Increase In DNA Binding Affinity Research Articles

Is Simultaneous Binding to DNA and Gyrase Important for the Antibacterial Activity of Cystobactamids?

Cystobactamids are aromatic oligoamides that exert their natural antibacterial properties by inhibition of bacterial gyrases. Such aromatic oligoamides were proposed to inhibit α-helix-mediated protein-protein interactions and may serve for specific recognition of DNA. Based on this suggestion, we designed new derivatives that have duplicated cystobactamid triarene units as model systems to decipher the specific binding mode of cystobactamids to double stranded DNA. Solution NMR analyses revealed that natural cystobactamids as well as their elongated analogues show an overall bent shape at their central aliphatic unit, with an average CX-CY-CZ angle of ~110 degrees. Our finding is corroborated by the target-bound structure of close analogues, as established by cryo-EM very recently. Cystobactamid CN-861-2 binds directly to the bacterial gyrase with an affinity of 9 μM, and also exhibits DNA-binding properties with specificity for AT-rich DNA. Elongation/dimerization of the triarene subunit of native cystobactamids is demonstrated to lead to an increase in DNA binding affinity. This implies that cystobactamids' gyrase inhibitory activity necessitates not just interaction with the gyrase itself, but also with DNA via their triarene unit.

Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.

Sequence Properties of An Intramolecular Interaction That Inhibits p53 DNA Binding.

An intramolecular interaction between the p53 transactivation and DNA binding domains inhibits DNA binding. To study this autoinhibition, we used a fragment of p53, referred to as ND WT, containing the N-terminal transactivation domains (TAD1 and TAD2), a proline rich region (PRR), and the DNA binding domain (DBD). We mutated acidic, nonpolar, and aromatic amino acids in TAD2 to disrupt the interaction with DBD and measured the effects on DNA binding affinity at different ionic strengths using fluorescence anisotropy. We observed a large increase in DNA binding affinity for the mutants consistent with reduced autoinhibition. The ΔΔG between DBD and ND WT for binding a consensus DNA sequence is -3.0 kcal/mol at physiological ionic strength. ΔΔG increased to -1.03 kcal/mol when acidic residues in TAD2 were changed to alanine (ND DE) and to -1.13 kcal/mol when all the nonpolar residues, including W53/F54, were changed to alanine (ND NP). These results indicate there is some cooperation between acidic, nonpolar, and aromatic residues from TAD2 to inhibit DNA binding. The dependence of DNA binding affinity on ionic strength was used to predict excess counterion release for binding both consensus and scrambled DNA sequences, which was smaller for ND WT and ND NP with consensus DNA and smaller for scrambled DNA overall. Using size exclusion chromatography, we show that the ND mutants have similar Stokes radii to ND WT suggesting the mutants disrupt autoinhibition without changing the global structure.

A germline STAT6 gain-of-function variant is associated with early-onset allergies

The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.

Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing.

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3–AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3–AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3–AG tetramer. SEP3 and SEP3–AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3–AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3–AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication.

Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

Alterations in Linker Histone Dynamics upon DNA Damage

Cells are continuously exposed to DNA damaging agents and have evolved numerous pathways to recognize and repair damage. Defects in the DNA damage response (DDR) pathways have been implicated in many human diseases, including cancers, and there are a handful of approved therapeutic drugs that target these pathways. Although DNA repair has been extensively studied, the initiation of these pathways, specifically the ability to sense DNA damage, remains elusive. In vivo, genomic DNA exists in the context of chromatin, and existing evidence suggests that chromatin proteins are directly involved in the DDR. We hypothesize that H1 linker histones play an important role in the recognition and initiation of the single‐strand break repair pathway. H1 binds to DNA between nucleosomes in a dynamic manner, continuously exchanging positions, with an average chromatin residence time of approximately one minute. This behavior is thought to mediate localized chromatin relaxation to allow transient access to DNA‐binding proteins involved in transcription, replication, or repair. To determine if DNA damage has a direct effect on H1 dynamics, we generated a stable murine fibroblast cell line expressing a photo‐convertible fluorescently tagged H1 (pSMOrange‐H1o) and measured H1 dynamics via live cell fluorescence microscopy. We observed that H1 was specifically depleted from chromatin in the vicinity of localized DNA damage induced by micro‐irradiation and that H1 from non‐irradiated chromatin was unable to enter the damaged region for at least ten minutes. By inducing damage and photoconverting H1 in the same region, we found that the off‐rate of H1 from chromatin in the vicinity of damaged DNA was significantly faster (p<0.001) than the off‐rate from chromatin in the vicinity of undamaged DNA. We further noted that the eviction of H1 from chromatin containing damaged DNA was temporally correlated with the recruitment of the early responding DDR protein, poly(ADP‐ribose) polymerase 1 (PARP1). There is evidence that PARP1 and H1 compete for chromatin binding sites and that H1 may be a target of poly ADP‐ribosylation by PARP1 upon DNA damage. By successive additions of C‐terminal DNA‐binding subdomains of H1, we created vectors capable of expressing mutant H1s with increased DNA binding affinity. These vectors were introduced into cell lines expressing GFP‐tagged PARP1. We observed that both the rate and extent of PARP1 recruitment to chromatin containing damaged DNA was significantly decreased upon the overexpression of mutant H1s with enhanced DNA‐binding affinity. Our results show that H1 dynamics are altered upon DNA damage, resulting in the active evcition and subsequent exclusion from chromatin containing damaged DNA. We also suggest that interactions between the linker histones and PARP1 may be an integral aspect of the DNA damage response.Support or Funding InformationNational Science Foundation, Department of Cell and Molecular Biology, School of Graduate Studies in the Health Sciences, SURE UMMCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism

The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed.Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.

The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins.

A central question important to understanding DNA repair is how certain proteins are able to search for, detect, and fix DNA damage on a biologically relevant time scale. A feature of many base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for lesions. In this paper, we establish the importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an O2-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying [4Fe4S]3+ clusters bind to DNA 550 times more tightly than those with [4Fe4S]2+ clusters. The measured increase in DNA-binding affinity matches the calculated affinity change associated with the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S] cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

BackgroundSequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. MethodsDNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. ResultsIntroduction of the N1-alkyl amino group reduced selectivity for the target sequence 5′-TACGAT-3′ on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1–2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. ConclusionsPolyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. General significancePharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

Peptidylprolyl Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen Receptor α

The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)(118)-Pro(119) in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.

Mycobacterium smegmatis Ku binds DNA without free ends

Ku is central to the non-homologous end-joining pathway of double-strand-break repair in all three major domains of life, with eukaryotic homologues being associated with more diversified roles compared with prokaryotic and archaeal homologues. Ku has a conserved central 'ring-shaped' core domain. While prokaryotic homologues lack the N- and C-terminal domains that impart functional diversity to eukaryotic Ku, analyses of Ku from certain prokaryotes such as Pseudomonas aeruginosa and Mycobacterium smegmatis have revealed the presence of distinct C-terminal extensions that modulate DNA-binding properties. We report in the present paper that the lysine-rich C-terminal extension of M. smegmatis Ku contacts the core protein domain as evidenced by an increase in DNA-binding affinity and a decrease in thermal stability and intrinsic tryptophan fluorescence upon its deletion. Ku deleted for this C-terminus requires free DNA ends for binding, but translocates to internal DNA sites. In contrast, full-length Ku can directly bind DNA without free ends, suggesting that this property is conferred by its C-terminus. Such binding to internal DNA sites may facilitate recruitment to sites of DNA damage. The results of the present study also suggest that extensions beyond the shared core domain may have independently evolved to expand Ku function.

Origins of specificity and cross‐talk in metal ion sensing by Bacillus subtilisFur

Fur (ferric uptake regulator) is the master regulator of iron homeostasis in many bacteria, but how it responds specifically to Fe(II) in vivo is not clear. Biochemical analyses of Bacillus subtilis Fur (BsFur) reveal that in addition to Fe(II), both Zn(II) and Mn(II) allosterically activate BsFur-DNA binding. Dimeric BsFur co-purifies with site 1 structural Zn(II) (Fur(2) Zn(2) ) and can bind four additional Zn(II) or Mn(II) ions per dimer. Metal ion binding at previously described site 3 occurs with highest affinity, but the Fur(2) Zn(2) :Me(2) form has only a modest increase in DNA binding affinity (approximately sevenfold). Metallation of site 2 (Fur(2) Zn(2) :Me(4) ) leads to a ~ 150-fold further enhancement in DNA binding affinity. Fe(II) binding studies indicate that BsFur buffers the intracellular Fe(II) concentration at ~ 1 μM. Both Mn(II) and Zn(II) are normally buffered at levels insufficient for metallation of BsFur site 2, thereby accounting for the lack of cross-talk observed in vivo. However, in a perR mutant, where the BsFur concentration is elevated, BsFur may now use Mn(II) as a co-repressor and inappropriately repress iron uptake. Since PerR repression of fur is enhanced by Mn(II), and antagonized by Fe(II), PerR may co-regulate Fe(II) homeostasis by modulating BsFur levels in response to the Mn(II)/Fe(II) ratio.

TWIST1 associates with NF-κB subunit RELA via carboxyl-terminal WR domain to promote cell autonomous invasion through IL8 production

BackgroundMetastasis is the primary cause of death for cancer patients. TWIST1, an evolutionarily conserved basic helix-loop-helix (bHLH) transcription factor, is a strong promoter of metastatic spread and its expression is elevated in many advanced human carcinomas. However, the molecular events triggered by TWIST1 to motivate dissemination of cancer cells are largely unknown.ResultsHere we show that TWIST1 induces the production of interleukin 8 (IL8), which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. In this novel mechanism, TWIST1-mediated IL8 transcription is induced through the TWIST1 carboxy-terminal WR (Trp-Arg) domain instead of the classic DNA binding bHLH domain. Co-immunoprecipitation analyses revealed that the WR domain mediates the formation of a protein complex comprised of TWIST1 and the nuclear factor-kappaB (NF-κB) subunit RELA (p65/NF-κB3), which synergistically activates the transcriptional activity of NF-κB. This activation leads to increased DNA binding affinity of RELA to the IL8 promoter and thus induces the expression of the cytokine. Blockage of IL8 signaling by IL8 neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that TWIST1 induces autonomous cell invasion by establishing an IL8 antocrine loop.ConclusionsOur data demonstrate that the TWIST1 WR domain plays a critical role in TWIST1-induced IL8 expression through interactions with and activation of NF-κB. The produced IL8 signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane.

PKM2 as a protein kinase

Pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme that mediates the conversion of phosphoenolpyruvate (PEP) to pyruvate. It has been suggested that PKM2 may have additional enzymatic activities and localizes to the nucleus to regulate transcription. Here, the authors show that PKM2 can act as a kinase in the nucleus. They found that nuclear PKM2 affected the expression of many genes; one of its targets was Mek5 (also known as Map2k5), the expression of which was increased following an interaction of PKM2 with signal transducer and activator of transcription 3 (STAT3). Specifically, PKM2 was found to phosphorylate STAT3 on Tyr705, leading to STAT3 activation and increased DNA binding affinity. Importantly, the authors observed that dimeric PKM2 localizes to the nucleus and is the active protein kinase, whereas the tetramer is found in the cytoplasm and acts as a pyruvate kinase.

Structural and Biophysical Insights into the Ligand-Free Pitx2 Homeodomain and a Ring Dermoid of the Cornea Inducing Homeodomain Mutant

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA binding affinity and a 5 °C decrease in thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in an only 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to those of other Pitx2 homeodomain mutations.

Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIalpha-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIalpha decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIalpha covalent complex indicating that they acted as topoisomerase IIalpha poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.

Abstract 3509: Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Abstract Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binder and intercalator functionalities, have the potential for increased DNA binding affinity and increased cytotoxicity due to their dual mode of DNA binding. In this study DNA minor groove binder netropsin analogs containing either a mono-N-methylpyrrole carboxamide or two units of N-methylpyrrole carboxamide linked to DNA intercalating anthrapyrazoles were synthesized. Two of the hybrid molecules which possessed bis-methylpyrrole moities and amine head groups displayed submicromolar cytotoxicity towards K562 human leukemia cells. The synthesized hybrids were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIα covalent complex, indicating that they acted as topoisomerase IIα poisons. The results indicate that the hybrid agents have higher affinity for DNA than the parent compounds. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazoles and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents. Support: CIHR, a Canada Research Chair in Drug Development, National Institutes of Health grant CA090787 to J.C.Y., the Dishman Foundation at Southwestern University and the Robert A. Welsh Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3509.