ABSTRACT
BACKGROUND: Choline deficiency has numerous negative health consequences; although the preponderance of the US population consumes less than the recommended Adequate Intake (AI), clinical assessment of choline status is difficult. Further, several pathways involved in primary metabolism of choline are estrogen-sensitive and the AI for premenopausal women is lower than that for men. OBJECTIVES: We sought to determine whether in vivo magnetic resonance spectroscopy (MRS) of liver and/or isotope-dilution MS of plasma could identify biomarkers reflective of choline intake (preregistered primary outcomes 1 and 2, secondary outcome 1). Determination of whether biomarker concentrations showed sex dependence was a post hoc outcome. This substudy is a component of a larger project to identify a clinically useful biomarker panel for assessment of choline status. METHODS: In a double-blind, randomized, crossover trial, people consumed 3 diets, representative of â¼100%, â¼50%, and â¼25% of the choline AI, for 2-wk periods. We measured the concentrations of choline and several metabolites using 1H single-voxel MRS of liver in vivo and using 2H-labeled isotope dilution MS of several choline metabolites in extracted plasma. RESULTS: Plasma concentrations of 2H9-choline, unlabeled betaine, and 2H9-betaine, and the isotopic enrichment ratio (IER) of betaine showed highly significant between-diet effects (q < 0.0001), with unlabeled betaine concentration decreasing 32% from highest to lowest choline intake. Phosphatidylcholine IER was marginally significant (q = 0.03). Unlabeled phosphatidylcholine plasma concentrations did not show between-diet effects (q = 0.34). 2H9 (trimethyl)-phosphatidylcholine plasma concentrations (q = 0.07) and MRS-measured total soluble choline species liver concentrations (q = 0.07) showed evidence of between-diet effects but this was not statistically significant. CONCLUSIONS: Although MRS is a more direct measure of choline status, variable spectral quality limited interpretation. MS analysis of plasma showed clear correlation of plasma betaine concentration, but not plasma phosphatidylcholine concentration, with dietary choline intake. Plasma betaine concentrations also correlate with sex status (premenopausal women, postmenopausal women, men).This trial was registered at clinicaltrials.gov as NCT03726671.
Subject(s)
Choline/administration & dosage , Choline/blood , Liver/diagnostic imaging , Magnetic Resonance Imaging/methods , Betaine/blood , Cross-Over Studies , Double-Blind Method , HumansABSTRACT
Folate (vitamin B9) is a common name for a group of coenzymes that function as carriers of chemical moieties called one-carbon groups in numerous biochemical reactions. The combination of these folate-dependent reactions constitutes one-carbon metabolism, the name synonymous to folate metabolism. Folate coenzymes and associated metabolic pathways are vital for cellular homeostasis due to their key roles in nucleic acid biosynthesis, DNA repair, methylation processes, amino acid biogenesis, and energy balance. Folate is an essential nutrient because humans are unable to synthesize this coenzyme and must obtain it from the diet. Insufficient folate intake can ultimately increase risk of certain diseases, most notably neural tube defects. More than 20 enzymes are known to participate in folate metabolism. Single-nucleotide polymorphisms (SNPs) in genes encoding for folate enzymes are associated with altered metabolism, changes in DNA methylation and modified risk for the development of human pathologies including cardiovascular diseases, birth defects, and cancer. ALDH1L1, one of the folate-metabolizing enzymes, serves a regulatory function in folate metabolism restricting the flux of one-carbon groups through biosynthetic processes. Numerous studies have established that ALDH1L1 is often silenced or strongly down-regulated in cancers. The loss of ALDH1L1 protein positively correlates with the occurrence of malignant tumors and tumor aggressiveness, hence the enzyme is viewed as a candidate tumor suppressor. ALDH1L1 has much higher frequency of non-synonymous exonic SNPs than most other genes for folate enzymes. Common SNPs at the polymorphic loci rs3796191, rs2886059, rs9282691, rs2276724, rs1127717, and rs4646750 in ALDH1L1 exons characterize more than 97% of Europeans while additional common variants are found in other ethnic populations. The effects of these SNPs on the enzyme is not clear but studies indicate that some coding and non-coding ALDH1L1 SNPs are associated with altered risk of certain cancer types and it is also likely that specific haplotypes define the metabolic response to dietary folate. This review discusses the role of ALDH1L1 in folate metabolism and etiology of diseases with the focus on non-synonymous coding ALDH1L1 SNPs and their effects on the enzyme structure/function, metabolic role and association with cancer.
ABSTRACT
Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA-binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective toward the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This mechanism of p53 activation is fundamentally different from the canonical p53 regulation through protein-protein interactions or posttranslational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as a natural small molecule activating p53 through the direct binding.
Subject(s)
Cell Nucleus/metabolism , Ceramides/metabolism , Stress, Physiological , Tumor Suppressor Protein p53/metabolism , A549 Cells , Active Transport, Cell Nucleus , Ceramides/chemistry , HCT116 Cells , HeLa Cells , Hep G2 Cells , Humans , Ligands , PC-3 Cells , Protein Binding , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
ALDH1L1, a member of the aldehyde dehydrogenase superfamily of enzymes, catalyzes the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. The enzyme is a tetramer of identical subunits, with each subunit consisting of three functional domains that originated from unrelated genes. The N- and C-terminal domains are catalytic, while the intermediate domain transfers the reaction intermediate from the N- to the C-terminal domain. The intermediate domain is an acyl carrier protein, possessing the covalently attached 4'-phosphopantetheine (4-PP) prosthetic group. This prosthetic group is known to function as a swinging arm transferring intermediates between enzymes in complex biosynthetic reactions. Here we have applied computer modeling using available structures of the three functional domains of ALDH1L1 to evaluate the extent of flexibility within the full-length protein. This approach allowed us to define positions of the 4-PP arm within the two catalytic domains and to predict N-terminal:intermediate and intermediate:C-terminal domain interfaces. Our models further suggested high degree of flexibility within the full-length enzyme.
Subject(s)
Aldehyde Dehydrogenase/chemistry , Molecular Docking Simulation , Aldehyde Dehydrogenase/metabolism , Binding Sites , Biocatalysis , Folic Acid/analogs & derivatives , Folic Acid/chemistry , Folic Acid/metabolism , Humans , Hydroxymethyl and Formyl Transferases/metabolism , Oxidoreductases Acting on CH-NH Group Donors , Protein Binding , Protein DomainsABSTRACT
Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.
Subject(s)
Multiprotein Complexes/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , YY1 Transcription Factor/metabolism , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Mechanistic Target of Rapamycin Complex 2 , Mice , Models, Biological , Peptides/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Binding , Protein Domains , Structure-Activity Relationship , YY1 Transcription Factor/chemistryABSTRACT
High-molecular-weight kininogen domain 5 (HK5) is an angiogenic modulator that is capable of inhibiting endothelial cell proliferation, migration, adhesion, and tube formation. Ferritin can bind to a histidine-glycine-lysine-rich region within HK5 and block its antiangiogenic effects. However, the molecular intricacies of this interaction are not well understood. Analysis of the structure of HK5 using circular dichroism and nuclear magnetic resonance [(1) H, (15) N]-heteronuclear single quantum coherence determined that HK5 is an intrinsically unstructured protein, consistent with secondary structure predictions. Equilibrium binding studies using fluorescence anisotropy were used to study the interaction between ferritin and HK5. The interaction between the two proteins is mediated by metal ions such as Co(2+) , Cd(2+) , and Fe(2+) . This metal-mediated interaction works independently of the loaded ferrihydrite core of ferritin and is demonstrated to be a surface interaction. Ferritin H and L bind to HK5 with similar affinity in the presence of metals. The ferritin interaction with HK5 is the first biological function shown to occur on the surface of ferritin using its surface-bound metals.
Subject(s)
Ferritins/chemistry , Intrinsically Disordered Proteins/chemistry , Kininogen, High-Molecular-Weight/chemistry , Metals, Heavy/chemistry , Ferritins/metabolism , Humans , Intrinsically Disordered Proteins/isolation & purification , Intrinsically Disordered Proteins/metabolism , Kininogen, High-Molecular-Weight/isolation & purification , Kininogen, High-Molecular-Weight/metabolism , Metals, Heavy/metabolism , Models, MolecularABSTRACT
Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjunction with [(1)H-(15)N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.
Subject(s)
Antibiotics, Antineoplastic/chemistry , Breast Neoplasms/pathology , DNA/chemistry , Doxorubicin/chemistry , Antibiotics, Antineoplastic/pharmacology , Cell Line, Tumor , Doxorubicin/pharmacology , Female , Humans , Magnetic Resonance SpectroscopyABSTRACT
The interaction between platelet integrin αIIbß3 and fibrin(ogen) plays a key role in blood clot formation and stability. Integrin antagonists, a class of pharmaceuticals used to prevent and treat cardiovascular disease, are designed to competitively interfere with this process. However, the energetics of the integrin-drug binding are not fully understood, potentially hampering further development of this class of pharmaceuticals. We integrated dynamic force spectroscopy (DFS) and surface plasmon resonance (SPR) to probe the energetics of complex formation between αIIbß3 and cHarGD, a cyclic peptide integrin antagonist. Analysis of αIIbß3:cHarGD DFS rupture force data at pulling rates of 14 000 pN/s, 42 000 pN/s and 70 000 pN/s yielded koff = 0.02-0.09 s-1, a dissociation energy barrier [Formula: see text] = 22-29 kJ/mol, and a potential well width x-1 = 0.5-0.8 nm. SPR kinetic data yielded an association rate constant kon = 7 × 103 L/mol-s and a dissociation rate constant koff = 10-2 s-1, followed by a slower stabilization step (τ ~ 400 s). Both DFS and SPR detected minimal interactions between αIIbß3 and cHarGA demonstrating a key role for electrostatic interactions between the ligand aspartate and the integrin metal ion-dependent adhesion site (MIDAS). Our work provides new insights into the energy landscape of αIIbß3's interactions with pharmacological and physiological ligands.
ABSTRACT
This study represents the first longitudinal, within-subject (1) H MRS investigation of the developing rat brain spanning infancy, adolescence and early adulthood. We obtained neurometabolite profiles from a voxel located in a central location of the forebrain, centered on the striatum, with smaller contributions for the cortex, thalamus and hypothalamus, on postnatal days 7, 35 and 60. Water-scaled metabolite signals were corrected for T1 effects and quantified using the automated processing software LCModel, yielding molal concentrations. Our findings indicate age-related concentration changes in N-acetylaspartate + N-acetylaspartylglutamate, myo-inositol, glutamate + glutamine, taurine, creatine + phosphocreatine and glycerophosphocholine + phosphocholine. Using a repeated measures design and analysis, we identified significant neurodevelopment changes across all three developmental ages and identified adolescence as a distinctive phase in normative neurometabolic brain development. Between postnatal days 35 and 60, changes were observed in the concentrations of N-acetylaspartate + N-acetylaspartylglutamate, glutamate + glutamine and glycerophosphocholine + phosphocholine. Our data replicate past studies of early neurometabolite development and, for the first time, link maturational profiles in the same subjects across infancy, adolescence and adulthood.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Prosencephalon/metabolism , Aging , Animals , Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Dipeptides/metabolism , Glutamic Acid/metabolism , Inositol/metabolism , Male , Phosphocreatine/metabolism , Prosencephalon/growth & development , Rats , Rats, Sprague-Dawley , Taurine/metabolismABSTRACT
The simultaneous binding of netropsin in the minor groove and Zn(2+) in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3'-terminus (3'FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn(2+)/netropsin: 3'FdU complex had very high thermal stability with aspects of the complex intact at 85 °C, conditions that result in complete dissociation of Mg(2+) complexes. CD and (19)F NMR spectroscopy were consistent with Zn(2+) binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 (1)H and netropsin (1)H resonances. The Zn(2+)/netropsin: 3'FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn(2+):3'FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:32.
Subject(s)
Antineoplastic Agents/chemistry , Coordination Complexes/chemistry , DNA/chemistry , Floxuridine/chemistry , Netropsin/chemistry , Zinc/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cations, Divalent , Circular Dichroism , Computer Simulation , Coordination Complexes/pharmacology , Cytotoxins/metabolism , Humans , Male , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Prostatic Neoplasms , Tumor Cells, CulturedABSTRACT
Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.
Subject(s)
Nuclear Matrix-Associated Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Transcription, Genetic , Vesicular stomatitis Indiana virus/metabolism , Viral Matrix Proteins/metabolism , Active Transport, Cell Nucleus , Cell Line , Gene Expression , HEK293 Cells , Humans , Nuclear Matrix-Associated Proteins/genetics , Nuclear Pore Complex Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Vesicular stomatitis Indiana virus/geneticsABSTRACT
A 15-LOX, it is proposed, suppresses the growth of prostate cancer in part by converting arachidonic, eicosatrienoic, and/or eicosapentaenoic acids to n-6 hydroxy metabolites. These metabolites inhibit the proliferation of PC3, LNCaP, and DU145 prostate cancer cells but only at ≥1-10 µM. We show here that the 15-LOX metabolites of docosahexaenoic acid (DHA), 17-hydroperoxy-, 17-hydroxy-, 10,17-dihydroxy-, and 7,17-dihydroxy-DHA inhibit the proliferation of these cells at ≥0.001, 0.01, 1, and 1 µM, respectively. By comparison, the corresponding 15-hydroperoxy, 15-hydroxy, 8,15-dihydroxy, and 5,15-dihydroxy metabolites of arachidonic acid as well as DHA itself require ≥10-100 µM to do this. Like DHA, the DHA metabolites a) induce PC3 cells to activate a peroxisome proliferator-activated receptor-γ (PPARγ) reporter, express syndecan-1, and become apoptotic and b) are blocked from slowing cell proliferation by pharmacological inhibition or knockdown of PPARγ or syndecan-1. The DHA metabolites thus slow prostate cancer cell proliferation by engaging the PPARγ/syndecan-1 pathway of apoptosis and thereby may contribute to the prostate cancer-suppressing effects of not only 15-LOX but also dietary DHA.
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Docosahexaenoic Acids/pharmacology , Prostatic Neoplasms/metabolism , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Male , PPAR gamma/metabolism , Signal Transduction/drug effects , Syndecan-1/metabolismABSTRACT
NOXO1ß [NOXO1 (Nox organizer 1) ß] is a cytosolic protein that, in conjunction with NOXA1 (Nox activator 1), regulates generation of reactive oxygen species by the NADPH oxidase 1 (Nox1) enzyme complex. NOXO1ß is targeted to membranes through an N-terminal PX (phox homology) domain. We have used NMR spectroscopy to solve the structure of the NOXO1ß PX domain and surface plasmon resonance (SPR) to assess phospholipid specificity. The solution structure of the NOXO1ß PX domain shows greatest similarity to that of the phosphatidylinositol 3-kinase-C2α PX domain with regard to the positions and types of residues that are predicted to interact with phosphatidylinositol phosphate (PtdInsP) head groups. SPR experiments identify PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) as preferred targets of NOXO1ß PX. These findings contrast with previous lipid overlay experiments showing strongest binding to monophosphorylated PtdInsP and phosphatidylserine. Our data suggest that localized membrane accumulation of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(2) may serve to recruit NOXO1ß and activate Nox1.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Binding Sites , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
Apoptosis inducing factor (AIF) is a mediator of caspase-independent cell death that is also necessary for mitochondrial energy production. How these seemingly opposite cellular functions of AIF are controlled is poorly understood. X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspases that also regulates several caspase-independent signaling pathways. The RING domain of XIAP possesses E3 ubiquitin ligase activity, though the importance of this function to signal regulation remains incompletely defined. XIAP binds and ubiquitinates AIF, and in this study, we determined the functional consequences of XIAP-mediated AIF ubiquitination. Unlike canonical ubiquitination, XIAP-dependent AIF ubiquitination did not lead to proteasomal degradation of AIF. Experiments using ubiquitin mutants demonstrated that the XIAP-dependent ubiquitin linkage was not formed through the commonly used lysine 48, suggesting a noncanonical ubiquitin linkage is employed. Further studies demonstrated that only lysine 255 of AIF was a target of XIAP-dependent ubiquitination. Using recombinant AIF, we determined that mutating lysine 255 of AIF interferes with the ability of AIF not only to bind DNA but also to degrade chromatin in vitro. These data indicate that XIAP regulates the death-inducing activity of AIF through nondegradative ubiquitination, further defining the role of XIAP in controlling AIF and caspase-independent cell death pathways.
Subject(s)
Apoptosis Inducing Factor/metabolism , Chromatin/metabolism , Lysine/metabolism , Ubiquitination , X-Linked Inhibitor of Apoptosis Protein/metabolism , Amino Acid Substitution , Apoptosis Inducing Factor/chemistry , Apoptosis Inducing Factor/genetics , Binding Sites , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cell Survival , Chromatin/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Mutant Proteins/chemistry , Mutant Proteins/metabolism , NAD/metabolism , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , RING Finger Domains , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , X-Linked Inhibitor of Apoptosis Protein/chemistryABSTRACT
We report, based on semi-empirical calculations, that Zn(2+) binds duplex DNA containing consecutive FdU-dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn(2+) complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn(2+) complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔT(m) > 15 °C). Mg(2+) neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn(2+). A lipofectamine preparation of the Zn(2+)-DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn(2+)-DNA complexes for cancer treatment.
Subject(s)
DNA/chemistry , Floxuridine/chemistry , Zinc/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Binding Sites , Cell Line, Tumor , Circular Dichroism , DNA/toxicity , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Zinc/toxicityABSTRACT
NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47(phox). NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1ß PX.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Nuclear Magnetic Resonance, Biomolecular , Adaptor Proteins, Signal Transducing , Carbon Isotopes , Nitrogen Isotopes , Protein Structure, Tertiary , Recombinant Proteins/chemistryABSTRACT
NADPH oxidase is essential in the human innate immune response. p47 (phox), a cytosolic NADPH oxidase component, plays a regulatory role in the activation of NADPH oxidase. Our manipulation of p47 (phox) by mutation and amino acid deletion shows that the linker region between the PX and N-terminal SH3 domain plays a role in blocking the binding of the phosphoinositide 3,4-bisphosphate [PI(3,4)P2], a lipid second messenger generated upon neutrophil activation. Replacement of linker residues 151-158 with glycine alters NMR-measured spin lattice relaxation rates and sedimentation velocity compared to those of the wild-type protein, suggesting that the PX domain is released from its autoinhibited conformation. Liposome binding and surface plasmon resonance experiments confirm this result, showing that this mutant has a similar binding affinity for the isolated PX domain toward PI(3,4)P2. However, an in vitro NADPH oxidase activity assay reveals that this glycine mutant of the full-length protein greatly reduced NADPH oxidase activity upon activation even though it displayed PI(3,4)P2 binding activity comparable to that of the isolated PX domain. Our results highlight an active role of the PX-SH3 linker region in maintaining p47 (phox) in its fully autoinhibited form and demonstrate that binding of p47 (phox) to membrane phospholipids is mechanistically distinct from NADPH oxidase activation.
Subject(s)
Mutation , NADPH Oxidases/metabolism , Phosphatidylinositols/metabolism , Amino Acid Sequence , Circular Dichroism , Glycine/chemistry , Glycine/genetics , Glycine/metabolism , Humans , Liposomes/metabolism , Magnetic Resonance Spectroscopy , Molecular Sequence Data , NADPH Oxidases/chemistry , NADPH Oxidases/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Surface Plasmon Resonance , src Homology Domains/geneticsABSTRACT
Integrin-dependent adhesion and signaling are regulated by conformational changes whose details remain controversial. Crystallography revealed bent shapes for resting and primed integrin ectodomains, whereas large, ligand-induced rearrangements in other constructs suggested extension, "opening," and tail separation. We have used experimental/computed hydrodynamics to discriminate among different alpha(v)beta(3) and alpha(IIb)beta(3) atomic models built on X-ray, NMR, and EM data. In contrast with X-ray structures and EM maps, hydrodynamics indicate that resting integrins are already extended. Furthermore, the hydrodynamics of an alpha(v)beta(3) ectodomain-fibronectin fragment complex support opening via additional head region conformational changes (hybrid domain swing-out), but without tail separation. Likewise, frictional changes induced by priming agents in full-length alpha(IIb)beta(3) correlate well with the swing-out coupled to a simple transmembrane helix shift in an extended, electron tomography-based model. Extension and immediate tail separation are then uncoupled from head region rearrangements following activation, thus underscoring integrins' delicate, finely tuned plasticity.
Subject(s)
Integrin alphaVbeta3/chemistry , Models, Molecular , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Crystallography, X-Ray , Fibronectins/chemistry , Integrin alphaVbeta3/ultrastructure , Intercellular Signaling Peptides and Proteins , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/ultrastructure , Protein Binding , Protein Structure, Tertiary , Water/chemistryABSTRACT
This investigation examined the molecular mechanisms that enable the alphaIIbbeta3 integrin to bind efficiently, tightly, and selectively to echistatin, an RGD disintegrin. We used surface plasmon resonance spectroscopy to measure the rate, extent, and stability of complexes formed between micellar alphaIIbbeta3 and recombinant echistatin (rEch) mutants, immobilized on the surface of a biosensor chip. alphaIIbbeta3 bound readily and tightly to wild-type RGD-rEch and RGDF-rEch but not to RGA-rEch or AGD-rEch, demonstrating that both of those charged moieties contribute to integrin recognition. van't Hoff analysis of the temperature dependence of the RGD-rEch K d data yielded an unfavorable enthalpy change, Delta H degrees = 14 +/- 3 kcal/mol, offset by a favorable entropy term, TDelta S degrees = 23 +/- 3 kcal/mol. Eyring analysis of the temperature dependence of the kinetic parameters yielded Delta H a degrees (++) = 9 +/- 2 kcal/mol and TDelta S a degrees (++) = -4 +/- 2 kcal/mol, indicating that a substantial activation enthalpy barrier and a smaller activation entropy hinder assembly of the encounter complex. Thus, equilibrium thermodynamic data demonstrate that entropy is the dominant factor stabilizing integrin:echistatin binding, while transition-state thermodynamic parameters indicate that the rate of complex formation is enthalpy-limited. When electrostatic contacts are the major source of receptor:ligand stability, theory and experiment indicate that entropy-favorable ion-pair desolvation often provides the driving force for molecular recognition.
Subject(s)
Entropy , Peptides/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Surface Plasmon Resonance/methods , Biosensing Techniques , Humans , Intercellular Signaling Peptides and Proteins , Kinetics , Peptides/chemistry , Peptides/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Temperature , ThermodynamicsABSTRACT
This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alphaIIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming--i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.