Mediate DNA Binding Research Articles

A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome

A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells

The thyroid hormone, 3, 3',5-triiodo-l-thyronine (T(3)), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T(3) are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T(3)-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TRα1 (HepG2-TRα1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T(3) target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions -327/-312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T(3) induced PAI-1 expression in J7-TRα1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T(3)/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T(3)-treated HepG2-TRα1 cells. The T(3)-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T(3)-associated tumor progression and prognosis.

An Arginine Residue Instead of a Conserved Leucine Residue in the Recognition Helix of the Finger 3 of Zif268 Stabilizes the Domain Structure and Mediates DNA Binding

The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.

Molecular Determinants for Small Maf Protein Control of Platelet Production

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition

The segregation of plasmid DNA typically requires three elements: a DNA centromere site, an NTPase, and a centromere-binding protein. Because of their simplicity, plasmid partition systems represent tractable models to study the molecular basis of DNA segregation. Unlike eukaryotes, which utilize the GTPase tubulin to segregate DNA, the most common plasmid-encoded NTPases contain Walker-box and actin-like folds. Recently, a plasmid stability cassette on Bacillus thuringiensis pBtoxis encoding a putative FtsZ/tubulin-like NTPase called TubZ and DNA-binding protein called TubR has been described. How these proteins collaborate to impart plasmid stability, however, is unknown. Here we show that the TubR structure consists of an intertwined dimer with a winged helix-turn-helix (HTH) motif. Strikingly, however, the TubR recognition helices mediate dimerization, making canonical HTH-DNA interactions impossible. Mutagenesis data indicate that a basic patch, encompassing the two wing regions and the N termini of the recognition helices, mediates DNA binding, which indicates an unusual HTH-DNA interaction mode in which the N termini of the recognition helices insert into a single DNA groove and the wings into adjacent DNA grooves. The TubZ structure shows that it is as similar structurally to eukaryotic tubulin as it is to bacterial FtsZ. TubZ forms polymers with guanine nucleotide-binding characteristics and polymer dynamics similar to tubulin. Finally, we show that the exposed TubZ C-terminal region interacts with TubR-DNA, linking the TubR-bound pBtoxis to TubZ polymerization. The combined data suggest a mechanism for TubZ-polymer powered plasmid movement.

Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

Structure and putative function of NFX1‐like proteins in plants

The human NFX1 transcription factor constitutes a group of NFX1-type zinc finger proteins. It forms a central Cys-rich region with several NFX1-type zinc finger domains that have been shown to mediate DNA binding. Proteins with NFX1-type zinc fingers are found in protists, fungi, animals and plants, and may be ubiquitous in eukaryotes. This review discusses the structure and putative roles of NFX1-like proteins, with a focus on human NFX1 and Arabidopsis NFXL1 proteins. By means of manual sequence analysis and application of hidden Markov models, we demonstrate that NFX1-like proteins form a specific RING finger motif with a C(4)HC(3) Zn ligand signature and additional distinct features, suggesting that these proteins function as E3 ubiquitin ligases. Phylogenetic analysis revealed different clades of NFX1-like proteins. The plant proteins group into two distinct clades. The genomes of plants such as rice, Arabidopsis, poplar and grapevine encode one member of each clade, suggesting that the presence of two NFX1-like factors is sufficient in flowering plants. The Arabidopsis proteins presumably fine-tune opposed biotic and abiotic stress response pathways.

Targeted mutagenesis induced by AID (144.10)

Abstract Activation-induced cytidine deaminase (AID) is an essential enzyme for immunoglobulin somatic hypermutation, gene conversion, and class switch recombination in immune cells. Somatic hypermutation is induced by AID mediated cytidine deamination around the V regions of immunoglobulin genes in germinal center B cells. It has been shown that AID also can induce somatic mutations outside Ig loci and in non-lymphoid cells. Our present study attempts to use AID as a target specific mutagen. We propose to confine the mutagenesis function of AID in certain DNA regions by fusing AID with a specific DNA binding protein. We choose the Cre recombinase as the DNA binding protein in the test system. It is hypothesized that Cre-AID fusion protein should promote AID mediated mutagenesis around the loxp site, the binding site of Cre. A reporter system was built by placing a single loxp site next to a GFP expression cassette, which contains stop condon in the middle of GFP coding sequence. If the stop codon is reversely mutated into non-stop codon by AID, GFP signal can be detected. This reporter system would allow us to evaluate whether Cre mediated DNA binding could enhance AID mediated mutagenesis around the loxp site. This experimental strategy has the potential to be used in the directed mutagenesis when Cre is further modified to inactivate the recombinase activity but retain loxp DNA binding activity.

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.

Drosophila Maelstrom Ensures Proper Germline Stem Cell Lineage Differentiation by Repressing microRNA-7

Nuage is a germline-unique perinuclear structure conserved throughout the animal kingdom. Maelstrom (Mael) is an unusual nuage component, as it is also found in the nucleus. Mael contains a High Mobility Group box, known to mediate DNA binding. We show that Mael nuclear function is required for proper differentiation in the Drosophila germline stem cell (GSC) lineage. In mael mutant testes, transit-amplifying cysts fail to differentiate into primary spermatocytes, instead breaking down into ectopic GSCs and smaller cysts, due to a depletion of Bag-of-marbles (Bam) protein. Mael regulates Bam via repression of miR-7. Mael binds the miR-7 promoter and is required for the local accumulation of HP1 and H3K9me3. miR-7 targets bam directly at its 3'UTR, and a reduction in miR-7 expression can rescue germline differentiation defects found in mael mutants by alleviating Bam repression. We propose that Mael ensures proper differentiation in the GSC lineage by repressing miR-7.

Epidermal growth factor increases prostaglandin E2 production via ERK1/2 MAPK and NF-κB pathway in fibroblast like synoviocytes from patients with rheumatoid arthritis

High concentration of epidermal growth factor (EGF) is found in the synovial fluid of rheumatoid arthritis (RA) that might imply the involvement of EGF in the pathogenesis of arthritic diseases. In order to investigate if EGF is involved in the regulation of cyclooxygenase-2 (COX-2) and the prostaglandin E(2) (PGE(2)) production in fibroblast like synoviocytes (FLS) from patients with RA. The levels of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed to investigate EGF mediated DNA binding activity of nuclear factor-kappaB (NF-kappaB). PGE(2) levels were analyzed by ELISA. EGF enhanced both COX-2 protein and mRNA expressions. mPGES-1 mRNA level was also increased by EGF treatment. EGF also stimulated ERK1/2 MAPK activity and the inhibition of ERK1/2 by PD098059 (ERK1/2 specific inhibitor) resulted in the suppression of EGF-induced COX-2 expression. The DNA binding activity of NF-kappaB was remarkably increased by EGF treatment and the pretreatment of PD098059 abolished EGF-stimulated NF-kappaB activity. We also observed that the level of PGE(2) was significantly elevated with the treatment of EGF in FLS, and the pretreatment of PD098059 abolished this stimulating effect. These results suggest that EGF is involved in the inflammatory process of RA by stimulating COX-2 expression and PGE(2) production. And EGF enhanced PGE(2) production appears to be mediated via ERK1/2 MAPK and NF-kappaB pathway in FLS.

Scavenger Chemokine (CXC Motif) Receptor 7 (CXCR7) Is a Direct Target Gene of HIC1 (Hypermethylated in Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.

The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

CBFβ is critical for AML1-ETO and TEL-AML1 activity

AbstractAML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor β (CBFβ) subunit. To determine whether CBFβ is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt domain that disrupt heterodimerization with CBFβ but not DNA binding. We show that CBFβ contributes to AML1-ETO’s inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFβR in generating acute myeloid leukemia in mice. Similarly, CBFβ is essential for TEL-AML1’s ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt domain/CBFβ interaction as a therapeutic target in core binding factor leukemias.

CXXC Domain of Human DNMT1 Is Essential for Enzymatic Activity

DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 (DeltaCXXC)) localized on replication foci. Both point mutant and DNMT1 (DeltaCXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 (DeltaCXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding approximately 2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further approximately 40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.

The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway, it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acid residues in UvrA that provide binding energy to DNA. Based on conservation among ∼300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3–37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA − strain of bacteria 10–100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA.

Synthesis, metal ion binding, and biological evaluation of new anticancer 2-(2′-hydroxyphenyl)benzoxazole analogs of UK-1

UK-1 is a bis(benzoxazole) natural product displaying activity against a wide range of human cancer cell lines. A simplified analog of UK-1, 4-carbomethoxy-2-(2′-hydroxyphenyl)benzoxazole, was previously found to be almost as active as UK-1 against cancer cell lines, and similar to the natural product, formed complexes with a variety of metal ions such as Mg 2+ and Zn 2+. A series of 4-substituted-2-(2′-hydroxyphenyl)benzoxazole analogs of this ‘minimal pharmacophore’ of UK-1 were prepared. The anti-cancer activity of these analogs was examined in breast and lung cancer cell lines. Spectrophotometric titrations in methanol were carried out in order to assess the ability of UK-1 and these analogs to coordinate with Mg 2+ and Cu 2+ ions. Although none of the new analogs were more cytotoxic than 4-carbomethoxy-2-(2′-hydroxyphenyl)benzoxazole, some analogs were identified that display similar cytotoxicity to this simplified UK-1 analog with improved water solubility. UK-1 and all of these new analogs bind Cu 2+ ions better than Mg 2+ ions, and the nature of the 4-substituent is important for the Mg 2+ ion binding ability of these 2-(2′-hydroxyphenyl)benzoxazoles. Previous studies of a limited number of UK-1 analogs demonstrated a correlation between Mg 2+ ion binding ability and cytotoxicity; however, within this series of 4-substituted-2-(2′-hydroxyphenyl)benzoxazoles the variations in cytotoxicity do not correlate with either Mg 2+ or Cu 2+ ion binding ability. These results, together with recent ESI-MS studies of Cu 2+-mediated DNA binding by UK-1 and analogs, indicate that UK-1 and analogs may exert their cytotoxic effects by interaction with Cu 2+ or other transition metal ions, rather than Mg 2+, and that metal ion-mediated DNA binding, rather than metal ion binding affinity, is important for the cytotoxic effect of these compounds. The potential role of Cu 2+ ions in the cytotoxic action of UK-1 is further supported by the observation that UK-1 in the presence of Cu 2+ displays enhanced cytotoxicity to MCF-7 and A549 cells when compared to UK-1 alone.

C1-inhibitor structure reveals a novel mechanism of heparin potentiation

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s129 Acta Cryst. (2007). A63, s129 motif, while the C-terminal domain shows a typical restriction endonuclease fold. By structural comparison and mutational analysis we showed that the active site of SdaI is located at the C-terminal domain and exhibits a new variation of the canonical PD...(D/E)XK active site motif. Mutational analysis of the residues from the predicted recognition helix of the wHTH motif suggests that SdaI determinants of sequence specificity are clustered at the N-terminal domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from the previously characterized restriction enzymes interacting with symmetric recognition sequences.