Inhibition by pesticides of the DJ-1/Park7 protein related to Parkinson disease.

Parkinson's disease is a severe neurodegenerative disease. Several environmental contaminants such as pesticides have been suspected to favor the appearance of this pathology. The protein DJ-1 (or Park7) protects against the development of Parkinson's disease. Thus, the possible inhibitory effects of about a hundred pesticides on human DJ-1 have been studied. We identified fifteen of them as strong inhibitors of DJ-1 with IC50 values between 0.02 and 30 µM. Thiocarbamates are particularly good inhibitors, as shown by thiram that acts as an irreversible inhibitor of an esterase activity of DJ-1 with an IC50 value of 0.02 µM. Thiram was also found as a good inhibitor of the protective activity of DJ-1 against glycation. Such inhibitory effects could be one of the various biological effects of these pesticides that may explain their involvement in the development of Parkinson's disease.

Polycyclic nitrogen heterocycles as potential thymidine phosphorylase inhibitors: synthesis, biological evaluation, and molecular docking study.

New polycyclic heterocycles were synthesised and evaluated as potential inhibitors of thymidine phosphorylase (TP). Inspired by the pharmacophoric pyrimidinedione core of the natural substrate, four series have been designed in order to interact with large empty pockets of the active site: pyrimidoquinoline-2,4-diones (series A), pyrimidinedione linked to a pyrroloquinoline-1,3-diones (series B and C), the polycyclic heterocycle has been replaced by a pyrimidopyridopyrrolidinetetraone (series D). In each series, the tricyclic nitrogen heterocyclic moiety has been synthesised by a one-pot multicomponent reaction. Compared to 7-DX used as control, 2d, 2l, 2p (series A), 28a (series D), and the open intermediate 30 showed modest to good activities. A kinetic study confirmed that the most active compounds 2d, 2p are competitive inhibitors. Molecular docking analysis confirmed the interaction of these new compounds at the active binding site of TP and highlighted a plausible specific interaction in a pocket that had not yet been explored.

Insights into the interaction of high potency inhibitor IRC-083864 with phosphatase CDC25.

CDC25 phosphatases play a crucial role in cell cycle regulation. They have been found to be over-expressed in various human tumours and to be valuable targets for cancer treatment. Here, we report the first model of binding of the most potent CDC25 inhibitor to date, the bis-quinone IRC-083864, into CDC25B obtained by combining molecular modeling and NMR studies. Our study provides new insights into key interactions of the catalytic site inhibitor and CDC25B in the absence of any available experimental structure of CDC25 with a bound catalytic site inhibitor. The docking model reveals that IRC-083864 occupies both the active site and the inhibitor binding pocket of the CDC25B catalytic domain. NMR saturation transfer difference and WaterLOGSY data indicate the binding zones of the inhibitor and support the docking model. Probing interactions of analogues of the two quinone units of IRC-083864 with CDC25B demonstrate that IRC-083864 competes with each monomer. Proteins 2017; 85:593-601. © 2016 Wiley Periodicals, Inc.

Recombinant overexpression of camel hepcidin cDNA in Pichia pastoris: purification and characterization of the polyHis-tagged peptide HepcD-His.

Hepcidin, a liver-expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis-tagged camel hepcidin (HepcD-His) in yeast Pichia pastoris. The use of this eukaryotic expression system, for the production of HepcD-His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichia coli. Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2 mg/L for the E coli system. Matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry of the purified HepcD-His showed 2 major peaks at m/z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5µM, against E coli, than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD-His, may be helpful for future therapeutic applications including bacterial infection diseases.

The Andromonoecious Sex Determination Gene Predates the Separation of Cucumis and Citrullus Genera.

Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera.

Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli.

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.

A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges.

Understanding the evolution of sex determination in plants requires identifying the mechanisms underlying the transition from monoecious plants, where male and female flowers coexist, to unisexual individuals found in dioecious species. We show that in melon and cucumber, the androecy gene controls female flower development and encodes a limiting enzyme of ethylene biosynthesis, ACS11. ACS11 is expressed in phloem cells connected to flowers programmed to become female, and ACS11 loss-of-function mutants lead to male plants (androecy). CmACS11 represses the expression of the male promoting gene CmWIP1 to control the development and the coexistence of male and female flowers in monoecious species. Because monoecy can lead to dioecy, we show how a combination of alleles of CmACS11 and CmWIP1 can create artificial dioecy.

Expression and purification of a new recombinant camel hepcidin able to promote the degradation of the iron exporter ferroportin1.

Hepcidin, a 25-amino-acid and highly disulfide bonded antimicrobial peptide, is the central regulator of iron homeostasis. This hormone is expressed in response to iron and inflammation and interacts with ferroportin1 (FPN1), the only known iron exporter in vertebrates, inducing its internalization and degradation. Thus, the export of iron from cells to plasma will be significantly diminished. Thereby, hepcidin has become the target of intense research studies due to its profound biomedical significance. This study describes the functional expression of recombinant camel hepcidin in Escherichia coli. Biologically active recombinant camel hepcidin was obtained thanks to the production of a hepcidin-thioredoxin fusion protein (TRX-HepcD) and a purified camel hepcidin, with an extra methionine at the N-terminus, was obtained after enterokinase cleavage of the fusion protein. Presence of the four disulfide bridges was verified using MALDI-ToF spectrometry. The recombinant camel hepcidin was compared to related synthetic bioactive peptides, including human hepcidin, and was found equally able to promote ferroportin degradation of mouse macrophages. Furthermore, camel hepcidins exhibits a high capacity to inhibit the growth of Leishmania major promastigotes. These results proved that production of functional camel hepcidin can be achieved in E. coli, this is a major interest for the production of cysteine rich peptides or proteins that can be purified under their functional form without the need of a refolding process.

Molecular characterization of a novel hepcidin (HepcD) from Camelus dromedarius. Synthetic peptide forms exhibit antibacterial activity.

Hepcidin is a cysteine-rich peptide widely characterized in immunological processes and antimicrobial activity in several vertebrate species. Obviously, this hormone plays a central role in the regulation of systemic iron homeostasis. However, its role in camelids' immune response and whether it is involved in antibacterial immunity have not yet been proven. In this study, we characterized the Arabian camel hepcidin nucleotide sequence with an open reading frame of 252 bp encoding an 83-amino acid preprohepcidin peptide. Eight cysteine key residues conserved in all mammalian hepcidin sequences were identified. The model structure analysis of hepcidin-25 peptide showed a high homology structure and sequence identity to the human hepcidin. Two different hepcidin-25 analogs manually synthesized by SPPS shared significant cytotoxic capacity toward the Gram-negative bacterium Escherichia coli American Type Culture Collection (ATCC) 8739 as well as the Gram-positive bacteria Bacillus subtilis ATCC 11779 and Staphylococcus aureus ATCC 6538 in vitro. The three disulfide bridges hepcidin analog demonstrated bactericidal activity, against B. subtilis ATCC 11779 and S. aureus ATCC 6538 strains, at the concentration of 15 µM (50 µg/ml) or above at pH 6.2. This result correlates with the revealed structural features suggesting that camel hepcidin is proposed to be involved in antibacterial process of innate immune response.

Hepcidin bound to α2-macroglobulin reduces ferroportin-1 expression and enhances its activity at reducing serum iron levels.

Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down-regulate Fpn1.

Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase.

We examined the kinetics of single-electron reduction of a large number of structurally diverse quinones and nitroaromatic compounds, including a number of antitumour and antiparasitic drugs, and nitroaromatic explosives by recombinant rat neuronal nitric oxide synthase (nNOS, EC 1.14.13.39), aiming to characterize the role of nNOS in the oxidative stress-type cytotoxicity of the above compounds. The steady-state second-order rate constants (kcat/Km) of reduction of the quinones and nitroaromatics varied from 10² M⁻¹s⁻¹ to 106 M⁻¹s⁻¹, and increased with an increase in their single-electron reduction potentials (E¹7). The presence of Ca²âº/calmodulin enhanced the reactivity of nNOS. These reactions were consistent with an 'outer sphere' electron-transfer mechanism, considering the FMNH∙/FMNH2 couple of nNOS as the most reactive reduced enzyme form. An analysis of the reactions of nNOS within the 'outer sphere' electron-transfer mechanism gave the approximate values of the distance of electron transfer, 0.39-0.47 nm, which are consistent with the crystal structure of the reductase domain of nNOS. On the other hand, at low oxygen concentrations ([O2] = 40-50 µM), nNOS performs a net two-electron reduction of quinones and nitroaromatics. This implies that NOS may in part be responsible for the bioreductive alkylation by two-electron reduced forms of antitumour aziridinyl-substituted quinones under a modest hypoxia.

Crystal structure of two anti-porphyrin antibodies with peroxidase activity.

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααß-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

Synthesis and biological activity of mouse hepcidin peptide analogs containing three disulfide bridges: manual and microwave-assisted solid-phase peptide synthesis.

Hepcidin, a 25 amino acid peptide hormone containing a complex network of four disulfide bonds is the hormone regulator of iron homeostasis. Three bridges synthetic peptide analogs have been prepared following two synthetic strategies and two oxidation procedures: i) a microwave-assisted solid phase synthesis followed by air oxidation of the six free cysteines ii) a manual solid phase synthesis followed by stepwise deprotection and oxidation of cysteine pairs. All the peptides with different connectivities have been characterized by MALDI ToF spectrometry, and tested for their ability to degrade the cellular iron exporter, ferroportin. While linear peptides are inactive, the one-bridge and two-bridge peptides retaining protected cysteines by bulky substituents are active. Similarly, the three-bridge peptides are active irrespective of their disulfide connectivities.

A new cytochrome P450 belonging to the 107L subfamily is responsible for the efficient hydroxylation of the drug terfenadine by Streptomyces platensis.

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450(terf), was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV-Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450-substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with k(m) values at the µM level, and k(cat) values around 30min(-1). Sequencing of the p450(terf) gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56-65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs.

A conserved ethylene biosynthesis enzyme leads to andromonoecy in two cucumis species.

Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.

Pro-hepcidin is unable to degrade the iron exporter ferroportin unless maturated by a furin-dependent process.

BACKGROUND/AIMS: The iron-regulatory peptide hepcidin is synthesized in the liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. This peptide regulates iron export from enterocytes and macrophages by binding the membrane iron exporter, ferroportin, leading to its degradation. Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an open question. However, the activity of the pro-peptide on ferroportin degradation has never been addressed. METHODS: To answer this question, we produced recombinant pro-hepcidin, both the wild-type form and a furin cleavage site mutant, and tested their activity on ferroportin levels in macrophagic J774 cells. Furin activity was also modulated using furin inhibitor or siRNA-mediated furin mRNA knockdown. RESULTS: We found that pro-hepcidin could fully induce ferroportin degradation, but only when processed by furin to generate the mature hepcidin-25 form. Pro-hepcidin activity was abolished in the presence of furin inhibitor and diminished after siRNA-mediated knockdown of furin mRNA. Furthermore, the mutated version of pro-hepcidin was completely inefficient at degrading ferroportin in macrophages. CONCLUSIONS: Our results demonstrate that pro-hepcidin lacks biological activity, unless fully maturated by a furin-dependent process to yield the bioactive 25-aa peptide.

A conserved mutation in an ethylene biosynthesis enzyme leads to andromonoecy in melons.

Andromonoecy is a widespread sexual system in angiosperms characterized by plants carrying both male and bisexual flowers. In melon, this sexual form is controlled by the identity of the alleles at the andromonoecious (a) locus. Cloning of the a gene reveals that andromonoecy results from a mutation in the active site of 1-aminocyclopropane-1-carboxylic acid synthase. Expression of the active enzyme inhibits the development of the male organs and is not required for carpel development. A causal single-nucleotide polymorphism associated with andromonoecy was identified, which suggests that the a allele has been under recent positive selection and may be linked to the evolution of this sexual system.

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone.

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.

LEF-1 recognition of platinated GG sequences within double-stranded DNA. Influence of flanking bases.

The lymphoid enhancer-binding factor 1 (LEF-1) recognizes a double-stranded 9 base-pairs (bp) long motif in DNA which is significantly bent upon binding. This bend is centered at two destacked adenines whose geometry closely resembles that of two adjacent guanines crosslinked by the antitumor drug cisplatin. It has been proposed that cisplatin-GG crosslinks could hijack high mobility group (HMG) box containing transcription factors such as LEF-1. In order to examine such a possibility, we used electrophoretic mobility shift assays to determine the affinity of the HMG box of LEF-1 for a series of 25 oligonucleotides containing a central GG sequence, free or site-specifically modified by cisplatin. The binding affinity of the GG-platinated oligonucleotides was 3-6-fold higher than that determined for the corresponding unplatinated oligonucleotides, however, the binding to all cisplatin-modified oligonucleotides was at least 1 order of magnitude weaker than that to the 25 bp oligonucleotide containing the recognition 9 bp motif. The binding affinity was dependent on the nature of bases flanking the cisplatin-crosslinked G(*)G(*) dinucleotide, the AG(*)G(*)T sequence displaying the strongest affinity and CG(*)G(*)T showing the strongest binding enhancement upon platination. In contrast, modification of the AGGT sequence with the third-generation platinum antitumor drug oxaliplatin did not enhance the affinity significantly. These results suggest that the cisplatin-caused bending of DNA does produce a target for LEF-1 binding, however, the cisplatinated DNA does not appear to be a strong competitor for the LEF-1 recognition sequence.