The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids

open5noBACKGROUND: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. METHODS: Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. RESULTS: The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. CONCLUSIONS: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. GENERAL SIGNIFICANCE: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis.openLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sara NLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sar

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

Heterologous expression of the NAD(+)-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD(+)/NADP(+) ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP(+) and NAD(+) for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance

Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways

Space, translations and media

The paper examines the role of media in the processes of the production of urban space in the case of Paolo Sarpi, a central neighborhood of Milan that is characterized by long–standing spatial conflicts between the residents and the Chinese migrants. The purpose of this complex case study is to highlight the many roles played by media in the processes of socio–spatial production, as well as the benefits of reading media within those same processes. To this end, we interrogate the space by drawing on concepts from science and technology studies and media studies. Finally, by analyzing the representations and practices enacted by users of three location–based social networks (TripAdvisor, Foursquare, and Facebook Places), we show that urban processes and media are in a relation of reciprocal shaping. We find that these representations and practices are informed by and feedback on the broader socio–spatial production patterns investing the area. Our conclusion is that extracting media from broader urban processes and focusing on them could be analytically counter–productive. Instead, investigations of the relationship of media and the city should take into account their reciprocal shaping

Introduction: Beyond the centrality of media and the centrality of space

Starting from the middle of the twentieth century human geography has allowed social sciences to escape the prison of Euclidean, abstract space. In that prison, social actors performed within an empty, static container known as “space,” which was more or less a background to their actions. This liberation had many fathers. We could quote Henri Lefebvre’s writings on spatial production (Lefebvre, 1991), Michel de Certeau’s notion of “space as practiced place” (de Certeau, 1984), and Yi–Fu Tuan’s (1976) treatment of “humanistic geography” among the most known “co–conspirators” of this escape. Breaking free of the notion of abstract space, meant to develop the powerful theoretical tool of socio–spatial production. Space emerged as a product of human interaction and at the same time as a context structuring those practices. By the mid–1980s, Massey expressed the circular relationship between space and the social in no ambiguous terms. “Space is a social construct ... [but] the spatial is not only an outcome: it is also part of the explanation [of social processes]” (Massey, 1984). Producing the space we inhabit meant that the more people were in a space, the more rapidly and often unpredictably it changed. And the more diverse the people, the more diverse the way they thought, and what they did in space. Therefore, the more rapid and unpredictable were the changes

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes

[Image: see text] HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses

A chimeric hydrolase-PTXD transgene enables chloroplast-based heterologous protein expression and non-sterile cultivation of Chlamydomonas reinhardtii

Photosynthetic microalgae hold great potential as light-driven heterologous protein expression hosts. In particular, the algal chloroplast is an ideal sub-cellular site for the compartmentalized synthesis and accumulation of high-value recombinant proteins. However, full integration of transplastomic algal biotechnology in the large-scale production of biocatalysts still suffers from major bottlenecks, such as genetic instability and pest contamination. To enhance the reliability of plastid-based algal expression platforms we developed a self-reinforcing genetic system in Chlamydomonas reinhardtii. We transformed the plastome with a bifunctional transgene encoding an in vivo cleavable fusion polypeptide composed of a hyperthermophilic cellulase and the phosphite dehydrogenase PTXD. The dual use of phosphite as a low-cost, environmentally friendly selective agent and fertilizer afforded axenic algal cultivation via mixotrophic metabolism and efficient expression of the hydrolytic enzyme. This study provides an example of chloroplast genetic engineering in which biosafety is integrated in the sustainable management of microalgal monocultures to produce enzymes with industrial applications