Molecular recognition of bioreceptors and enzymes relies on orthogonal interactions with small molecules within their cavity. To date, Chinese scientists have developed three types of strategies for introducing active...Molecular recognition of bioreceptors and enzymes relies on orthogonal interactions with small molecules within their cavity. To date, Chinese scientists have developed three types of strategies for introducing active sites inside the cavity of macrocyclic arenes to better mimic molecular recognition of bioreceptors and enzymes.The editorial aims to enlighten scientists in this field when they develop novel macrocycles for molecular recognition, supramolecular assembly, and applications.展开更多
Accurate identification of RNA-ligand binding sites is essential for elucidating RNA function and advancing structurebased drug discovery.Here,we present RLsite,a novel deep learning framework that integrates energy-,...Accurate identification of RNA-ligand binding sites is essential for elucidating RNA function and advancing structurebased drug discovery.Here,we present RLsite,a novel deep learning framework that integrates energy-,structure-and sequence-based features to predict nucleotide-level binding sites with high accuracy.RLsite leverages energy-based threedimensional representations,obtained from atomic probe interactions using a pre-trained ITScore-NL potential,and models their contextual features through a 3D convolutional neural network(3D-CNN)augmented with self-attention.In parallel,structure-based features,including network properties,Laplacian norm,and solvent-accessible surface area,together with sequence-based evolutionary constraint scores,are mapped along the RNA sequence and used as sequential descriptors.These descriptors are modeled using a bidirectional long short-term memory(BiLSTM)network enhanced with multihead self-attention.By effectively fusing these complementary modalities,RLsite achieves robust and precise binding site prediction.Extensive evaluations across four diverse RNA-ligand benchmark datasets demonstrate that RLsite consistently outperforms state-of-the-art methods in terms of precision,recall,Matthews correlation coefficient(MCC),area under the curve(AUC),and overall robustness.Notably,on a particularly challenging test set composed of RNA structures containing junctions,RLsite surpasses the second-best method by 7.3%in precision,3.4%in recall,7.5%in MCC,and 10.8%in AUC,highlighting its potential as a powerful tool for RNA-targeted molecular design.展开更多
The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS...The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS). This study offers supporting data of the interaction among drug and amino acids and peptides, which could potentially explain the cytotoxic and mutagenic effects of the drug. Collision-induced dissociation(CID) experiment demonstrated that under the same collision energy, the amino group combined with NL-101 adducts are sensitive and often produce more fragment ions; the carboxyl group combined with NL-101 adducts are hard to break and display fewer fragment ions. In addition, when other group(like sulfhydryl group) of amino acids binds to NL-101, CID spectra show different fragmentation pattern. These differences could display structural information about the drug adducts and be utilized as location of the authentic binding sites.展开更多
Understanding the functional effects of genetic variants is crucial in modern genomics and genetics. Transcription factor binding sites （TFBSs） are one of the most important cis-regulatory elements. While multiple t...Understanding the functional effects of genetic variants is crucial in modern genomics and genetics. Transcription factor binding sites （TFBSs） are one of the most important cis-regulatory elements. While multiple tools have been developed to assess functional effects of genetic variants at TFBSs, they usually assume that each variant works in isolation and neglect the potential ＂interference＂ among multiple variants within the same TFBS. In this study, we presented COPE-TFBS （Context-Oriented Predictor for variant Effect on Transcription Factor Binding Site）, a novel method that considers sequence context to accurately predict variant effects on TFBSs. We systematically re-analyzed the sequencing data from both the 1000 Genomes Project and the Genotype-Tissue Expression （GTEx） Project via COPE-TFBS, and identified numbers of novel TFBSs, transformed TFBSs and discordantly annotated TFBSs resulting from multiple variants, further highlighting the necessity of sequence context in accurately annotating genetic variants.展开更多
In this paper a new continuous variable called core-ratio is defined to describe the probability for a residue to be in a binding site, thereby replacing the previous binary description of the interface residue using ...In this paper a new continuous variable called core-ratio is defined to describe the probability for a residue to be in a binding site, thereby replacing the previous binary description of the interface residue using 0 and 1. So we can use the support vector machine regression method to fit the core-ratio value and predict the protein binding sites. We also design a new group of physical and chemical descriptors to characterize the binding sites. The new descriptors are more effective, with an averaging procedure used. Our test shows that much better prediction results can be obtained by the support vector regression （SVR） method than by the support vector classification method.展开更多
AIM To identify punitive transcriptional factor binding sites(TFBS) from regulatory single nucleotide polymorphisms(rS NPs) that are significantly associated with disease.METHODS The genome-wide association studies ha...AIM To identify punitive transcriptional factor binding sites(TFBS) from regulatory single nucleotide polymorphisms(rS NPs) that are significantly associated with disease.METHODS The genome-wide association studies have provided us with nearly 6500 disease or trait-predisposing SNPs where 93% are located within non-coding regions such as gene regulatory or intergenic areas of the genome. In the regulatory region of a gene, a SNP can change the DNA sequence of a transcriptional factor(TF) motif and in turn may affect the process of gene regulation. SNP changes that affect gene expression and impact gene regulatory sequences such as promoters, enhancers, and silencers are known as rS NPs. Computational tools can be used to identify unique punitive TFBS created by rS NPs that are associated with disease or sickness. Computational analysis was used to identify punitive TFBS generated by the alleles of these rS NPs.RESULTS r SNPs within nine genes that have been significantly associated with disease or sickness were used to illustrate the tremendous diversity of punitive unique TFBS that can be generated by their alleles. The genes studied are the adrenergic, beta, receptor kinase 1, the v-akt murine thymoma viral oncogene homolog 3, the activating transcription factor 3, the type 2 demodkinase gene, the endothetal Per-Arnt-Sim domain protein 1, the lysosomal acid lipase A, the signal Transducer and Activator of Transcription 4, the thromboxane A2 receptor and the vascular endothelial growth factor A. From this sampling of SNPs among the nine genes, there are 73 potential unique TFBS generated by the common alleles comparedto 124 generated by the minor alleles indicating the tremendous diversity of potential TFs that are capable of regulating these genes.CONCLUSION From the diversity of unique punitive binding sites for TFs, it was found that some TFs play a role in the disease or sickness being studied.展开更多
In this study platelet 3H-paroxetine binding site was studied in 16 depressed pa-tients and 16 healthy volunteers. We found that the mean Bmax of 3H-paroxetine binding on theplatelets of depressed patients was signifi...In this study platelet 3H-paroxetine binding site was studied in 16 depressed pa-tients and 16 healthy volunteers. We found that the mean Bmax of 3H-paroxetine binding on theplatelets of depressed patients was significantly lower than that of normal controls. After treated withamitriptyline or electro-acupuncture for 6 weeks, the density of paroxetine binding sites increased to-wards normal in well responded patients. But no significant difference was found between electro-acupuncture and amitriptyline as compared in their effects on 3H-paroxetine binding sites.展开更多
Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hya...Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the specificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of kcat/KM remained moderately constant around 8 mM-1s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.展开更多
A method for analyzing the protein site similarity was devised aiming at understanding selectivity of homologous proteins and guiding the design of new drugs. The method is based on calculating Cα distances between s...A method for analyzing the protein site similarity was devised aiming at understanding selectivity of homologous proteins and guiding the design of new drugs. The method is based on calculating Cα distances between selected pocket residues and subsequent analysis by multivariate methods. Five closely related serine proteases, the coagulation factors II, VII, IX, X, and XI, were studied and their pocket similarity was illustrated by PCA clustering. OPLS-DA was then applied to identify the residues responsible for the variation. By combining these two multivariate methods, we could successfully cluster the different proteases according to class and identify the important residues responsible for the observed variation.展开更多
This study focuses on bioinformatics search for new regulatory structures in the non-coding DNA, located around the patterns of gene expression levels changed significantly in response to oxidative stress. Hypothesize...This study focuses on bioinformatics search for new regulatory structures in the non-coding DNA, located around the patterns of gene expression levels changed significantly in response to oxidative stress. Hypothesized that all of the genes increase the expression in response to oxidative stress may have the same motifs in non-coding DNA. To search for motifs created an integrated collection database of transcription binding sites - JASPAR, TRANSFAC, Hocomoco TF Homo sapiens, Uniprobe TF Mus musculus. Two types of regulatory regions: the promoter region and the sequence with the capture of potential cis-regulatory modules. In the regulatory regions of genes increase the expression in response to oxidative stress, in contrast to the gene expression level did not change, families of transcription factors identified SOX (1-30) and HX (A, B, C, D).展开更多
Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein...Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein-protein interactions occur on comparatively fiat regions whereas protein-ligand bindings involve crevices. In the search for such sites various approaches have been designed and developed each of which is algorithmically unique. The use of grid units or voxels has been demonstrated in early studies with relatively good results obtained. We present here an approximated approach comprising of the use of voxels and computer vision methods in the search for ligand-binding areas. Each test protein is modelled and analysed in 2D with all corresponding residues graphically presented for successfully identified sites. The study was carried out on 2 sets of proteins： FK506-bound proteins and heme-bound proteins with promising results obtained for all test cases.展开更多
BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin...BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.展开更多
Herein we investigated the effect of primer binding site polymorphisms in achieving correct genotyping when a mismatch occurs in distinct positions of the primer sequence. For that purpose primer sequences were design...Herein we investigated the effect of primer binding site polymorphisms in achieving correct genotyping when a mismatch occurs in distinct positions of the primer sequence. For that purpose primer sequences were designed in order to carry either allelic form at the 3’ end and at 3 bp, 5 bp and 7 bp apart from the 3’ end of an intronic polymorphism (rs2247836) observed in phenylalanine hydroxylase (PAH) gene. For one of the alleles annealing failure was obtained when the mismatch occurs at all the four primer-site locations. Primer sequences carrying the alternative SNP allele resulted to be less specific as the distance to the primer-3’ end was increased. Altogether, these results revealthat effects in the extension of the annealing failure is allele and mismatch-position dependent.展开更多
With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measu...With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.展开更多
AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect an...AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.展开更多
OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues i...OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues in GABAAreceptors(GABAARs) were also investigated.METHODS Using a zebrafish larvae locomotion model,we investigated the detailed dose-dependent effects of diazepam and other BZDs on zebrafish larvae behaviors,with a focus on their high-dose effects.We then evaluated the influence of the classical BZD antagonist flumazenil,GABAARs antagonist bicuculline,and the antagonist of a proposed BZD binding site in α4/6β3δ subtype receptor Ro15-4513 on BZDs induced immobility.Using wholecell patch clamp electrophysiological recordings on recombinant GABAARs,we investigated the modulation of diazepam alone or combined with flumazenil on GABA-elicited current in wildtype and mutated receptors.RESULTS Diazepam dose-dependently decreased the locomotor activities of zebrafish larvae at doses of 0.4,2,10,20,30,50 and 75 mg·L^(-1).The hypolocomotion(sedation-like state) induced by diazepam at10 and 20 mg·L^(-1) were effectively antagonized by flumazenil with EC150 of 0.086 mg·L-and1.295 mg·L^(-1),while the immobility(anesthesialike state) induced by diazepam at 30 mg·L^(-1) was abolished by bicuculline(3 mg·L^(-1)),but not affected by flumazenil(even at concentration up to150 mg·L^(-1)) or Ro15-4513(100 mg·L^(-1)).The immobility induced by clonazepam and lorazepam(100 mg·L^(-1)) was also resistant to flumazenil(100 mg·L^(-1)).In the α1β2γ2 subtype receptor expressed in HEK293 T cells,diazepam dose-dependently potentiated GABA-elicited current,and this potentiation was effectively antagonized by flumazenil(100 μmol·L^(-1)).However,in α1β2 subtype receptor,diazepam(150 μmol·L^(-1)) induced potentiation was insensitive to flumazenil(100 μmol·L^(-1)),but was abolished by the mutation of β2 N265 I.CONCLUSION These results provide direct in vivo evidence for the nonclassical binding sites,which may be located at the second transmembrane domain of GABAAR,mediate BZD-induced anesthesia.展开更多
The orientation of the biological molecule immobi-lized on a solid surface has been critical in devel-opment of various applications. In this study, ori-entation of antibody was retained by protecting the antigen-bind...The orientation of the biological molecule immobi-lized on a solid surface has been critical in devel-opment of various applications. In this study, ori-entation of antibody was retained by protecting the antigen-binding site of the antibody prior to immo-bilization to -functionalized mixed self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 1-heptanethiol. More importantly, the number of immobilization bonds formed between each an-tigen-binding site protected antibody molecule and the solid surface was controlled by optimizing the mole fraction of the activated carboxyl group of the linker molecules in the mixed SAM. The amount of antibody used in this study was approximately equivalent to the amount for one monolayer surface coverage. The resulting activity of protected immo-bilized antibody was about 10 fold higher than that of random immobilized展开更多
The aim of this study was to compare the binding profile of a range of β2-adrenoceptor (β2-AR) agonists and antagonists in human lung tissue. Radioligand saturation and competition binding experiments were performed...The aim of this study was to compare the binding profile of a range of β2-adrenoceptor (β2-AR) agonists and antagonists in human lung tissue. Radioligand saturation and competition binding experiments were performed by filtration with a β2-AR antagonist ([3H]propranolol) or agonist ([3H]vilanterol) radioligand and membrane fragments generated from lung parenchyma in the presence of 100 μM guanosine 5’-[β,γ-imido]triphosphate (Gpp(NH)p). In membranes prepared from human lung parenchyma, carmoterol, formoterol, ICI118551, propranolol and salbutamol resulted in inhibition of [3H]vilanterol binding to levels that were significantly different from indacaterol, salmeterol and vilanterol (ANOVA, Bonferroni post-test, P < 0.001 except formoterol vs indacaterol where P < 0.01). Indacaterol and salmeterol resulted in inhibition of [3H]vilanterol binding to levels that were not significantly different from vilanterol (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol resulted in full inhibition of [3H]propranolol binding to levels not significantly different from ICI118551 (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol bind to an additional site in human lung parenchyma membranes that is distinct from the β2-AR.展开更多
Transcription factors (TFs) are the core sentinels of gene regulation functioning by binding to highly specific DNA sequences to activate or repress the recruitment of RNA polymerase. The ability to identify transcrip...Transcription factors (TFs) are the core sentinels of gene regulation functioning by binding to highly specific DNA sequences to activate or repress the recruitment of RNA polymerase. The ability to identify transcription factor binding sites (TFBSs) is necessary to understand gene regulation and infer regulatory networks. Despite the fact that bioinformatics tools have been developed for years to improve computational identification of TFBSs, the accurate prediction still remains changeling as DNA motifs recognized by TFs are typically short and often lack obvious patterns. In this study we introduced a new attribute-motif distribution pattern (MDP) to assist in TFBS prediction. MDP was developed using a TF distribution pattern curve generated by analyzing 25 yeast TFs and 37 of their experimentally validated binding motifs, followed by calculating a scoring value to quantify the reliability of each motif prediction. Finally, MDP was tested using another set of 7 TFs with known binding sites to in silico validate the approach. The method was further tested in a non-yeast system using the filamentous fungus Magnaporthe oryzae transcription factor MoCRZ1. We demonstrate superior prediction reranking results using MDP over the commonly used program MEME and the other four predictors. The data showed significant improvements in the ranking of validated TFBS and provides a more sensitive statistics based approach for motif discovery.展开更多
文摘Molecular recognition of bioreceptors and enzymes relies on orthogonal interactions with small molecules within their cavity. To date, Chinese scientists have developed three types of strategies for introducing active sites inside the cavity of macrocyclic arenes to better mimic molecular recognition of bioreceptors and enzymes.The editorial aims to enlighten scientists in this field when they develop novel macrocycles for molecular recognition, supramolecular assembly, and applications.
基金supported by the National Natural Science Foundation of China(Grant No.12204118)the Guizhou University Talent Fund(Grant No.[2022]30).
文摘Accurate identification of RNA-ligand binding sites is essential for elucidating RNA function and advancing structurebased drug discovery.Here,we present RLsite,a novel deep learning framework that integrates energy-,structure-and sequence-based features to predict nucleotide-level binding sites with high accuracy.RLsite leverages energy-based threedimensional representations,obtained from atomic probe interactions using a pre-trained ITScore-NL potential,and models their contextual features through a 3D convolutional neural network(3D-CNN)augmented with self-attention.In parallel,structure-based features,including network properties,Laplacian norm,and solvent-accessible surface area,together with sequence-based evolutionary constraint scores,are mapped along the RNA sequence and used as sequential descriptors.These descriptors are modeled using a bidirectional long short-term memory(BiLSTM)network enhanced with multihead self-attention.By effectively fusing these complementary modalities,RLsite achieves robust and precise binding site prediction.Extensive evaluations across four diverse RNA-ligand benchmark datasets demonstrate that RLsite consistently outperforms state-of-the-art methods in terms of precision,recall,Matthews correlation coefficient(MCC),area under the curve(AUC),and overall robustness.Notably,on a particularly challenging test set composed of RNA structures containing junctions,RLsite surpasses the second-best method by 7.3%in precision,3.4%in recall,7.5%in MCC,and 10.8%in AUC,highlighting its potential as a powerful tool for RNA-targeted molecular design.
基金supported by the National Natural Science Foundation of China (Nos. 21327010, 21372199)
文摘The binding between NL-101, a novel nitrogen mustard anti-cancer drug, with amino acids and peptides has been investigated by high performance liquid chromatography electrospray tandem mass spectrometry(HPLC/ESI-MS/MS). This study offers supporting data of the interaction among drug and amino acids and peptides, which could potentially explain the cytotoxic and mutagenic effects of the drug. Collision-induced dissociation(CID) experiment demonstrated that under the same collision energy, the amino group combined with NL-101 adducts are sensitive and often produce more fragment ions; the carboxyl group combined with NL-101 adducts are hard to break and display fewer fragment ions. In addition, when other group(like sulfhydryl group) of amino acids binds to NL-101, CID spectra show different fragmentation pattern. These differences could display structural information about the drug adducts and be utilized as location of the authentic binding sites.
基金supported by funds from the National Key R&D Program of China (2016YFC0901603)the China 863 Program (2015AA020108)+1 种基金the State Key Laboratory of Protein and Plant Gene Researchsupported in part by the National Program for Support of Top-notch Young Professionals
文摘Understanding the functional effects of genetic variants is crucial in modern genomics and genetics. Transcription factor binding sites （TFBSs） are one of the most important cis-regulatory elements. While multiple tools have been developed to assess functional effects of genetic variants at TFBSs, they usually assume that each variant works in isolation and neglect the potential ＂interference＂ among multiple variants within the same TFBS. In this study, we presented COPE-TFBS （Context-Oriented Predictor for variant Effect on Transcription Factor Binding Site）, a novel method that considers sequence context to accurately predict variant effects on TFBSs. We systematically re-analyzed the sequencing data from both the 1000 Genomes Project and the Genotype-Tissue Expression （GTEx） Project via COPE-TFBS, and identified numbers of novel TFBSs, transformed TFBSs and discordantly annotated TFBSs resulting from multiple variants, further highlighting the necessity of sequence context in accurately annotating genetic variants.
基金Project supported by the National Natural Science Foundation of China (Grant Nos. 10674172 and 10874229)
文摘In this paper a new continuous variable called core-ratio is defined to describe the probability for a residue to be in a binding site, thereby replacing the previous binary description of the interface residue using 0 and 1. So we can use the support vector machine regression method to fit the core-ratio value and predict the protein binding sites. We also design a new group of physical and chemical descriptors to characterize the binding sites. The new descriptors are more effective, with an averaging procedure used. Our test shows that much better prediction results can be obtained by the support vector regression （SVR） method than by the support vector classification method.
文摘AIM To identify punitive transcriptional factor binding sites(TFBS) from regulatory single nucleotide polymorphisms(rS NPs) that are significantly associated with disease.METHODS The genome-wide association studies have provided us with nearly 6500 disease or trait-predisposing SNPs where 93% are located within non-coding regions such as gene regulatory or intergenic areas of the genome. In the regulatory region of a gene, a SNP can change the DNA sequence of a transcriptional factor(TF) motif and in turn may affect the process of gene regulation. SNP changes that affect gene expression and impact gene regulatory sequences such as promoters, enhancers, and silencers are known as rS NPs. Computational tools can be used to identify unique punitive TFBS created by rS NPs that are associated with disease or sickness. Computational analysis was used to identify punitive TFBS generated by the alleles of these rS NPs.RESULTS r SNPs within nine genes that have been significantly associated with disease or sickness were used to illustrate the tremendous diversity of punitive unique TFBS that can be generated by their alleles. The genes studied are the adrenergic, beta, receptor kinase 1, the v-akt murine thymoma viral oncogene homolog 3, the activating transcription factor 3, the type 2 demodkinase gene, the endothetal Per-Arnt-Sim domain protein 1, the lysosomal acid lipase A, the signal Transducer and Activator of Transcription 4, the thromboxane A2 receptor and the vascular endothelial growth factor A. From this sampling of SNPs among the nine genes, there are 73 potential unique TFBS generated by the common alleles comparedto 124 generated by the minor alleles indicating the tremendous diversity of potential TFs that are capable of regulating these genes.CONCLUSION From the diversity of unique punitive binding sites for TFs, it was found that some TFs play a role in the disease or sickness being studied.
文摘In this study platelet 3H-paroxetine binding site was studied in 16 depressed pa-tients and 16 healthy volunteers. We found that the mean Bmax of 3H-paroxetine binding on theplatelets of depressed patients was significantly lower than that of normal controls. After treated withamitriptyline or electro-acupuncture for 6 weeks, the density of paroxetine binding sites increased to-wards normal in well responded patients. But no significant difference was found between electro-acupuncture and amitriptyline as compared in their effects on 3H-paroxetine binding sites.
文摘Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the specificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of kcat/KM remained moderately constant around 8 mM-1s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.
文摘A method for analyzing the protein site similarity was devised aiming at understanding selectivity of homologous proteins and guiding the design of new drugs. The method is based on calculating Cα distances between selected pocket residues and subsequent analysis by multivariate methods. Five closely related serine proteases, the coagulation factors II, VII, IX, X, and XI, were studied and their pocket similarity was illustrated by PCA clustering. OPLS-DA was then applied to identify the residues responsible for the variation. By combining these two multivariate methods, we could successfully cluster the different proteases according to class and identify the important residues responsible for the observed variation.
文摘This study focuses on bioinformatics search for new regulatory structures in the non-coding DNA, located around the patterns of gene expression levels changed significantly in response to oxidative stress. Hypothesized that all of the genes increase the expression in response to oxidative stress may have the same motifs in non-coding DNA. To search for motifs created an integrated collection database of transcription binding sites - JASPAR, TRANSFAC, Hocomoco TF Homo sapiens, Uniprobe TF Mus musculus. Two types of regulatory regions: the promoter region and the sequence with the capture of potential cis-regulatory modules. In the regulatory regions of genes increase the expression in response to oxidative stress, in contrast to the gene expression level did not change, families of transcription factors identified SOX (1-30) and HX (A, B, C, D).
文摘Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein-protein interactions occur on comparatively fiat regions whereas protein-ligand bindings involve crevices. In the search for such sites various approaches have been designed and developed each of which is algorithmically unique. The use of grid units or voxels has been demonstrated in early studies with relatively good results obtained. We present here an approximated approach comprising of the use of voxels and computer vision methods in the search for ligand-binding areas. Each test protein is modelled and analysed in 2D with all corresponding residues graphically presented for successfully identified sites. The study was carried out on 2 sets of proteins： FK506-bound proteins and heme-bound proteins with promising results obtained for all test cases.
基金Supported by Recurrent Grants from Institut National de la Santéet de la Recherche Médicale to the INSERM U1048.
文摘BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.
文摘Herein we investigated the effect of primer binding site polymorphisms in achieving correct genotyping when a mismatch occurs in distinct positions of the primer sequence. For that purpose primer sequences were designed in order to carry either allelic form at the 3’ end and at 3 bp, 5 bp and 7 bp apart from the 3’ end of an intronic polymorphism (rs2247836) observed in phenylalanine hydroxylase (PAH) gene. For one of the alleles annealing failure was obtained when the mismatch occurs at all the four primer-site locations. Primer sequences carrying the alternative SNP allele resulted to be less specific as the distance to the primer-3’ end was increased. Altogether, these results revealthat effects in the extension of the annealing failure is allele and mismatch-position dependent.
文摘With a view to finding out precisely how small peptides recognize a particular binding site of DNA, we have accomplished DNA binding studies of two peptides, H-Tyr-Arg-OH (YR) and H-Gly-Gly-His-OH (GGH) by using measurements in comparison with the binding between DNA and Hoechst 33258. The inhibition mode by YR and GGH to DNA binding of Hoechst 33258 was analyzed by Lineweaver-Burk plot which shows the plot of typical competitive inhibition at concentration of Hoechst 33258 from 3.66 ( 10-9 mol / L to 1.09 ( 10-8 mol / L. And it is concluded that YR binds to DNA in its minor groove (AT rich regions) with a binding constant K = 1.02 ( 108 (mol / L)-1. The GGH(s specificity is reduced at high concentration because it can also bind GC base pair.
基金Supported by The National Cancer InstituteNo.R01-CA104729Jayne Haines Center for Pharmacogenomics and Drug Safety of Temple University School of Pharmacy and Temple University Summer Research Award(to Evgeny Krynetskiy)
文摘AIM:To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase(GAPDH),and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS:We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters(diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching(FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.RESULTS:Using MALDI-TOF analysis,we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94,S98,and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH,we demonstrated accumulation of phospho-T99-GAPDH inthe nuclear fractions of A549,HCT116,and SW48 cancer cel s after cytotoxic stress. We performed site-mutagenesis,and estimated enzymatic properties,intranuclear distribution,and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+(Km = 741 ± 257 μmol/L in T99 I vs 57 ± 11.1 μmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP(fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION:Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.
基金Foundation for Young Scientists of Beijing Institute of Pharmacology and Toxicology.
文摘OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues in GABAAreceptors(GABAARs) were also investigated.METHODS Using a zebrafish larvae locomotion model,we investigated the detailed dose-dependent effects of diazepam and other BZDs on zebrafish larvae behaviors,with a focus on their high-dose effects.We then evaluated the influence of the classical BZD antagonist flumazenil,GABAARs antagonist bicuculline,and the antagonist of a proposed BZD binding site in α4/6β3δ subtype receptor Ro15-4513 on BZDs induced immobility.Using wholecell patch clamp electrophysiological recordings on recombinant GABAARs,we investigated the modulation of diazepam alone or combined with flumazenil on GABA-elicited current in wildtype and mutated receptors.RESULTS Diazepam dose-dependently decreased the locomotor activities of zebrafish larvae at doses of 0.4,2,10,20,30,50 and 75 mg·L^(-1).The hypolocomotion(sedation-like state) induced by diazepam at10 and 20 mg·L^(-1) were effectively antagonized by flumazenil with EC150 of 0.086 mg·L-and1.295 mg·L^(-1),while the immobility(anesthesialike state) induced by diazepam at 30 mg·L^(-1) was abolished by bicuculline(3 mg·L^(-1)),but not affected by flumazenil(even at concentration up to150 mg·L^(-1)) or Ro15-4513(100 mg·L^(-1)).The immobility induced by clonazepam and lorazepam(100 mg·L^(-1)) was also resistant to flumazenil(100 mg·L^(-1)).In the α1β2γ2 subtype receptor expressed in HEK293 T cells,diazepam dose-dependently potentiated GABA-elicited current,and this potentiation was effectively antagonized by flumazenil(100 μmol·L^(-1)).However,in α1β2 subtype receptor,diazepam(150 μmol·L^(-1)) induced potentiation was insensitive to flumazenil(100 μmol·L^(-1)),but was abolished by the mutation of β2 N265 I.CONCLUSION These results provide direct in vivo evidence for the nonclassical binding sites,which may be located at the second transmembrane domain of GABAAR,mediate BZD-induced anesthesia.
文摘The orientation of the biological molecule immobi-lized on a solid surface has been critical in devel-opment of various applications. In this study, ori-entation of antibody was retained by protecting the antigen-binding site of the antibody prior to immo-bilization to -functionalized mixed self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 1-heptanethiol. More importantly, the number of immobilization bonds formed between each an-tigen-binding site protected antibody molecule and the solid surface was controlled by optimizing the mole fraction of the activated carboxyl group of the linker molecules in the mixed SAM. The amount of antibody used in this study was approximately equivalent to the amount for one monolayer surface coverage. The resulting activity of protected immo-bilized antibody was about 10 fold higher than that of random immobilized
文摘The aim of this study was to compare the binding profile of a range of β2-adrenoceptor (β2-AR) agonists and antagonists in human lung tissue. Radioligand saturation and competition binding experiments were performed by filtration with a β2-AR antagonist ([3H]propranolol) or agonist ([3H]vilanterol) radioligand and membrane fragments generated from lung parenchyma in the presence of 100 μM guanosine 5’-[β,γ-imido]triphosphate (Gpp(NH)p). In membranes prepared from human lung parenchyma, carmoterol, formoterol, ICI118551, propranolol and salbutamol resulted in inhibition of [3H]vilanterol binding to levels that were significantly different from indacaterol, salmeterol and vilanterol (ANOVA, Bonferroni post-test, P < 0.001 except formoterol vs indacaterol where P < 0.01). Indacaterol and salmeterol resulted in inhibition of [3H]vilanterol binding to levels that were not significantly different from vilanterol (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol resulted in full inhibition of [3H]propranolol binding to levels not significantly different from ICI118551 (ANOVA, Bonferroni post-test, P > 0.05). Indacaterol, salmeterol and vilanterol bind to an additional site in human lung parenchyma membranes that is distinct from the β2-AR.
文摘Transcription factors (TFs) are the core sentinels of gene regulation functioning by binding to highly specific DNA sequences to activate or repress the recruitment of RNA polymerase. The ability to identify transcription factor binding sites (TFBSs) is necessary to understand gene regulation and infer regulatory networks. Despite the fact that bioinformatics tools have been developed for years to improve computational identification of TFBSs, the accurate prediction still remains changeling as DNA motifs recognized by TFs are typically short and often lack obvious patterns. In this study we introduced a new attribute-motif distribution pattern (MDP) to assist in TFBS prediction. MDP was developed using a TF distribution pattern curve generated by analyzing 25 yeast TFs and 37 of their experimentally validated binding motifs, followed by calculating a scoring value to quantify the reliability of each motif prediction. Finally, MDP was tested using another set of 7 TFs with known binding sites to in silico validate the approach. The method was further tested in a non-yeast system using the filamentous fungus Magnaporthe oryzae transcription factor MoCRZ1. We demonstrate superior prediction reranking results using MDP over the commonly used program MEME and the other four predictors. The data showed significant improvements in the ranking of validated TFBS and provides a more sensitive statistics based approach for motif discovery.
