离子交换色谱法研究蛋白质与RNA的结合特异性

RNA是以磷酸二酯键相连的核苷酸组成的聚阴离子,而蛋白质在PH小于它的等电点的缓冲溶液中带有正电性.因此,它们在离子交换色谱分子时能出现在差异很大的洗脱组分中.据此.我们设计了用离子交换色谱法研究蛋白质与RNA的结合特异性方法,用以研究位于T细胞核内的具有RNA结合活性的蛋白质人环素33(hCyP33)与不同RNA的结合特性.结果表明,人亲环素33只有ply(A)尾序列结构的mRNA,即poly(A)^ RNA发生特异性结合.     

RNA binding and thiolytic stability of a quinoline-containing helix-threading peptide

Helix-threading peptides (HTPs) bind selectively to sites predisposed to intercalation in folded RNA molecules placing peptide functional groups into the dissimilar grooves of the duplex. Here we report the design and synthesis of new HTPs with quinoline as the intercalation domain. A quinoline-containing HTP is shown to bind selectively to duplex RNA binding sites. Furthermore, the affinity cleavage pattern generated using an EDTA.Fe modified derivative is consistent with minor groove localization of its N-terminus. This compound binds base-pair steps flanked by single nucleotide bulges on the 3' side on both strands, whereas bulges on the 5' side of the intercalation site do not support binding. Furthermore, unlike acridine HTPs, the quinoline compound is resistant to thiolytic degradation that leads to loss of RNA-binding activity. The RNA-binding selectivity and stability observed for quinoline-containing HTPs make them excellent candidates for further development as regulators of intracellular RNA function.     

分子信标研究5-氟尿嘧啶对鼻咽癌HNE1细胞p33ING1基因转录水平的影响

p33ING1作为一种重要的抑癌基因，在乳腺癌及胃癌等恶性肿瘤细胞中的mRNA表达水平显著低于癌旁正常组织细胞中的表达，并导致这些肿瘤细胞的侵袭转移能力增强，对化疗药物及r射线和紫外线处理的敏感性降低。     

A comprehensive in Silico analysis of the functional and structural impact of single nucleotide polymorphisms (SNPs) in the human IL-33 gene

Interleukin 33 (IL-33) is the latest member of the IL-1 cytokine family, which plays both pro - and anti-inflammatory functions. Numerous Single-nucleotide polymorphisms (SNPs) in the IL-33 gene have been recognized to be associated with a vast variety of inflammatory disorders. SNPs associated studies have become a crucial approach in uncovering the genetic background of human diseases. However, distinguishing the functional SNPs in a disease-related gene from a pool of both functional and neutral SNPs is a major challenge and needs multiple experiments of hundreds or thousands of SNPs in candidate genes. This study aimed to identify the possible deleterious SNPs in the IL-33 gene using bioinformatics predictive tools. The nonsynonymous SNPs (nsSNPs) were analyzed by SIFT, PolyPhen, PROVEAN, SNP&GO, MutPred, SNAP, PhD SNP, and I-Mutant tools. The Non-coding SNPs (ncSNPs) were also analyzed by SNPinfo and RegulomeDB tools. In conclusion, our in-silico analysis predicted 5 nsSNPs and 22 ncSNPs as potential candidates in the IL-33 gene for future genetic association studies.     

Design and synthesis of artificial ribonucleases based on 1,4-diazabicyclo[2.2.2]octane and imidazole

The review surveys the results of our studies devoted to the design of highly efficient catalysts of hydrolysis of the phosphodiester bonds in RNA. These catalysts contain the imidazole residue in the catalytic domain, one or several bis-quaternized rings of 1,4-diazabicyclo[2.2.2]octane as a polycationic RNA-binding domain, and a lipophilic radical. A versatile approach to artificial ribonucleases of this type was proposed, which allows one to vary not only the number of positive charges in the RNA-binding domain, the structure of the catalytic site, and their mutual arrangement but also the domain structure of the molecule as a whole. Analysis of the catalytic properties of the synthesized constructs makes it possible to optimize the domain structure and the geometry of the molecule ensuring its maximum ribonuclease activity.     

DNA and RNA noncovalent interaction of platinum(II) polypyridine complexes

A comparative investigation of the noncovalent interaction of the platinum(II) polypyridine complexes [Pt(dipy)(n-Rpy)2]2+ and [Pt(4,4'-Me2dipy)(2-Rpy)2]2+ (dipy = 2,2'-dipyridine; Me = CH3; n = 2-4; R = H or CH3) with double-helical DNA (calf thymus) and RNA [poly(A).poly(U)] has been conducted. With the exception of [Pt(dipy)(2-Mepy)2]2+, all of the complexes interact strongly, by intercalation, with both nucleic acids giving rise to large changes in the electronic spectra and induced circular dichroism signals; in addition, viscosity experiments on rodlike DNA and RNA show that both biopolymers elongate upon interaction with the complexes. The binding constant values, KB, determined at 25 degrees C, indicate that, at 0.101 M ionic strength, the affinity for poly(A).poly(U) is strongly dependent on the complexes nature, while for DNA it is leveled off. [Pt(dipy)(2-Mepy)2]2+ binds to DNA but does not interact appreciably with poly(A).poly(U).     

Rational Design,Synthesis and Evaluation of Oxazolo[4,5-c]-quinolinone Analogs as Novel Interleukin-33 Inhibitors

Interleukin-33 (IL-33) is an epithelial-derived cytokine that plays an important role in immune-mediated diseases such as asthma, atopic dermatitis, and rheumatoid arthritis. Although IL-33 is considered a potential target for the treatment of allergy-related diseases, no small molecule that inhibits IL-33 has been reported. Based on the structure-activity relationship and in vitro 2D NMR studies employing ¹⁵N-labeled IL-33, we identified that the oxazolo[4,5-c]-quinolinone analog 7 c binds to the interface region of IL-33 and IL-33 receptor (ST2), an orphan receptor of the IL-1 receptor family. Compound 7 c effectively inhibited the production of IL-6 in human mast cells in a dose-dependent manner. Compound 7 c is the first low molecular weight IL-33 inhibitor and may be used as a prototype molecule for structural optimization and investigation of the IL-33/ST2 signaling pathway.     

Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.     

Unusually short RNA sequences: design of a 13-mer RNA that selectively binds and recognizes theophylline

RNA plays critical roles in numerous biological processes and constitutes valuable therapeutic targets. RNA is significant not only for its roles in transmitting the genetic code but also for its enzymatic functions in ribozymes and in peptide bond formation in ribosomes. Recent studies have shown that RNAs containing as few as 22 nucleotides can be key elements in cellular functions. This suggests the possibility of using short RNAs as regulatory elements. Here, we show that ligand recognition and selectivity by RNA molecules can occur with only the presence of a binding pocket and as few as six additional scaffolding nucleotides holding the binding pocket in place. A 13-mer RNA truncation of a 33-mer aptamer for theophylline preserves the ability to bind to theophylline and to discriminate against the structurally similar compound caffeine. The truncated aptamer retains nearly all of the same structural elements in its binding site as those present in the original aptamer. This is the first demonstration of selective ligand binding by a 13-mer RNA.     

RNA-binding residues in sequence space: Conservation and interaction patterns

RNA-binding proteins (RBPs) perform fundamental and diverse functions within the cell. Approximately 15% of proteins sequences are annotated as RNA-binding, but with a significant number of proteins without functional annotation, many RBPs are yet to be identified. A percentage of uncharacterised proteins can be annotated by transferring functional information from proteins sharing significant sequence homology. However, genomes contain a significant number of orphan open reading frames (ORFs) that do not share significant sequence similarity to other ORFs, but correspond to functional proteins. Hence methods for protein function annotation that go beyond sequence homology are essential. One method of annotation is the identification of ligands that bind to proteins, through the characterisation of binding site residues. In the current work RNA-binding residues (RBRs) are characterised in terms of their evolutionary conservation and the patterns they form in sequence space. The potential for such characteristics to be used to identify RBPs from sequence is then evaluated.In the current work the conservation of residues in 261 RBPs is compared for (a) RBRs vs. non-RBRs surface residues, and for (b) specific and non-specific RBRs. The analysis shows that RBRs are more conserved than other surface residues, and RBRs hydrogen-bonded to the RNA backbone are more conserved than those making hydrogen bonds to RNA bases. This observed conservation of RBRs was then used to inform the construction of RBR sequence patterns from known protein–RNA structures. A series of RBR patterns were generated for a case study protein aspartyl-tRNA synthetase bound to tRNA; and used to differentiate between RNA-binding and non-RNA-binding protein sequences. Six sequence patterns performed with high precision values of >80% and recall values 7 times that of an homology search. When the method was expanded to the complete dataset of 261 proteins, many patterns were of poor predictive value, as they had not been manipulated on a family-specific basis. However, two patterns with precision values ≥85% were used to make function predictions for a set of hypothetical proteins. This revealed a number of potential RBPs that require experimental verification.     

p‐Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic‐Binding Proteins

Identification of arsenic‐binding proteins is important for understanding arsenic health effects and for developing arsenic‐based therapeutics. We report here a strategy for the capture and identification of arsenic‐binding proteins in living cells. We designed an azide‐labeled arsenical, p‐azidophenylarsenoxide (PAzPAO), to serve bio‐orthogonal functions: the trivalent arsenical group binds to cellular proteins in situ, and the azide group facilitates click chemistry with dibenzylcyclooctyne. The selective and efficient capture of arsenic‐binding proteins enables subsequent enrichment and identification by shotgun proteomics. Applications of the technique are demonstrated using the A549 human lung carcinoma cells and two in vitro model systems. The technique enables the capture and identification of 48 arsenic‐binding proteins in A549 cells incubated with PAzPAO. Among the identified proteins are a series of antioxidant proteins (e.g., thioredoxin, peroxiredoxin, peroxide reductase, glutathione reductase, and protein disulfide isomerase) and glyceraldehyde‐3‐phosphate dehydrogenase. Identification of these functional proteins, along with studies of arsenic binding and enzymatic inhibition, points to these proteins as potential molecular targets that play important roles in arsenic‐induced health effects and in cancer treatment.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

Segmental,Domain‐Selective Perdeuteration and Small‐Angle Neutron Scattering for Structural Analysis of Multi‐Domain Proteins

Multi‐domain proteins play critical roles in fine‐tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi‐domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA‐1 using Sortase A mediated protein ligation. We show that domain‐selective perdeuteration combined with contrast‐matched small‐angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi‐domain proteins and changes induced by ligand binding.     

A quantitative study of the enzymatic activity of horseradish peroxidase at a planar poly(acrylic acid) brush

The enzymatic activity of horseradish peroxidase (HRP) has been quantified at a planar poly(acrylic acid) (PAA) brush. A PAA brush is known to exhibit an unusual protein binding affinity, since proteins adsorb at low ionic strength only. At elevated ionic strengths of a few 100 mM proteins desorb, and the PAA brush becomes largely protein resistant. In this study, HRP was used to catalyze the oxidation of Amplex Red to resorufin by hydrogen peroxide. The fluorescence of resorufin was recorded using a microplate reader. As compared to a bare silica surface, the enzymatic activity of HRP is higher by more than one order of magnitude at a PAA brush. This increase results from a higher degree of adsorption and a reasonable preservation of the HRP activity. Upon adsorption at a PAA brush from a 20 mU/mL (0.1 μg/mL) solution, the molecular enzymatic activity of HRP is reduced to about 11%. However, when a HRP molecule is desorbed from a PAA brush by increasing the ionic strength, a gain of the molecular enzymatic activity by only 52% can be observed. Overall, this study illustrates the potential applicability of a PAA brush as a biocompatible material coating.     

Sanglifehrin-cyclophilin interaction: degradation work,synthetic macrocyclic analogues,X-ray crystal structure,and binding data

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.     

Crystallographic and spectroscopic evidence for high affinity binding of FeEDTA(H2O)- to the periplasmic nickel transporter NikA

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.