Benzo[a]pyrene-derived DNA lesions decrease DNA methylation by murine methyltransferase DNMT3A2

We have studied the impact of DNA damage by one of the most common carcinogens, benzo[a]pyrene, on the functioning of the truncated isoform of DNA methyltransferase Dnmt3a (Dnmt3a2). It is revealed with 30-mer model DNA substrates that DNA methylation rates are drastically reduced when the lesions disturb the structure of the Dnmt3a recognition site or hinder the interaction of the enzyme catalytic loop with the DNA minor groove. Under the chosen conditions, the PWWP domain of Dnmt3a possessing lower affinity to DNA in comparison with the catalytic domain does not influence catalysis.     

Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity

Background

Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.

Results

Mutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.

Conclusions

We conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

     

The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1

Background  

Though Dnmt1 is considered the primary maintenance methyltransferase and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals, these three enzymes may work together in maintaining as well as establishing DNA methylation patterns. It has been proposed that Dnmt1 may carry out de novo methylation at sites in the genome with transient single-stranded regions, such as replication origins, and then spread methylation from these nucleation sites in vivo, even though such activity has not been reported.     

Homology modeling,docking and structure-based pharmacophore of inhibitors of DNA methyltransferase

DNA methyltransferase 1 (DNMT1) is an emerging epigenetic target for the treatment of cancer and other diseases. To date, several inhibitors from different structural classes have been published. In this work, we report a comprehensive molecular modeling study of 14 established DNTM1 inhibitors with a herein developed homology model of the catalytic domain of human DNTM1. The geometry of the homology model was in agreement with the proposed mechanism of DNA methylation. Docking results revealed that all inhibitors studied in this work have hydrogen bond interactions with a glutamic acid and arginine residues that play a central role in the mechanism of cytosine DNA methylation. The binding models of compounds such as curcumin and parthenolide suggest that these natural products are covalent blockers of the catalytic site. A pharmacophore model was also developed for all DNMT1 inhibitors considered in this work using the most favorable binding conformations and energetic terms of the docked poses. To the best of our knowledge, this is the first pharmacophore model proposed for compounds with inhibitory activity of DNMT1. The results presented in this work represent a conceptual advance for understanding the protein–ligand interactions and mechanism of action of DNMT1 inhibitors. The insights obtained in this work can be used for the structure-based design and virtual screening for novel inhibitors targeting DNMT1.     

An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.     

Global DNA Methylation Level Monitoring by methyl-CpG Binding Domain-Fused Luciferase

DNA methylation is an epigenetic modification that represses gene expression. In cancer cells, alterations of the DNA methylation state in promoter regions and repetitive DNA sequences are observed; therefore, DNA methyltransferase inhibitors have been the focus of interest as potential anticancer drugs. We previously reported a simple global DNA methylation level-sensing assay using methyl-CpG binding domain (MBD) fused to luciferase (MBD-luciferase). In the assay, the MBD-luciferase binds to methyl-CpG sites on genomic DNA. Subsequently, bioluminescence resonance energy transfer (BRET) between the luciferase and a fluorescent DNA intercalating dye generates a signal that is dependent on DNA methylation level. In this study, we investigated whether global DNA hypomethylation induced by a DNA methyltransferase inhibitor or nutrient can be monitored by the BRET assay. 5-Aza-2′-deoxycytidine and folic acid were utilized as the DNA-methyltransferase inhibitor and nutrient that affect DNA methylation in cells. The HeLa cells were cultured with the inhibitor or in folic acid-deficient medium and their global DNA methylation levels measured. Both time- and concentration-dependent hypomethylation were detected by the BRET assay. These results demonstrate that global DNA hypomethylation can be monitored by the BRET assay, indicating that the assay is applicable to cell-based screening of DNA-methyltransferase inhibitors.     

Discovery of a small-molecule inhibitor of Dvl–CXXC5 interaction by computational approaches

The Wnt/β-catenin signaling pathway plays a significant role in the control of osteoblastogenesis and bone formation. CXXC finger protein 5 (CXXC5) has been recently identified as a negative feedback regulator of osteoblast differentiation through a specific interaction with Dishevelled (Dvl) protein. It was reported that targeting the Dvl–CXXC5 interaction could be a novel anabolic therapeutic target for osteoporosis. In this study, complex structure of Dvl PDZ domain and CXXC5 peptide was simulated with molecular dynamics (MD). Based on the structural analysis of binding modes of MD-simulated Dvl PDZ domain with CXXC5 peptide and crystal Dvl PDZ domain with synthetic peptide–ligands, we generated two different pharmacophore models and applied pharmacophore-based virtual screening to discover potent inhibitors of the Dvl–CXXC5 interaction for the anabolic therapy of osteoporosis. Analysis of 16 compounds selected by means of a virtual screening protocol yielded four compounds that effectively disrupted the Dvl–CXXC5 interaction in the fluorescence polarization assay. Potential compounds were validated by fluorescence spectroscopy and nuclear magnetic resonance. We successfully identified a highly potent inhibitor, BMD4722, which directly binds to the Dvl PDZ domain and disrupts the Dvl–CXXC5 interaction. Overall, CXXC5–Dvl PDZ domain complex based pharmacophore combined with various traditional and simple computational methods is a promising approach for the development of modulators targeting the Dvl–CXXC5 interaction, and the potent inhibitor BMD4722 could serve as a starting point to discover or design more potent and specific the Dvl–CXXC5 interaction disruptors.     

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.     

Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase

We report the 2.6 A X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.     

Weak lignin-binding enzymes

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

羰基还原酶辅酶结合域位点突变对其不对称催化性能的影响

基于同源模型的比较和分析,发现羰基还原酶SCR1辅酶结合域P124和W125位点对辅酶NADPH的结合形成了一定的空间位阻效应.通过对该位点进行小侧基氨基酸的取代突变,该酶的底物专一性和立体选择性均发生了不同程度的改变,表明该位点是酶与辅酶有效结合的关键位点,而且它与辅酶结合的空间效应进一步影响了底物结合域活性中心对不同构型的底物及其对映体产物的亲和作用.在底物专一性方面,野生型酶对2-羟基苯乙酮和2-溴苯乙酮及其衍生物等底物表现出较高的催化活性,而突变株W125A,W125G,P124A/W125A和P124G/W125G对苯乙酮及其部分衍生物和2-辛酮等底物的催化活性均有所提高.对于酶的立体选择性,部分突变株发生了转化产物对映体构型反转的现象,突变株P124A/W125A和P124G/W125G催化还原2-羟基苯乙酮和4-氯乙酰乙酸乙酯均生成了(R)-型产物.     

Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification(HRCA) and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.     

Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design

Summary We report structural models of the full-length integrase enzyme (IN) of the human immunodeficiency virus type 1 (HIV-1) and its complex with viral and human DNA. These were developed by means of molecular modeling techniques using all available experimental evidence, including X-ray crystallographic and NMR structures of portions of the full-length protein. Special emphasis was placed on obtaining a model of the enzyme’s active site with the viral DNA apposed to it, based on the hypothesis that such a model would allow structure-based design of inhibitors that retain activity in vivo. This was because bound DNA might be present in vivo after 3’-processing but before strand transfer. These structural models were used to study the potential binding modes of various diketo-acid HIV-1 IN inhibitors (many of them preferentially inhibiting strand transfer) for which no experimentally derived complexed structures are available. The results indicate that the diketo-acid IN inhibitors probably chelate the metal ion in the catalytic site and also prevent the exposure of the 3’-processed end of the viral DNA to human DNA.     

Analysis of DNA methylation markers for tissue identification in individuals with different clinical phenotypes

Deoxyribonucleic acid (DNA) methylation patterns can be used to identify the type of tissue or body fluid found at a crime scene. However, tissue-related methylation levels have not been analyzed in individuals with different illnesses and medical conditions in forensic-specific studies. The primary goal of this study was to investigate if certain clinical phenotypes can alter the methylation levels of CpG sites in genes involved in tissue typing. Four studies with focus on DNA methylation analysis on individuals with different clinical conditions were selected from the Gene Expression Omnibus database. Then, a list of 137 CpG sites was compiled for further investigation. Statistical tests were performed to compare the beta-values results obtained for the control groups and the individuals affected by medical conditions. For each study, CpG sites that presented significant statistical differences between patients and control group were identified and it was possible to notice that DNA methylation levels can be affected in sites with potential forensic use. Although the observed DNA methylation variation (less than 10% difference) in this study would likely not cause any issues in body fluid identification, the results are important to show that this type of analysis should be taken into consideration when investigating and further validating body fluid markers. The CpG sites identified in this study should be further investigated by future studies on body fluids identification, and due to the significant difference in methylation levels in samples from affected individuals, caution must be taken before including these sites in tissue identification investigations.     

Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme

We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme--an RNA-cleaving catalytic DNA motif previously reported--from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans, and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine tuning when different dinucleotide junctions are given as cleavage sites.     

From Natural Products to Drugs for Epimutation Computer-Aided Drug Design

The epimutational event, i.e., ectopic methylation in tumor suppressor genes, can lead to gene silencing, thus promoting prognosis of cancer. The progression of DNA methylation is a cycle of demethylation, de novo methylation, and maintenance methylation. The enzyme responsible for maintenance of methylation status is DNA methyltransferase 1 (DNMT1), the continuous activity of which is required to maintain the pattern of epimutation; thus, its inhibition is a promising strategy for the treatment of cancer. To the best of our knowledge, this study is the first to focus on the recently developed crystal structure of the catalytic site of DNMT1. Here in this study, we have used the crystal structure for the development of non-nucleoside DNMT1 inhibitors using virtual screening (VS), absorption, distribution, metabolism, elimination/toxicology analysis, and molecular docking studies. In this study, VS was carried out on 48,531 natural products to create a subset of lead-like natural products. Three of them were found to form hydrogen bonds with the catalytic site of the DNMT1 (Cys 1226). Thus, this study adumbrates potential lead compounds for treatment of epimutation.