G-ruption: The third international meeting on G-quadruplex and G-assembly

A three and a half day conference focusing on nucleic acid structures called G-quadruplexes (G4s) and other guanine-based assemblies was held in Sorrento, Italy (June 28–July 1, 2011) and featured 35 invited talks and over 89 posters. The G-quadruplex field continues to expand at an explosive rate with the emergence of new connections to biology, chemistry, physics, and nanotechnology. Following the trend established by the previous two international G4 meetings, the conference touched upon all these areas and facilitated productive exchanges of ideas between researchers from all over the world.     

In search of a cytostatic agent derived from the alkaloid lycorine: synthesis and growth inhibitory properties of lycorine derivatives

As a continuation of our studies aimed at the development of a new cytostatic agent derived from an Amaryllidaceae alkaloid lycorine, we synthesized 32 analogues of this natural product. This set of synthetic analogues included compounds incorporating selective derivatization of the C1 versus C2 hydroxyl groups, aromatized ring C, lactamized N6 nitrogen, dihydroxylated C3-C3a olefin functionality, transposed olefin from C3-C3a to C2-C3 or C3a-C4, and C1 long-chain fatty esters. All synthesized compounds were evaluated for antiproliferative activities in vitro in a panel of tumor cell lines including those exhibiting resistance to proapoptotic stimuli and representing solid cancers associated with dismal prognoses, such as melanoma, glioblastoma, and non-small-cell lung cancer. Most active analogues were not discriminatory between cancer cells displaying resistance or sensitivity to apoptosis, indicating that these compounds are thus able to overcome the intrinsic resistance of cancer cells to pro-apoptotic stimuli. 1,2-Di-O-allyllycorine was identified as a lycorine analogue, which is 100 times more potent against a U373 human glioblastoma model than the parent natural product. Furthermore, a number of synthetic analogues were identified as promising for the forthcoming in vivo studies.     

Sus Scrofa immune tissues as a new source of bioactive substances for skin wound healing

Influence of a new protein-peptide complex on promoting skin wound healing in male BALB/c mice was studied. Protein-peptide complex, extracted from Sus scrofa immune organs, was percutaneously administered using two methods: by lecithin gel-like liquid crystals and by liquid microemulsion. On the fifth day, wound closure in mice with a linear wound model become faster in group (less 2 days comparison to other ones), which was treated with lecithin liquid crystals carrying the protein-peptide complex. This promoting healing can be caused by resorption of bioactive high-molecular compounds the animal skin. In mice with the linear wound model, the tensile strength of the scars were respectively higher both in mice, treated using lecithin liquid crystals with protein-peptide complex, and in mice, treated using microemulsion containing protein-peptide complex, by 215.4% and 161.5% relative to the animals, which did not receive bioactive substances for wound treatment. It was associated with the regeneratory effects of tissue- and species-specific protein-peptide complexes, including α-thymosin Sus scrofa (C3VVV8_PIG, m/z 3802.8) and other factors, which were described as parts of the new extracted complex. This reveals that percutaneous administration of the complex reliably activates local regenerative processes in animals.     

The first crystal structures of hybrid and parallel four-tetrad intramolecular G-quadruplexes

G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized G-tetrads. GQs play an important role in a variety of biological processes and may form at telomeres and oncogene promoters among other genomic locations. Here, we investigate nine variants of telomeric DNA from Tetrahymena thermophila with the repeat (TTGGGG)_n. Biophysical data indicate that the sequences fold into stable four-tetrad GQs which adopt multiple conformations according to native PAGE. Excitingly, we solved the crystal structure of two variants, TET25 and TET26. The two variants differ by the presence of a 3′-T yet adopt different GQ conformations. TET25 forms a hybrid [3 + 1] GQ and exhibits a rare 5′-top snapback feature. Consequently, TET25 contains four loops: three lateral (TT, TT, and GTT) and one propeller (TT). TET26 folds into a parallel GQ with three TT propeller loops. To the best of our knowledge, TET25 and TET26 are the first reported hybrid and parallel four-tetrad unimolecular GQ structures. The results presented here expand the repertoire of available GQ structures and provide insight into the intricacy and plasticity of the 3D architecture adopted by telomeric repeats from T. thermophila and GQs in general.     

Cu(I) binding and transfer by the N terminus of the Wilson disease protein

Wilson and Menkes diseases are genetic disorders of copper metabolism caused by mutations in the Wilson (WND) and Menkes (MNK) copper-transporting P1B-type ATPases. The N termini of these ATPases consist of six metal binding domains (MBDs). The MBDs interact with the copper chaperone Atox1 and are believed to play roles in catalysis and in copper-mediated cellular relocalization of WND and MNK. Although all six MBDs have similar folds and bind one Cu(I) ion via a conserved CXXC motif, biochemical and genetic data suggest that they have distinct functions. Most studies aimed at characterizing the MBDs have employed smaller polypeptides consisting of one or two domains. The role of each MBD is probably defined by its environment within the six-domain N terminus, however. To study the properties of the individual domains within the context of the intact Wilson N terminus (N-WND), a series of variants in which five of the six metal binding CXXC motifs are mutated to SXXS was generated. For each variant, the Cu(I) binding affinity and the ability to exchange Cu(I) with Atox1 were investigated. The results indicate that Atox1 can deliver Cu(I) to and remove Cu(I) from each MBD, that each MBD has stronger Cu(I) retention properties than Atox1, and that all of the MBDs as well as Atox1 have similar K(Cu) values of (2.2-6.3) x 10(10) m(-1). Therefore, the specific role of each MBD is not conferred by its position within the intact N-WND but may be related to interactions with other domains and partner proteins.     

The M.EcoRV DNA-(adenine N6)-methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys11 of M.EcoRV is involved in recognition of Gua1 and Arg128 contacts the Gua in base pair 6. These residues correspond to Lys9 and Arg124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59+/-4 degrees) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua6-Arg128 contact forms early followed by DNA bending and, finally, the formation of the Lys11-Gua1 contact.     

Cellobiose dehydrogenase of Chaetomium sp. INBI 2-26(-): structural basis of enhanced activity toward glucose at neutral pH

Cellobiose dehydrogenase (CDH) is an extracellular fungal flavocytochrome specifically oxidizing cellooligosaccharides and lactose to corresponding (-lactones by a variety of electron acceptors. In contrast to basidiomycetous CDHs, CDHs of ascomycetes also display certain activity toward glucose. The objective of this study was to establish the structural reasons of such an activity of CDH from mesophilic ascomycete Chaetomium sp. INBI 2-26 (ChCDH). The complete amino acid sequence of ChCDH displayed high levels of similarity with the amino acid sequences of CDHs from the thermophilic fungi Thielavia heterotallica and Myriococcum thermophilum. Peptide mass fingerprinting of purified ChCDH provided evidence for the oxidation of methionine residues in the FAD-domain. Comparative homology modeling of the structure of the ChCDH FAD-domain in complex with the transition state analog based on the structure of the same complex of basidiomycetous CDH (1NAA) as template indicated possible structural reasons for the enhanced activity of ascomycetous CDHs toward glucose at neutral pH, which is a prerequisite for application of CDH in a variety of biocompatible biosensors and biofuel cells.     

High affinity cation-binding sites in Complex I from Escherichia coli

Studies on the activity of Complex I from Escherichia coli in the presence of different metal cations revealed at least two high affinity metal-binding sites. Membrane-bound or isolated Complex I was activated by K⁺ (apparent binding constant ∼ 125 μM) and inhibited by La³⁺ (IC₅₀ = 1 μM). K⁺ and La³⁺ do not occupy the same site. Possible localization of these metal-binding sites and their implication in catalysis are discussed.     

Synthesis, enantioresolution, and activity profile of chiral 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones as potent N-formyl peptide receptor agonists

A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures and purified enantiomers to stimulate intracellular Ca(2+) flux in FPR-transfected HL-60 cells and human neutrophils and to induce β-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Furthermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(-)-forms over the S-(+)-enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active compounds generally increased biological activity. Thus, these studies provide important new information regarding molecular features involved in FPR ligand preference and report the identification of a novel series of FPR agonists.