RESUMO
Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (â¼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.
Assuntos
Fibroínas , Aranhas , Animais , Fibroínas/química , Seda/química , Dimerização , Espectroscopia de Ressonância Magnética , Aranhas/metabolismoRESUMO
Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Luminol/metabolismo , Mioglobina/metabolismo , Peroxidase/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Simulação por Computador , Heme/metabolismo , Luminescência , Luminol/química , Ressonância Magnética Nuclear Biomolecular , Técnica de Seleção de Aptâmeros , Especificidade por SubstratoRESUMO
Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Mitocôndrias/genética , Peptídeos/genética , Endossomos/química , Endossomos/genética , Vetores Genéticos , Humanos , Mitocôndrias/patologia , Peptídeos/química , TransfecçãoRESUMO
The stereochemical theory claims that primitive coded translation initially occurred in the RNA world by RNA-directed amino acid coupling. In this study, we show that the HIV Tat aptamer RNA is capable of recognizing two consecutive arginine residues within the Tat peptide, thus demonstrating how RNA might be able to position two amino acids for sequence-specific coupling. We also show that this RNA can act as a template to accelerate the coupling of a single arginine residue to the N-terminal arginine residue of a peptide primer. The results might have implications for our understanding of the origin of translation.
Assuntos
Arginina/metabolismo , RNA Viral/metabolismo , Sequência de Aminoácidos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arginina/química , Sítios de Ligação , HIV/genética , HIV/metabolismo , Humanos , Conformação de Ácido Nucleico , Peptídeos/química , Peptídeos/metabolismo , RNA Viral/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Human APOBEC3G exhibits anti-human immunodeficiency virus-1 (HIV-1) activity by deaminating cytidines of the minus strand of HIV-1. Here, we report a solution structure of the C-terminal deaminase domain of wild-type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real-time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3' --> 5' order. Virus infectivity factor (Vif) of HIV-1 counteracts the anti-HIV-1 activity of APOBEC3G. The structure of the N-terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species-specific sensitivity of APOBEC3G to Vif action.
Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , HIV-1/metabolismo , Desaminase APOBEC-3G , Animais , Códon , Citidina/química , DNA de Cadeia Simples/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Mutação , Estrutura Terciária de Proteína , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismoRESUMO
INTRODUCTION: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained. METHODS: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation. RESULTS: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies. CONCLUSION: In particular, the flexible structure was induced by the A30P mutation.
Assuntos
alfa-Sinucleína , alfa-Sinucleína/química , Espectroscopia de Ressonância Magnética , MutaçãoRESUMO
A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP. A combination of NMR measurements, enzymatic and chemical mapping, and nucleotide mutation studies of the RNP-adenosine complex show that RNP interacts with the adenine ring of adenosine by forming a U:A:U triple with two invariant U nucleotides. The observed recognition mode for the adenine ring is different from those of RNA aptamers for ATP derivatives reported previously. The RNP-adenosine complex is folded into a particular structure by formation of the U:A:U triple and a Hoogsteen type A:U base pair. This recognition mechanism was successfully utilized to convert the substrate-binding specificity of RNP from ATP- to GTP-binding with a C(+):G:C triple recognition mode.
Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Aptâmeros de Nucleotídeos/química , Espectroscopia de Ressonância Magnética , Conformação ProteicaRESUMO
RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its beta-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad-hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer's disease.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Príons/química , Animais , Sítios de Ligação , Bovinos , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Príons/antagonistas & inibidores , Príons/metabolismoRESUMO
HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more ß-structures at the ß2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.
Assuntos
Substituição de Aminoácidos , Proteína do Núcleo p24 do HIV/química , HIV-1/química , Modelos Moleculares , Mutação de Sentido Incorreto , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
Fully modified 4'-thioDNA, an oligonucleotide only comprising 2'-deoxy-4'-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4'-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4'-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4'-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3'-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4'-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4'-thioDNA showed that partially modified 4'-thioDNA that contained some 2'-deoxy-4'-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4'-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4'-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.
Assuntos
DNA Forma A/química , Tionucleotídeos/química , Configuração de Carboidratos , DNA/química , DNA Forma A/metabolismo , Endodesoxirribonucleases/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Soluções , Tionucleotídeos/metabolismoRESUMO
We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1-telomerase RNA-telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1-telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA.
Assuntos
DNA/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , RNA/química , Telomerase/química , Telômero/química , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , RNA/metabolismo , Telomerase/metabolismoRESUMO
The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.
Assuntos
Amidas/química , Proteínas Intrinsicamente Desordenadas/genética , Prótons , alfa-Sinucleína/química , Humanos , Proteínas Intrinsicamente Desordenadas/química , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Proteínas Mutantes/química , Mutação , Doença de Parkinson/genética , Estrutura Secundária de Proteína , Temperatura , alfa-Sinucleína/genéticaRESUMO
Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300 mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Fibroínas/química , Espectroscopia de Prótons por Ressonância Magnética , Aranhas/metabolismo , Sequência de Aminoácidos , Estruturas Animais , Animais , Concentração de Íons de Hidrogênio , Isótopos de Nitrogênio , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.
Assuntos
Cátions/química , Elementos da Série dos Lantanídeos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Sítios de Ligação , Calorimetria/métodos , Cátions/metabolismo , Cristalografia por Raios X , Elementos da Série dos Lantanídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Peptídeos/metabolismo , Termodinâmica , Água/químicaRESUMO
Human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The single-stranded telomere terminus can adopt the structure of a G-quadruplex, which is of particular important for anticancer drug discovery many researchers have reported various G-quadruplex structures in the human telomere. Although the human telomere consists of a number of tandem repeats, higher-order G-quadruplex structures are less discussed due to the complexity of the structures. Here we examined the orientation of the ends of the G-quadruplex structures with consideration given to higher-order structures. We prepared end-extended and (Br)G-substituted oligonucleotides. Native PAGE analysis, CD measurements and NMR spectroscopy showed that the ends of stable G-quadruplex structures point in opposite directions. Our results indicate that the human telomere DNA is likely to form rod-like higher-order structures. This may provide important information for understanding telomere structure and the development of telomere G-quadruplex-binding molecules as telomerase inhibitors.
Assuntos
Quadruplex G , Oligonucleotídeos/química , Telômero/química , Sequência de Bases , Dicroísmo Circular , Eletroforese em Gel de Ágar , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for ß-sheet formation.
Assuntos
Conformação Proteica em Folha beta , Seda/química , Animais , Cloretos/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Sódio/química , Solubilidade , AranhasRESUMO
The objective of this study is to control and elucidate the mechanism of molecular degradation in a polysaccharide hydrogel. Glycidyl methacrylate (GMA) immobilized dextran (Dex-GMA) was oxidized by periodate to introduce aldehyde groups (oxidized Dex-GMA). The hydrogel was formed by the addition of dithiothreitol to the oxidized Dex-GMA solution through thiol Michael addition with the preservation of the aldehyde group for degradation points. It was experimentally determined that the degradation of this hydrogel can be controlled by the addition of amino groups and the speed of degradation can be controlled independently of mechanical properties because crosslinking and degradation points are different. In addition, the molecular mechanism of the crosslinking between the thiol and aldehyde groups was found to control the degradation of dextran derivatives. It is expected that these results will be beneficial in the design of polymer materials in which the speed of degradation can be precisely controlled. In addition, the cytotoxicity of oxidized Dex-GMA was approximately 3000 times lower than that of glutaraldehyde. The low cytotoxicity of the aldehyde in oxidized Dex-GMA was the likely reason for the harmless functionalized polysaccharide material. Possible future clinical applications include cell scaffolds in regenerative medicine and carriers for drug delivery systems.
RESUMO
The ß-sheet is the key structure underlying the excellent mechanical properties of spider silk. However, the comprehensive mechanism underlying ß-sheet formation from soluble silk proteins during the transition into insoluble stable fibers has not been elucidated. Notably, the assembly of repetitive domains that dominate the length of the protein chains and structural features within the spun fibers has not been clarified. Here we determine the conformation and dynamics of the soluble precursor of the repetitive domain of spider silk using solution-state NMR, far-UV circular dichroism and vibrational circular dichroism. The soluble repetitive domain contains two major populations: ~65% random coil and ~24% polyproline type II helix (PPII helix). The PPII helix conformation in the glycine-rich region is proposed as a soluble prefibrillar region that subsequently undergoes intramolecular interactions. These findings unravel the mechanism underlying the initial step of ß-sheet formation, which is an extremely rapid process during spider silk assembly.
Assuntos
Conformação Proteica em Folha beta/fisiologia , Seda/química , Aranhas/metabolismo , Animais , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Estresse Mecânico , Resistência à Tração/fisiologiaRESUMO
The structure of human telomeric DNA is controversial; it depends upon the sequence contexts and the methodologies used to determine it. The solution structure in the presence of K(+) is particularly interesting, but the structure is yet to be elucidated, due to possible conformational heterogeneity. Here, a unique strategy is applied to stabilize one such structure in a K(+) solution by substituting guanosines with 8-bromoguanosines at proper positions. The resulting spectra are cleaner and led to determination of the structure at a high atomic resolution. This demonstrates that the application of 8-bromoguanosine is a powerful tool to overcome the difficulty of nucleic acid structure determination arising from conformational heterogeneity. The obtained structure is a mixed-parallel/antiparallel quadruplex. The structure of telomeric DNA was recently reported in another study, in which stabilization was brought about by mutation and resultant additional interactions [Luu KN, Phan AT, Kuryavyi V, Lacroix L & Patel DJ (2006) Structure of the human telomere in K(+) solution: an intramolecular (3+1) G-quadruplex scaffold. J Am Chem Soc 128, 9963-9970]. The structure of the guanine tracts was similar between the two. However, a difference was seen for loops connecting guanine tracts, which may play a role in the higher order arrangement of telomeres. Our structure can be utilized to design a small molecule which stabilizes the quadruplex. This type of molecule is supposed to inhibit a telomerase and thus is expected to be a candidate anticancer drug.
Assuntos
DNA/química , DNA/genética , Guanosina/análogos & derivados , Conformação de Ácido Nucleico , Potássio/química , Telômero/genética , Sequência de Bases , Cátions/química , Guanina/química , Guanosina/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Potássio/farmacologia , SoluçõesRESUMO
Human vaults are the largest cytoplasmic ribonucleoprotein and are overexpressed in cancer cells. Vaults reportedly function in the extrusion of xenobiotics from the nuclei of resistant cells, but the interactions of xenobiotics with the vault-associated proteins or non-coding RNAs have never been directly observed. In the present study, we show that vault RNAs (vRNAs), specifically the hvg-1 and hvg-2 RNAs, bind to a chemotherapeutic compound, mitoxantrone. Using an in-line probing assay (spontaneous transesterification of RNA linkages), we have identified the mitoxantrone binding region within the vRNAs. In addition, we analyzed the interactions between vRNAs and mitoxantrone in the cellular milieu, using an in vitro translation inhibition assay. Taken together, our results clearly suggest that vRNAs have the ability to bind certain chemotherapeutic compounds and these interactions may play an important role in vault function, by participating in the export of toxic compounds.