Veronica Esposito

MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

Background Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Conclusions/Significance Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

A new modified thrombin binding aptamer containing a 5′–5′ inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(3′GGT5′-5′TGGTGTGGTTGG3′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3′GGT5′-5′TGGTGTGGTTGG3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(3′GGT5′-5′TGGTGTGGTTGG3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.

Understanding the Molecular Basis for the Inhibition of the Alzheimer's Aβ-Peptide Oligomerization by Human Serum Albumin Using Saturation Transfer Difference and Off-Resonance Relaxation NMR Spectroscopy

Human serum albumin (HSA) inhibits the formation of amyloid beta-peptide (Abeta) fibrils in human plasma. However, currently it is not known how HSA affects the formation of the highly toxic soluble diffusible oligomers that occur in the initial stages of Abeta fibrillization. We have therefore investigated by solution NMR the interaction of HSA with the Abeta(12-28) peptide, which has been previously shown to provide a reliable and stable model for the early prefibrillar oligomers as well as to contain key determinants for the recognition by albumin. For this purpose we propose a novel NMR approach based on the comparative analysis of Abeta in its inhibited and filtrated states monitored through both saturation transfer difference and recently developed nonselective off-resonance relaxation experiments. This combined NMR strategy reveals a mechanism for the oligomerization inhibitory function of HSA, according to which HSA targets preferentially the soluble oligomers of Abeta(12-28) rather than its monomeric state. Specifically, HSA caps the exposed hydrophobic patches located at the growing and/or transiently exposed sites of the Abeta oligomers, thereby blocking the addition of further monomers and the growth of the prefibrillar assemblies. The proposed model has implications not only for the pharmacological treatment of Alzheimers disease specifically but also for the inhibition of oligomerization in amyloid-related diseases in general. In addition, the proposed NMR approach is expected to be useful for the investigation of the mechanism of action of other oligomerization inhibitors as well as of other amyloidogenic systems.

Cold denaturation of yeast frataxin offers the clue to understand the effect of alcohols on protein stability

Although alcohols are well-known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. In this paper, we present a study of the effects of alcohols on protein stability using Yfh1, the yeast ortholog of the human protein frataxin. Exploiting the unusual property of this protein of undergoing cold denaturation around 0 degrees C without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol, ethanol, and methanol. In all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. On the basis of simple thermodynamic considerations, we are able to interpret the literature on the effects of alcohols on proteins and to generalize our findings. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. Our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation.

Configuration assignment in small organic molecules via residual dipolar couplingsElectronic supplementary information (ESI) available: Listing of the C program RDC_AX, tridimensional models of compounds 1, 3-epi-1, 7-epi-1, and 12-epi-1 in PDB format, and the command files for 1, 3-epi-1, 7-epi-1, and 12-epi-1. See http://www.rsc.org/suppdata/cc/b2/b210454g/

Here we propose a new method to assign relative configurations of stereocenters in small organic molecules by using residual dipolar couplings; the main advantage of this method is that spatial proximity of the stereocenters is not required.

Understanding cold denaturation: the case study of Yfh1.

All globular proteins undergo transitions from their native to unfolded states if exposed either to cold or to heat perturbation. While the heat-induced transition is well described for a large number of proteins, in media compatible with natural environments, the limited number of examples of cold denatured states concern proteins artificially destabilized, for instance, by the presence of denaturants, ad hoc point mutations, or both. Here, we provide a characterization of the low temperature unfolded state of Yfh1, a natural protein that undergoes cold denaturation around water freezing temperature, in the absence of any denaturant. By achieving nearly full assignment of the NMR spectrum, we show that at -1 °C, Yfh1 has all the features of an unfolded protein, although retaining some local, residual secondary structure. The effect is not uniform along the sequence and does not merely reflect the secondary structural features of the folded species. The N-terminus seems to be dynamically more flexible, although retaining some nascent helix character. Interestingly, this region is the one containing functionally important hot-spots. The β-sheet region and the C-terminal helix are completely unfolded, although experiencing some conformational exchange, partly due to the presence of several prolines. Ours is the first step toward a full characterization of the low temperature unfolded state of a natural protein, reached without the aid of any destabilizing agent. We discuss the implications of our findings for understanding cold denatured states.

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.

Interaction of distamycin A and netropsin with quadruplex and duplex structures: a comparative 1H-NMR study.

ABSTRACT Homonuclear NMR techniques have been used to investigate the interactions of the minor groove binding agents distamycin A (Dist-A) and the related drug netropsin (Net) with three quadruplexes characterized by different groove widths: [d(TGGGGT)]4 (Q1), [d(GGGGTTTTGGGG)]2 (Q2), and d(GGGGTTGGGGTGTGGGGTTGGGG) (Q3). Netropsin has been found to be in a fast chemical exchange with all three kinds of quadruplexes, whereas Dist-A interacts tightly with Q1 and, at a less extent, with Q2. In order to determine the degree of selectivity of Dist-A for two- rather than four-stranded DNA, we titrated with Dist-A an equimolar solution of Q1 and the duplex d(CGCAAATTTGCG)2 (D). This comparative 1H-NMR study allowed us to conclude that Dist-A and, consequently, Net possess higher affinity for duplex DNA.

Structure of the C-terminal Domain of Neisseria Heparin Binding Antigen (NHBA), One of the Main Antigens of a Novel Vaccine against Neisseria meningitidis

Background: NHBA, a surface-exposed lipoprotein from Neisseria meningitidis, is part of a multicomponent vaccine against serogroup B meningitis. Results: We have solved the structure of a conserved C-terminal domain that adopts a β-barrel fold and seems to be the only independently folded region of the protein. Conclusion: We observed a significant structural similarity with other Nesseria proteins. Significance: Our data represent the first step toward understanding the structure/immunology relationship of NHBA. Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand β-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the β-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.

The insertion of two 8-methyl-2′-deoxyguanosine residues in tetramolecular quadruplex structures: trying to orientate the strands

In this article, we report a structural study, based on NMR and CD spectroscopies, and molecular modelling of all possible d(TG3T) and d(TG4T) analogues containing two 8-methyl-2′-deoxyguanosine residues (M). Particularly, the potential ability of these modified residues to orientate the strands and then to affect the folding topology of tetramolecular quadruplex structures has been investigated. Oligodeoxynucleotides (ODNs) TMMGT (T12) and TMMGGT (F12) form parallel tetramolecular quadruplexes, characterized by an all-syn M-tetrad at the 5′-side stacked to all-anti M- and G-tetrads. ODNs TMGMT (T13) and TMGGMT (F14) form parallel tetramolecular quadruplexes, in which an all-anti G core is sandwiched between two all-syn M-tetrads at the 5′- and the 3′-side. Notably, the quadruplex formed by T13 corresponds to an unprecedented structure in which the syn residues exceed in number the anti ones. Conversely, ODN TGMGMT (F24) adopts a parallel arrangement in which all-anti G-tetrads alternate with all-syn M-tetrads. Most importantly, all data strongly suggest that ODN TMGMGT (F13) forms an unprecedented anti-parallel tetramolecular quadruplex in which G and M residues adopt anti and syn glycosidic conformations, respectively. This article opens up new understandings and perspectives about the intricate relationship between the quadruplex strands orientation and the glycosidic conformation of the residues.