DNA accessibility to regulatory proteins is significantly affected by nucleosome structure and dynamics. FACT (facilitates chromatin transcription) increases the accessibility of nucleosomal DNA but the mechanism and extent of this nucleosome reorganization are unknown. We report here the effects of FACT on single nucleosomes revealed with spFRET microscopy. FACT binding results in a dramatic, ATP-independent, and reversible uncoiling of DNA that affects at least 70% of the DNA in a nucleosome. A mutated version of FACT is defective in this uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this is an important function of FACT in vivo.

This study presents purification, activity characterization, and 1 H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized ␣-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.Antimicrobial peptides (AMPs) 4 are a structurally diverse group of generally small, positively charged peptides produced by various living organisms and demonstrating a wide spectrum of antimicrobial activity (1, 2). Natural sources of AMPs range from prokaryotes to higher animals, and their targets include bacteria, fungi, protozoa, and viruses. The mechanism of action of most known AMPs involves their direct or receptor-mediated interaction with microbial membranes (3-5). It has been generally accepted that membrane-disruptive AMPs kill microorganisms by provoking in different ways an increase in plasma membrane permeability. Non-membrane-disruptive peptides have been shown to target cell wall formation or traverse membranes and affect various internal cellular processes, for example, RNA, DNA, and/or protein biosynthesis. Some AMPs can combine disruptive and non-disruptive mechanisms of action (6). Moreover, mechanisms of action of the same peptide may differ depending on the target. Recent studies have also indicated that AMPs are multifunctional molecules; they can interact with host membrane receptors and influence diverse intracellular processes modulating the immune response of the host organism (7,8).Essential variety in detailed mechanisms of action and multifunctionality imply structural diversity among AMPs. The following structural groups are usually recognized: (i) linear peptides that form ␣-helices in contact with membranes; (ii) disulfide-containing with predominance of ␤-structural elements; and (iii) linear non-␣-helix-forming, usually with a high content of certain amino acid residues (1, 2, 9). Most of the approximately 200 AMP spatial structures known at present (see the Antimicrobial Peptide Database v2.26 (10)) fall into one of the first two groups. Further classification is based on unique features in the sequences and/or structures of AMPs. For example, thionins, defensins, nonspecific lipid transfer proteins, and hevein-and knottin-like peptides have been identified in plants (11-13).To characterize the array of AMPs produced by a plant under certain physiological conditions, we have carried out a systematic analysis of these peptides from...

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC . We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization.

Protumor factor FACT is removed from transcribed genes by anticancer drugs curaxins.

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (Kd=7.2+/-0.4 microM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (DeltaG=-15.4+/-0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.

The essential histone chaperone FACT (cilitates hromatinranscription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization , indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

Background: Isolated Eph transmembrane domains (TMD) dimerize in membrane mimetics, but the functional significance of these interactions is unclear. Results: Mutations introduced into the alternative dimerization motifs of the EphA2 TMD induced an opposite effect on receptor activity. Conclusion: Alternative TMD interactions promote either the active or inactive EphA2 conformation. Significance: The involvement of TMD interactions in Eph receptor activity is discovered.The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/ oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L 535 X 3 G 539 X 2 A 542 X 3 V 546 X 2 L 549 rather than through the alternative glycine zipper motif A 536 X 3 G 540 X 3 G 544(typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr 588 and/or Tyr 594) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.Receptor tyrosine kinases of the Eph family and their ephrin ligands are key regulators of cell-cell and cell-matrix adhesion, coordinating cell migration and positioning in various adult and embryonic tissues of human organisms (1, 2). The EphA2 receptor, a representative of the human 14-member Eph family, controls such diverse processes as capillary stabilization by pericytes (3), keratinocyte movement out of the basal layer (2), blastocyst entry into the endometrial layer (4), and cardiac stem cell mobilization from the niche (5). Activation of EphA2 leads to cell detachment (mobilization), loss of intercellular contacts (an increase in cell layer permeability), or cell repulsion (guidance).A classical model of EphA2 activation assumes the binding of a li...