Stoichiometry Of Interaction Research Articles

A simple naphthalimide-based receptor for selective recognition of fluoride anion

Synthesis of a simple fluorescent naphthalimide based receptor 4-aminobenzyl-N-methyl-1,8naphthalimide 3 was carried out as a selective fluoride ion sensor. In acetonitrile, interaction of 3 with different anions such as, AcO – , F – , Cl – , Br – , I – , and SCN – revealed significant fluorescence quenching only with the F – anion. In the presence of the fluoride anion, the color of the solution changed from a fluorescent green to violet and this was visible to the naked eye. The probable mode of sensing mechanism by photo-induced electron transfer (PET) reaction is attributed to deprotonation of acidic NH proton in the presence of fluoride which was confirmed by change in optical properties intramolecular charge transfer (ICT) and 1 H NMR spectral data analysis. The Job’s plot analysis displayed a 1:1 stoichiometry for interaction between 3 and F – . The extent of fluorescence quenching was estimated by Stern-Volmer plot.

Chromatin Modification by PSC Occurs at One PSC per Nucleosome and Does Not Require the Acidic Patch of Histone H2A

Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC’s ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity of mechanisms used to modify chromatin architecture.

Time to Face the Fats: What Can Mass Spectrometry Reveal about the Structure of Lipids and Their Interactions with Proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.

Synthesis, computational, spectroscopic, thermal and antimicrobial activity studies on some metal–urate complexes

New sixteen uric acid metal complexes of different stoichiometry, stereo-chemistries and modes of interactions were synthesized using different metals Cr, Mn, Fe, Co, Ni, Cu, Cd, UO2, Na and K. The synthesized complexes were characterized by elemental analysis, spectral (IR, UV–Vis and ESR) methods, thermal analysis (TG, DTA and DSC) and magnetic susceptibility studies. Molecular modeling calculations were used to characterize the ligation sites of the free ligand. Furthermore, quantum chemical parameters of uric acid such as the energies of highest occupied molecular orbital (EHOMO), energies of lowest unoccupied molecular orbital (ELUMO), the separation energy (ΔE=ELUMO−EHOMO), the absolute electronegativity, χ, the chemical potential, Pi, the absolute hardness, η and the softness (σ) were obtained for uric acid. Eight different microbial categories were used to study the antimicrobial activity of the free ligand and ten of its complexes. The results indicate that the ligand and its metal complexes possess antimicrobial properties. The stoichiometry of iron–uric acid complex was studied by using different spectrophotometric methods.

Single Molecule Studies of Interaction Between Alzheimer's Amyloid-β Peptides of different Lengths

The oligomers formed by amyloid-β (Aβ) peptides are thought to play a causative role in Alzheimer's disease, possibly due to membrane permeabilization by small Aβ oligomers. Two primary variants of Aβ, of lengths 40 (Aβ40) and 42 (Aβ42) amino acids are produced at defined ratios in a normal individual. Changes in the ratio of Aβ40/Aβ42 have been shown to correlate strongly with the disease. Further, a mixture of the two forms of the peptide has been shown to exhibit different fibrillization kinetics in vitro and to elicit different extents of cellular toxicity. Thus, mixed Aβ40/Aβ42 oligomers can have different characteristics compared to oligomers formed by either one of the peptides. However, most research in the field focuses on one of the peptides at a time. Further, the Aβ oligomers are heterogenous, metastable and physiologically occur at low nanomolar concentrations, which makes it difficult for the use of conventional techniques to identify the toxic oligomers. In our work, we use single molecule methods to overcome these obstacles and study the formation and the evolution kinetics of mixed oligomers of Aβ40 and Aβ42 at different ratios in solution. We employ single molecule FRET and photobleaching of two different fluorophores attached to the N-terminal of Aβ40 and Aβ42 to determine the oligomer size and composition. Extending these studies to oligomer formation and binding kinetics of the two peptides on model membranes constructed from brain lipid extracts will provide a critical new understanding of how the stoichiometry of interaction of the two peptides both in solution and on the membrane surface affects the composition and permeabilizing potency of the resulting mixed oligomers.

Compelling Advantages of Negative Ion Mode Detection in High-Mass MALDI-MS for Homomeric Protein Complexes

Chemical cross-linking in combination with high-mass MALDI mass spectrometry allows for the rapid identification of interactions and determination of the complex stoichiometry of noncovalent protein-protein interactions. As the molecular weight of these complexes increases, the fraction of multiply charged species typically increases. In the case of homomeric complexes, signals from multiply charged multimers overlap with singly charged subunits. Remarkably, spectra recorded in negative ion mode show lower abundances of multiply charged species, lower background, higher reproducibility, and, thus, overall cleaner spectra compared with positive ion mode spectra. In this work, a dedicated high-mass detector was applied for measuring high-mass proteins (up to 200 kDa) by negative ion mode MALDI-MS. The influences of sample preparation and instrumental parameters were carefully investigated. Relative signal integrals of multiply charged anions were relatively independent of any of the examined parameters and could thus be approximated easily for the spectra of cross-linked complexes. For example, the fraction of doubly charged anions signals overlapping with the signals of singly charged subunits could be more precisely estimated than in positive ion mode. Sinapinic acid was found to be an excellent matrix for the analysis of proteins and cross-linked protein complexes in both ion modes. Our results suggest that negative ion mode data of chemically cross-linked protein complexes are complementary to positive ion mode data and can in some cases represent the solution phase situation better than positive ion mode.

Electrochemical and spectroscopic investigations of isoniazide and its analogs with ds.DNA at physiological pH: Evaluation of biological activities

Interaction and binding of isonicotinic acid hydrazide (INH) and its two analogs; pyrazine carboxylic acid hydrazide (PCH) and 2,4-dihydroxy benzoic acid hydrazide (2,4-DHBAH) with DNA has been investigated by UV-spectroscopy and cyclic voltammetry (CV) at physiological conditions of pH and temperature. Experimental results from both techniques were in good agreement and indicated stronger binding and formation of hydrazides–DNA complexes via intercalation. Among three hydrazides, 2,4-DHBAH showed greater interaction toward DNA at stomach pH (4.7) as evident from its comparatively greater binding constant, { K b ; 2.02 × 10 4 M −1 (UV), 3.13 × 10 4 M −1 (CV)}. The greater binding site size ( n = 3) for 2,4-DHBAH at stomach pH inferred 3:1 binding stoichiometry and possibility of electrostatic interactions or hydrogen bonding along with intercalative mode of interaction between 2,4-DHBAH and DNA. The free energies of hydrazides–DNA complexes indicated the spontaneity of their binding. 2,4-DHBAH has shown promising anti-bacterial activities while anti-oxidant and cytotoxic potentials were exhibited by all three hydrazides.

Diversity in the supramolecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole with modified cyclodextrins: Implications for physicochemical properties and antiparasitic activity.

The molecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole (G2), an antiprotozoa with poor aqueous solubility, with 2-hydroxypropyl-α-cyclodextrin (HPαCD), methyl-β-cyclodextrin (MβCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) were examined. The aqueous solubility enhancement by cyclodextrins (CDs) was evidenced in phase-solubility diagrams, and the stoichiometry of G2/CD systems was determined by Job's plots. Two-dimensional NMR spectroscopic data revealed that a different mode of interaction took place between G2 and CDs in solution. With HPαCD, a non-inclusion complex was generated. In the case of MβCD, a typical host-guest system was obtained and with HPβCD a partial inclusion complex through the narrow side of the macrocycle was formed. ESI-mass spectrometric data confirmed the stoichiometry and mode of interaction of these systems in solution. Solid-state characterization (scanning calorimetry and powder X-ray diffraction) supported the inclusion complex formation. The leishmanicidal activity, trypanocidal activity and non-toxic profile of G2/MβCD showed the advantages of using this inclusion complex to promote the biological assays extension of G2.

Abstract 1382: Use of polarized light spectroscopy (CD) to study STAT3 folding and STAT3:ligand interactions

Abstract STAT3 is a homodimeric transcription factor that is over-expressed in variety of solid tumours. It is known to trigger transcription of a range of genes associated with tumour growth and metastasis. In order to use the STAT3/STAT3 dimer for drug discovery, the factors affecting dimerisation of the monomeric protein and the interaction with ligands must be understood. In this study, we have demonstrated that Circular Dichroism (CD) can be used as a tool to study these processes. We have found that formation of the dimeric STAT3/STAT3 complex results in an induced CD signal which is absent in monomeric protein. Thus, CD can be used to optimize conditions for the production and storage of un-phosphorylated (un-P-) and phosphorylated (P-) STAT3. A similar induced signal was observed when a small molecule (or peptide ligand) interacted with either un-P- or P-STAT3 due to chiral perturbation of the protein structure or electron rearrangements. Crucially, the intensity of the induced CD signal was found to depend on the strength and geometry of the interaction of the binding partner. CD was also used to determine the secondary structure of un-P- and P-STAT3. HIS- and FLAG-tagged STAT3 were examined using the far-UV spectral region (190-250 nm), and the regular folded environment of the peptidic bonds was used to determine the alpha-helix, beta-sheet, and random coil content of different batches of STAT3 protein. For batches of P-STAT3, the composition was found to be 28.3% α-helix, 22.3% β-sheet and 49.4% random coils. For un-P-STAT3, the β-sheet content was slightly higher (23.2%) while the α-helix content was lower (26.8%). Next, protein:ligand interactions which alter the conformation of the protein were studied. Titration of un-P-STAT monomer with different small-molecule inhibitors produced a significant change in the CD spectrum consistent with modification of secondary structure due to altered folding. In particular, significant effects on the secondary structures of both un-P- and P-STAT3 were observed upon binding of the natural peptidic ligands LKTKF and LPQTV. Crucially, the different complex geometries adopted by the proteins upon interaction with ligands could be used to determine the stoichiometry of interaction and the orientation of the ligands in the binding pocket of the protein. These results demonstrate that CD spectroscopy can be used to evaluate STAT3 structure and ligand interactions at the molecular level, and thus represents a useful analytical tool in the development of small molecule inhibitors for potential therapeutic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1382. doi:10.1158/1538-7445.AM2011-1382

Binding of Chitosan to Phospholipid Vesicles Studied with Isothermal Titration Calorimetry

We thermodynamically characterize the interaction of chitosan with small liposomes and the binding and organization of the polysaccharide on the membrane of the vesicles. By means of isothermal titration calorimetry (ITC), we obtain the enthalpy variations arising from binding of the positively ionized chitosan to neutral and negatively charged liposomes. The strong electrostatic interaction of the polysaccharide with the negative charges at the membrane gives rise to highly exothermic signal until charge compensation is reached. The equilibrium constant, the interaction stoichiometry, and the molar enthalpy of binding chitosan monomers to phospholipids from the external leaflet of the vesicle membrane are obtained from the isotherm curve fitting assuming independent binding sites. The strong exothermic signal indicates that the electrostatically driven binding of chitosan to the membrane is energetically favored, leading to further stabilization of the vesicle suspension. The higher the net negative charge of the vesicles, the more pronounced the adsorption of chitosan is, leading to weaker chain organization of the adsorbed chitosan at the membrane. At the point of charge saturation, vesicle aggregation takes place and we show that this behavior does not always lead to charge reversal at the membrane. Models for the binding behavior and structural organization of chitosan are proposed based on the experimental results from ITC, ζ-potential, and dynamic light scattering.

Critical aspects in analysis of cellular DNA content.

This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin-embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and "mass action law" characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units.

Double-shell gold nanoparticle-based DNA-carriers with poly- l-lysine binding surface

In view of the prospective applications of polyamine coatings in functional gold nanoparticles for use as carriers in gene delivery systems, in tissue repair and as bactericidal and virucidal non-toxic vehicle, we have investigated the interactions of poly- l-lysine (PLL) with gold nanoparticles (AuNP). Since direct binding of PLL to AuNP is not strong at neutral pH, we have focused on PLL interactions with carboxylated self-assembled monolayers (SAM) on AuNP, such as the citrate-capped AuNP. The double-shell nanoparticles AuNP@Cit/PLL thus produced do not contain any toxic thiols. We have observed strong electrostatic interactions between polycationic chains of PLL and AuNP@Cit in weakly acidic to weakly alkaline solutions (pH 5–9), as evidenced by the bathochromic shift of the local surface plasmon (SP) band and strong increase in resonance elastic light scattering (RELS) intensity. The stoichiometry of interactions evaluated on the basis of RELS data indicates on a hyper-Langmuirian type of interactions with stoichiometric coefficient n = 1.35 (PLL : AuNP@Cit). From the RELS titration data, a shift of the deprotonation constant for the bound PLL has been determined ( pK a = 11.6 for the bound PLL vs. 10.48 for the free PLL). The deprotonation of PLL leads to AuNP aggregate disassembly, evidenced by sharp RELS decline and hypsochromic shift of SP band. We have found that under these conditions, a residual aggregation due to the interparticle interactions between β-sheets of PLL overcoat become predominant. The molecular dynamics simulations indicate that multiple hydrogen bonds can also be formed between the PLL linker and the shell molecules of AuNP@Cit. The double-shell nanoparticles, AuNP@Cit/PLL, have been shown to attract DNA molecules using highly sensitive RELS measurements presenting the proof-of-concept for the suitability of this non-toxic nanostructured material for gene delivery applications. The advantage of the proposed material is no toxicity related to the ligand release in gene delivery processes in contrast to the thiol-functionalized AuNP.

Functional Identification of Toxin-Antitoxin Molecules from Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Mass spectrometric studies of dissociation constants of noncovalent complexes

Specific interactions among biomolecules to form noncovalently bound complexes play a pivotal role in key cellular processes such as cell division, cell signalling, gene transcription and translation. The propensity of noncovalently bound complexes to dissociate into their components can be quantified and constants of dissociation (Kd) can be obtained by various methods. Mass spectrometry has become an important method to measure Kd. The advent of soft ionisation techniques, in particular electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) has established mass spectrometry as a viable technique for investigating noncovalent interactions and for quantifying their binding strengths. Under carefully chosen experimental and instrumental conditions, it is possible to observe intact noncovalent complexes in the gas phase using ESI and MALDI, and to use the mass spectra as a read-out for determining solution-phase Kd. Compared to other biophysical methods, mass spectrometry is highly sensitive and fast, and gives additional information about the stoichiometry and specificity of noncovalent interactions. This review focuses on recent MS-based methodologies for quantification of binding strengths, in particular those that promise to complement conventional biophysical methods.

Stoichiometric interpretation of thermoplastic starch water sorption and relation to mechanical behavior

The characterization of glycerol plasticized starch by water sorption was interpreted in terms of interaction stoichiometry. The saturation of starch was found to correspond to a 2:1 anhydroglucose unit/glycerol stoichiometric ratio. The introduction of cellulose into the formulation of plasticized starch (TPS) decreased the quantity of bonded glycerol, due to the strong interaction between cellulose and starch. The systematic study of the TPS properties in the whole range of relative humidities showed transitions which were not connected to the “saturation transition” or to the glass transition. Breaking properties probably depended on a complex combination of different system characteristics.

Survey of the year 2009: applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is now an established and invaluable method for determining the thermodynamic constants, association constant and stoichiometry of molecular interactions in aqueous solutions. The technique has become widely used by biochemists to study protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids and carbohydrates; and nucleic acid interaction with small molecules. The drug discovery industry has utilized this approach to measure protein (or nucleic acid) interaction with drug candidates. ITC has been used to screen candidates, guide the design of potential drugs and validate the modelling used in structure-based drug design. Emerging disciplines including nanotechnology and drug delivery could benefit greatly from ITC in enhancing their understanding and control of nano-particle assembly, and drug binding and controlled release.

A General Mechanism for Network-Dosage Compensation in Gene Circuits

Coping with variations in network dosage is crucial for maintaining optimal function in gene networks. We explored how network structure facilitates network-level dosage compensation. By using the yeast galactose network as a model, we combinatorially deleted one of the two copies of its four regulatory genes and found that network activity was robust to the change in network dosage. A mathematical analysis revealed that a two-component genetic circuit with elements of opposite regulatory activity (activator and inhibitor) constitutes a minimal requirement for network-dosage invariance. Specific interaction topologies and a one-to-one interaction stoichiometry between the activating and inhibiting agents were additional essential elements facilitating dosage invariance. This mechanism of network-dosage invariance could represent a general design for gene network structure in cells.

Cross‐linking FtsZ polymers into coherent Z rings

A key event in bacterial cytokinesis is the formation of the Z ring, which serves as a mechanical scaffold that recruits other cytokinetic proteins to establish functional divisomes. This scaffolding function of Z rings is essential throughout cytokinesis, but the underlying molecular interactions are poorly understood. Here we report that a widely conserved FtsZ binding protein, ZapA, has cytological, biochemical and biophysical properties that argue for the importance of cross-linking interactions between FtsZ polymers in the coherence of Z rings. Escherichia coli zapA null mutant cells have Z rings that are structurally looser and many helical precursors of Z rings fail to coalesce into coherent rings. Biophysical behaviour of FtsZ in the presence of ZapA reveals that ZapA not only bundles, but also cross-links FtsZ polymers, which makes it the first cross-linking protein of the bacterial cytoskeleton. Cross-linking in vitro occurs at the stoichiometry of FtsZ-ZapA interaction at the Z rings in vivo, where nearly all intracellular ZapA is dynamically associated. ZapA also stabilizes longitudinal bonds between FtsZ monomers since it promotes the polymerization of FtsZ mutants with lesions at the polymerization interface and since it reverses the inhibitory effects of SulA, a known antagonist of FtsZ longitudinal interactions.

MECHANISM OF INTERACTION OF SOME ANTIMICROBIAL AGENTS WITH BOVINE SERUM ALBUMIN

The mechanism of interaction of five antibiotic drugs with bovine serum albumin has been studied using fluorescence spectrophotometric technique. The stoichiometry of interaction was 1:1 and association constants were of the order of 10 4 . Thermodynamic parameters for the binding suggested hydrophobic interactions for enrofoxacin and ciprofloxacin HCl; hydrogen bonding and electrostatic interactions for levofloxacin and hydrogen bonding, hydrophobic and electrostatic interactions for sparfloxacin. Although hydrophobic interactions are predicted in most cases, the drug binding region was found to be different from that of hydrophobic probe (ANS). Stern-Volmer analysis of the data showed that the quenching mechanism was predominantly dynamic in the case of ciprofloxacin HCl and enrofloxacin, while both static and dynamic quenching mechanisms are operative in the case of levofloxacin and sparfloxacin.

The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27(kip1) gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27(kip1) transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27(kip1) transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70(-/-) ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 μM, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage.