Insights on the disruption of the complex between human positive coactivator 4 and p53 by small molecules.

Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of ∼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.

Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4.

Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA-protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations.

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.

The triterpenoid ursolic acid ameliorates stress in Caenorhabditis elegans by affecting the depression-associated genes skn-1 and prdx2.

INTRODUCTION: Depression is one of the leading causes of death worldwide. Lower antioxidant concentrations and increased oxidative stress levels contribute to the development of depression. Effective and tolerable medications are urgently needed. Nrf2 and PRDX2 are promising targets in the treatment of oxidative stress and, therefore, promising for the development of novel antidepressants. Ursolic acid (UA), a natural triterpenoid found in various plants is known to exert neuroprotective and antioxidant effects. Skn-1 (which corresponds to human Nrf2) and prdx2 deficient mutants of the nematode Caenorhabditis elegans are suitable models to study the effect of UA on these targets. Additionally, stress assays are used to mimic stress or depressed state. METHODS: We examined the antioxidant activity of UA in Caenorhabditis elegans wildtype and skn-1- and prdx2-deficient strains by H2DCF-DA and juglone assays as well as osmotic and heat stress assays. Additionally, we analyzed the binding of UA to human PRDX2 and Skn-1 proteins by molecular docking and microscale thermophoresis. RESULTS: UA exerted strong antioxidant activities. Additionally, induction of stress resistance towards osmotic and heat stress was observed. qRT-PCR revealed that UA upregulated the gene expression of skn-1 and prdx2. Molecular docking studies supported these findings. CONCLUSION: Our findings implicate that the strong antioxidant activity of UA may exert anti-depressive effects by its interaction with the Skn-1 transcription factor, which is part of a detoxification network, and the antioxidant PRDX2 protein, which protects the organism from the detrimental effects of radical oxygen species.

ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling.

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.

High-Throughput Screening to Identify Inhibitors of SSB-Protein Interactions.

The bacterial single-stranded DNA-binding protein (SSB) uses an acidic C-terminal tail to interact with over a dozen proteins, acting as a genome maintenance hub. These SSB-protein interactions are essential, as mutations to the C-terminal tail that disrupt these interactions are lethal in Escherichia coli. While the roles of individual SSB-protein interactions have been dissected with mutational studies, small-molecule inhibitors of these interactions could serve as valuable research tools and have potential as novel antimicrobial agents. This chapter describes a high-throughput screening campaign used to identify inhibitors of SSB-protein interactions. A screen targeting the PriA-SSB interface from Klebsiella pneumoniae is presented as an example, but the methods may be adapted to target nearly any SSB interaction.

Functional Characterization of BRASSINAZOLE-RESISTANT 1 in Panax Ginseng (PgBZR1) and Brassinosteroid Response during Storage Root Formation.

Brassinosteroids (BRs) play crucial roles in the physiology and development of plants. In the model plant Arabidopsis, BR signaling is initiated at the level of membrane receptors, BRASSINOSTEROIDS INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) complex, thus activating the transcription factors (TFs) BRASSINAZOLE RESISTANT 1/BRI1-EMS-SUPPRESSOR 1 (BZR1/BES1) to coordinate BR responsive genes. BRASSINOSTEROIDS INSENSITIVE 2 (BIN2), glycogen synthase kinase 3 (GSK3) like-kinase, negatively regulates BZR1/BES1 transcriptional activity through phosphorylation-dependent cytosolic retention and shuttling. However, it is still unknown whether this mechanism is conserved in Panax ginseng C. A. Mayer, a member of the Araliaceae family, which is a shade-tolerant perennial root crop. Despite its pharmacological and agricultural importance, the role of BR signaling in the development of P. ginseng and characterization of BR signaling components are still elusive. In this study, by utilizing the Arabidopsisbri1 mutant, we found that ectopic expression of the gain of function form of PgBZR1 (Pgbzr1-1D) restores BR deficiency. In detail, ectopic expression of Pgbzr1-1D rescues dwarfism, defects of floral organ development, and hypocotyl elongation of bri1-5, implying the functional conservation of PgBZR1 in P. ginseng. Interestingly, brassinolide (BL) and BRs biosynthesis inhibitor treatment in two-year-old P. ginseng storage root interferes with and promotes, respectively, secondary growth in terms of xylem formation. Altogether, our results provide new insight into the functional conservation and potential diversification of BR signaling and response in P. ginseng.

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1.

BACKGROUND: Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. METHODS: We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. RESULTS: Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. CONCLUSIONS: The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations.

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein (YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH-NPAT and YARP-NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple α -helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single α -helix that makes multiple contacts with α -helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Antimicrobial peptides as a promising treatment option against Acinetobacter baumannii infections.

BACKGROUND: With the increasing rate of antibiotic resistance in Acinetobacter, the World Health Organization introduced the carbapenem-resistant isolates in the priority pathogens list for which innovative new treatments are urgently needed. Antimicrobial peptides (AMPs) are one of the antimicrobial agents with high potential to produce new anti-Acinetobacter drugs. This review aims to summarize recent advances and compare AMPs with anti-Acinetobacter baumannii activity. METHODS: Active AMPs against Acinetobacter were considered, and essential features, including structure, mechanism of action, anti-A. baumannii potent, and other prominent characteristics, were investigated and compared to each other. In this regard, the Google Scholar search engine and databases of PubMed, Scopus, and Web of Science were used. RESULTS: Forty-six anti-Acinetobacter peptides were identified and classified into ten groups: Cathelicidins, Defensins, Frog AMPs, Melittin, Cecropins, Mastoparan, Histatins, Dermcidins, Tachyplesins, and computationally designed AMPs. According to the Minimum Inhibitory Concentration (MIC) reports, six peptides of Melittin, Histatin-8, Omega76, AM-CATH36, Hymenochirin, and Mastoparan have the highest anti-A. baumannii power against sensitive and antibiotic-resistant isolates. All anti-Acinetobacter peptides except Dermcidin have a net positive charge. Most of these peptides have alpha-helical structure; however, ß-sheet and other structures have been observed among them. The mechanism of action of these antimicrobial agents is divided into two categories of membrane-based and intracellular target-based attack. CONCLUSION: Evidence from this review indicates that AMPs would be likely among the main anti-A. baumannii drugs in the post-antibiotic era. Also, the application of computer science to increase anti-A. baumannii activity and reduce toxicity could be helpful.

Balance between asymmetry and abundance in multi-domain DNA-binding proteins may regulate the kinetics of their binding to DNA.

DNA sequences are often recognized by multi-domain proteins that may have higher affinity and specificity than single-domain proteins. However, the higher affinity to DNA might be coupled with slower recognition kinetics. In this study, we address this balance between stability and kinetics for multi-domain Cys2His2- (C2H2-) type zinc-finger (ZF) proteins. These proteins are the most prevalent DNA-binding domain in eukaryotes and C2H2 type zinc-finger proteins (C2H2-ZFPs) constitute nearly one-half of all known and predicted transcription factors in human. Extensive contact with DNA via tandem ZF domains confers high stability on the sequence-specific complexes. However, this can limit target search efficiency, especially for low abundance ZFPs. Earlier, we found that asymmetrical distribution of electrostatic charge among the three ZF domains of the low abundance transcription factor Egr-1 facilitates its DNA search process. Here, on a diverse set of 273 human C2H2-ZFP comprised of 3-15 tandem ZF domains, we find that, in many cases, electrostatic charge and binding specificity are asymmetrically distributed among the ZF domains so that neighbouring domains have different DNA-binding properties. For proteins containing 3-6 ZF domains, we show that the low abundance proteins possess a higher degree of non-specific asymmetry and vice versa. Our findings suggest that where the electrostatics of tandem ZF domains are similar (i.e., symmetrical), the ZFPs are more abundant to optimize their DNA search efficiency. This study reveals new insights into the fundamental determinants of recognition by C2H2-ZFPs of their DNA binding sites in the cellular landscape. The importance of electrostatic asymmetry with respect to binding site recognition by C2H2-ZFPs suggests the possibility that it may also be important in other ZFP systems and reveals a new design feature for zinc finger engineering.

New structural insights into the recognition of undamaged splayed-arm DNA with a single pair of non-complementary nucleotides by human nucleotide excision repair protein XPA.

XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.

Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation.

PURPOSE: The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia. METHODS: Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4-11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2'-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element. RESULTS: We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser588 of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4. CONCLUSIONS: From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.

New series of fused pyrazolopyridines: Synthesis, molecular modeling, antimicrobial, antiquorum-sensing and antitumor activities.

New series of fused pyrazolopyridines were prepared and assessed for antimicrobial, antiquorum-sensing and antitumor activities. Antimicrobial evaluation toward selected Gram-positive bacteria, Gram-negative bacteria and fungi indicated that 5-phenylpyrazolopyridotriazinone 4a has good and broad-spectrum antimicrobial activity. In addition, 5-(4-chlorophenyl)pyrazolopyridotriazinone 4b and 5-(4-(dimethylamino)phenyl)pyrazolopyridotriazinone 4c exhibited good activity against the selected Gram-positive bacteria and A. fumigatus, whereas 5-amino-4-phenylpyrazolopyridopyrimidine 6a demonstrated good activity against B. cereus and P. aeruginosa. Furthermore, 6-amino-5-imino-4-phenylpyrazolopyridopyrimidine 7a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b demonstrated promising activity against the tested Gram-negative bacteria and fungi, and moderate activity against Gram-positive bacteria. Antiquorum-sensing screening over C. violaceum illustrated that 4a, 6a and 7a-c have strong activity. In vitro antiproliferative assessment of the new derivatives against HepG2, HCT-116 and MCF-7 cancer cells revealed that 7a is the most active analog against all tested cell lines. Likewise, 3,7-dimethyl-4-phenylpyrazolopyridopyrimidinone 2a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b manifested strong activity against all examined cell lines. In vivo antitumor testing of 2a, 7a and 7b against EAC cells in mice indicated that 7a has the highest activity. Cytotoxicity toward WI38 and WISH normal cells was also assessed and results assured that all of the investigated analogs have lower cytotoxicity than doxorubicin. DNA-binding affinity and topoisomerase IIß inhibitory activity were evaluated, and results revealed that 5b, 7a and 7b bind strongly to DNA; in addition, 2a, 4a, 7a and 7b manifested higher topoisomerase IIß inhibitory activity than that of doxorubicin. Analogs 5b, 7a and 7b were docked into topoisomerase IIß, and results indicated that 7a and 7b have the highest binding affinity toward topoisomerase IIß. In silico simulation studies referred that most of the new analogs comply with the optimum needs for good oral absorption. Also, computational carcinogenicity evaluation was predicted.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic ß-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.

Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is one among the deadliest pathogens in the world. Due to long treatment regimen, HIV co-infection, persistence of bacilli in latent form and development of XDR and TDR strains of Mtb, tuberculosis has posed serious concerns for managing the disease, and calls for discovery of new drugs and drug targets. Using a computational pipeline involving analysis of the structural models of the Mtb proteome and an analysis of the ATPome, followed by a series of filters to identify druggable proteins, solubility and length of the protein, several candidate proteins were shortlisted. From this, Rv3405c, a tetR family of DNA binding protein involved in antibiotic resistance, was identified as one of the good drug targets. Rv3405c binds to the upstream non-coding region of Rv3406 and causes repression of Rv3406 activity there by affecting the downstream processes involved in antibiotic resistance was further characterized. The Rv3405c gene was cloned; the gene product was over-expressed in E. coli and purified by Ni NTA chromatography. DNA binding studies by EMSA showed that the recombinant Rv3405c protein binds to the DNA sequence corresponding to the promoter region of Rv3406 and upon addition of tetracycline, the DNA binding activity was lost. ß-galactosidase reporter assay in E. coli using both wild type and a DNA binding defective mutant protein indeed proved that Rv3405c acts as a repressor.

TTF1­NP induces protective autophagy during apoptosis by inhibiting the Akt/mTOR pathway and activating JNK in human liver cancer cells.

TTF1­NP is a flavonoid nanoparticle based on 5,2',4'­trihydroxy­6,7,5'­trimethoxyflavone (TTF1), which is derived from the medicinal plant Sorbaria sorbifolia that grows in the Changbai Mountain. We previously demonstrated antitumor effects of TTF1­NP in human hepatoma including induction of apoptosis and inhibition of angiogenesis, migration and invasion. Herein, we examined the effects of TTF1­NP on autophagy and its relationship with apoptosis, and explored potential underlying mechanisms in human hepatoma cell lines. We conducted cell viability assays, Annexin V/propidium iodide double staining, Hoechst staining, monodansylcadaverine staining, transmission electron microscopy, green fluorescent protein­light chain 3 plasmid transfection and western blots. We found that TTF1­NP induced apoptosis and autophagy in HepG2 and SMMC­7721 cells. Pretreatment with the autophagy inhibitor 3­methyladenine promoted TTF1­NP­induced apoptosis. TTF1­NP decreased levels of phosphorylated (p)­Akt, p­mTOR and p­ERK1/2 and increased p­JNK levels in the two cell lines. Treating cells with insulin, SP600125 and U0126 indicated that the Akt/mTOR pathway and JNK were involved in TTF1­NP­induced autophagy. Together, these findings suggest that TTF1­NP induced protective apoptosis­related autophagy by modulating the Akt/mTOR and JNK pathways in HepG2 and SMMC­7721 cells. Therefore, autophagy may be a potential target for TTF1­NP in human hepatoma therapy.

Near-Infrared Plasmonic Assemblies of Gold Nanoparticles with Multimodal Function for Targeted Cancer Theragnosis.

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (<5 nm) that can be easily cleared from the body through renal excretion without causing accumulation/toxicity problems. This DMA-based novel approach offers a promising platform for targeted cancer theragnosis based on simultaneous PA imaging and PT therapy.

Physical Interaction of Human Yin Yang 1 Protein with DNA.

Yin Yang 1 (YY1)'s interaction with DNA can result in various, even contradicting, effects on transcription in the form of initiation, activation, or repression. This surprising activity can be explained in the context of the YY1-DNA's complex structure. YY1's DNA-binding domain is formed by four zinc finger motifs. However, the sequence of both the zinc fingers and the linkers is non-canonical, which impairs their docking to the DNA duplex. Short linkers between the zinc fingers impose a concerted binding mechanism. Analysis of the sequences known to be recognized by YY1 suggests different contributions of particular zinc fingers in specific recognition of activated versus repressed promoters. Thermodynamic and kinetic studies show that, although the YY1's N-terminal fragment does not itself bind to DNA, it might regulate the interaction because its presence influences the binding parameters. Meta-analysis of YY1-DNA binding allowed us to observe that YY1's avidity to multiple binding sites is crucial in providing high-affinity specific sequence recognition. Alternatively, other trans-acting factors can modulate the YY1-DNA interaction and influence its outcome. This complex mechanism causes great sensitivity for individual point mutations, an increasing number of which are found in YY1 in cancer tissues.

An acridine derivative, [4,5-bis{(N-carboxy methyl imidazolium)methyl}acridine] dibromide, shows anti-TDP-43 aggregation effect in ALS disease models.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with aggregation of TAR DNA-binding protein-43 (TDP-43) in neuronal cells and manifests as motor neuron dysfunction &muscle atrophy. The carboxyl-terminal prion-like domain of TDP-43 can aggregate in vitro into toxic ß-sheet rich amyloid-like structures. So far, treatment options for ALS are very limited and Riluzole, which targets glutamate receptors, is the only but highly ineffective drug. Therefore, great interest exists in developing molecules for ALS treatment. Here, we have examined certain derivatives of acridine containing same side chains at position 4 &5, for inhibitory potential against TDP-43 aggregation. Among several acridine derivatives examined, AIM4, which contains polar carboxyl groups in the side arms, significantly reduces TDP-43-YFP aggregation in the powerful yeast model cell and also abolishes in vitro amyloid-like aggregation of carboxyl terminal domain of TDP-43, as observed by AFM imaging. Thus, AIM4 can be a lead molecule potentiating further therapeutic research for ALS.