also regulated by thiol oxidation. We generated a model of IRP2 bound to RNA and found that two cysteines (C512 and C516) are predicted to lie in the RNA-binding cleft. Site-directed mutagenesis and thiol modification show that, while IRP2 C512 and C516 do not directly interact with RNA, both cysteines are located within the RNA-binding cleft and must be unmodified/reduced for IRP2-RNA interactions. Oxidative stress induced by cellular glucose deprivation reduces the RNA-binding activity of IRP2 but not IRP2-C512S or IRP2-C516S, consistent with the formation of a disulfide bond between IRP2 C512 and C516 during oxidative stress. Decreased IRP2 RNA binding is correlated with reduced transferrin receptor 1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.

scholarly journals TP53 Mutation Status Influences Iron Regulatory Protein RNA Binding Activity and Sensitivity to Ferroptotic Cell Death

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1277-1277
Author(s):  
Laurie Thompson ◽  
Thais Oliveira ◽  
Evan Hermann ◽  
Mckale Montgomery ◽  
Winyoo Chowanadisai ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Tp53 Mutation ◽  
Rna Binding ◽  
Human Cancer ◽  
Regulatory Protein ◽  
Tp53 Gene ◽  
Binding Activity ◽  
Iron Regulatory Protein ◽  
Mutation Status

Abstract Objectives The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer, but mutations in TP53 do not just result in loss of tumor suppressor function, they can also promote cancer progression by altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the mediation of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis, a form of iron mediated cell death. The purpose of this study was to determine the extent to which TP53 mutation status effects iron-mediated cell death in response to ferroptosis induction. We also measured TP53 dependent differences in iron regulatory protein (IRP) RNA binding activity to begin to clarify the mechanisms by which TP53 mutation status may influence sensitivity to ferroptosis. Methods Using H1299 cells, which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type TP53 gene, or a representative mutated TP53 gene from exemplary “hotspot” mutations in the DNA binding domain (R248, R273, R282, G245, R249 and R175). These six mutation types were selected because they represent 25% of all TP53 mutations in human cancer. To determine the influence of TP53 mutation status on sensitivity to ferroptotic cell death, we treated cells with erastin, a potent inducer of ferroptosis and measured differences in cell viability between these cell lines using PrestoBlue cell viability reagent. To assess mutant TP53-depenent differences in IRP RNA binding activity during ferroptosis we measured differences in IRP RNA binding activity via Electrophoretic Mobility-Shift Assay. Results We found that TP53 mutants (R273, R248, R175, G245, and R249) were significantly less viable (P < 0.05) after initiation of ferroptosis compared to cells expressing WT TP53. Following ferroptosis induction, we observed a significant (P < 0.05) increase in IRP RNA binding in G245, R248, and R175 mutants. Conclusions Our preliminary analyses indicate that TP53 mutants may be more sensitive to ferroptosis, but IRPs do not seem to be solely responsible for the increase in iron during ferroptotic cell death. Furthermore, ferroptosis may be a potential therapeutic target for cancers with these TP53 mutations but further investigation is warranted. Funding Sources Internal funding at Oklahoma State University.

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