Erythroid differentiation ability of butyric acid analogues: identification of basal chemical structures of new inducers of foetal haemoglobin.

Several investigations have demonstrated a mild clinical status in patients with ß-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including ß-thalassaemia and sickle cell anaemia.

Chemical synthetic biology.

Although both the most popular form of synthetic biology (SB) and chemical synthetic biology (CSB) share the biotechnologically useful aim of making new forms of life, SB does so by using genetic manipulation of extant microorganism, while CSB utilises classic chemical procedures in order to obtain biological structures which are non-existent in nature. The main query concerning CSB is the philosophical question: why did nature do this, and not that? The idea then is to synthesise alternative structures in order to understand why nature operated in such a particular way. We briefly present here some various examples of CSB, including those cases of nucleic acids synthesised with pyranose instead of ribose, and proteins with a reduced alphabet of amino acids; also we report the developing research on the "never born proteins" (NBP) and "never born RNA" (NBRNA), up to the minimal cell project, where the issue is the preparation of semi-synthetic cells that can perform the basic functions of biological cells.

Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach.

In this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the chemical viewpoint, macromolecules are formed by chemical pathways leading to the condensation of building blocks (amino acids, or nucleotides) in long-chain copolymers (proteins and nucleic acids, respectively). The second difficulty deals with a conceptual problem, namely with the emergence of specific sequences among a vast array of possible ones, the huge "sequence space", leading to the question "why these macromolecules, and not the others?" We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides, like Ser-His, with respect to peptide- and nucleotides-condensation, as a realistic model of primitive organocatalysis. We have also set up a strategy for exploring the sequence space of random proteins and RNAs (the so-called "never born biopolymer" project) with respect to the production of folded structures. Being still far from solved, the main aspects of these "open questions" are discussed here, by commenting on recent results obtained in our groups and by providing a unifying view on the problem and possible solutions. In particular, we propose a general scenario for macromolecule formation via fragment-condensation, as a scheme for the emergence of specific sequences based on molecular growth and selection.

Chemical synthetic biology: a mini-review.

Chemical synthetic biology (CSB) is a branch of synthetic biology (SB) oriented toward the synthesis of chemical structures alternative to those present in nature. Whereas SB combines biology and engineering with the aim of synthesizing biological structures or life forms that do not exist in nature - often based on genome manipulation, CSB uses and assembles biological parts, synthetic or not, to create new and alternative structures. A short epistemological note will introduce the theoretical concepts related to these fields, whereas the text will be largely devoted to introduce and comment two main projects of CSB, carried out in our laboratory in the recent years. The "Never Born Biopolymers" project deals with the construction and the screening of RNA and peptide sequences that are not present in nature, whereas the "Minimal Cell" project focuses on the construction of semi-synthetic compartments (usually liposomes) containing the minimal and sufficient number of components to perform the basic function of a biological cell. These two topics are extremely important for both the general understanding of biology in terms of function, organization, and development, and for applied biotechnology.

Recombinant expression and insecticidal properties of a Conus ventricosus conotoxin-GST fusion protein.

A novel conotoxin, conotoxin Vn2, was recently isolated from the venom of Conus ventricosus, a worm-hunting cone snail species living in the Mediterranean Sea. Analysis of conotoxin Vn2 amino acid sequence suggested that it is a member of the O1 superfamily of conotoxins. Conotoxin Vn2 displays quite a high degree of sequence similarity with bioactive peptides targeting calcium channels and in particular with the ω conotoxin PnVIB, extracted from the venom of the molluscivorous cone snail Conus pennaceus. In this work we describe the development of a heterologous expression system to obtain a glutathione-S-transferase (GST) fusion product of conotoxin Vn2 in a pure form and in a sufficient amount to characterize its bioactivity. The fusion product has been expressed in recombinant form in Escherichia coli cells, purified, and its neurotoxic activity has been assayed on the larvae of the moth Galleria mellonella, a simple experimental model to test the toxicity of compounds in insects. Moreover the conotoxin Vn2 Asp2His mutant has been produced to analyse the role of this aspartic acid residue in the toxin bioactivity, as an acidic amino acid is conserved in this position in all the O1 superfamily C. ventricosus conotoxins. Results obtained indicate that indeed conotoxin Vn2 has strong insecticidal properties at a dose of only 100 pmol/g of body weight. Surprisingly, mutation of Asp2 to His leads to enhanced toxicity in the larvae model system opening up interesting possibilities for the use of conotoxin Vn2 variants in environmental friendly crop protection applications.

Approaches to chemical synthetic biology.

Synthetic biology is first represented in terms of two complementary aspects, the bio-engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the "Never Born Proteins" and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology.

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2.

BACKGROUND: HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNß to activate STAT1, -2 and -3. METHODOLOGY/PRINCIPAL FINDINGS: Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. CONCLUSIONS: Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNß.

Chemical approaches to synthetic biology.

The term chemical synthetic biology defines that part of the field that, instead of assuming an engineering approach based on genome manipulation, is oriented towards the synthesis of chemical structures alternative to those present in nature. Several different literature projects will be illustrated, including the two of our group. One is concerned with the 'Never Born Proteins' (NBPs), namely polypeptide sequences that are not present in nature, the other is concerned with the notion of 'minimal cells', semi-synthetic compartments (usually liposomes) containing the minimal and sufficient number of components to perform the basic function of a biological cell.

Question 3: the worlds of the prebiotic and never born proteins.

Starting from the statement that no reliable methods are known to produce high molecular weight polypeptides under prebiotic conditions, a possible approach, at least to understand the differences between extant proteins and the possible large number of never born proteins, could be biological. Using the phage display method a large library of totally random amino acidic sequences was obtained. Consequently, different experiments to directly consider the frequency of stable folds were performed, and the interesting results obtained from such new approach are discussed in terms of contingency, contributing to the discussion on the selection mechanism of extant proteins.

Question 5: on the chemical reality of the RNA world.

The discovery of catalytic RNA has revolutionised modern molecular biology and bears important implications for the origin of Life research. Catalytic RNA, in particular self-replicating RNA, prompted the hypothesis of an early "RNA world" where RNA molecules played all major roles such information storage and catalysis. The actual role of RNA as primary actor in the origin of life has been under debate for a long time, with a particular emphasis on possible pathways to the prebiotic synthesis of mononucleotides; their polymerization and the possibility of spontaneous emergence of catalytic RNAs synthesised under plausible prebiotic conditions. However, little emphasis has been put on the chemical reality of an RNA world; in particular concerning the chemical constrains that such scenario should have met to be feasible. This paper intends to address those concerns with regard to the achievement of high local RNA molecules concentration and the aetiology of unique sequence under plausible prebiotic conditions.

Investigation of de novo totally random biosequences, Part I: A general method for in vitro selection of folded domains from a random polypeptide library displayed on phage.

This paper reports the initial phase of a research aimed at investigating the folding frequency within a large library of polypeptides generated with a totally random sequence by phage-display technique. Resistance to proteolytic digestion has been used as a first, rudimentary folding criterion. The present paper describes, in particular, the development of a phage-display vector which has a selectable N-terminal affinity tag so that, after controlled proteolysis, the tag is cleaved from the phage. This enables the positive selection of phages that carry proteolytically resistant proteins. To test this system, avian pancreatic polypeptide (APP), one of the smallest proteins with a known structure, was chosen as a model, and its gene was inserted in a plasmid that was then used for phage display. A sequence of three amino acids, corresponding to a substrate for thrombin, was introduced at different locations within the APP sequence without significantly modifying the tertiary structure, as determined by circular dichroism (CD) analysis. These sequences were then used to show that the target tripeptide sequence was protected against proteolysis by the overall folding of the chain. Thus, these results show that the method permits the discrimination between folded and unfolded protein domains displayed on phage. The application of this protocol to a large library of totally random polypeptide chains is discussed as a preliminary to successive work, dealing with the production of totally random polypeptide sequences.

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display.

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.

Investigation of de novo totally random biosequences, Part III: RNA Foster: A novel assay to investigate RNA folding structural properties.

Fold is essential to RNA properties, and, in particular, its thermodynamic stability can be used to monitor RNA-protein or RNA-ligand interactions, and to engineer RNA with novel or improved properties. While clearly valuable, experimental determination of RNA folding stability by traditional biophysical techniques requires substantial amounts of pure sample and rather expensive equipment. In this paper, we report a new, simple approach to the determination of RNA folding stability by coupling enzymatic digestion and temperature denaturation. The assay, named RNA folding stability Test (RNA Foster), is designed to probe the fraction of folded RNA (f(fold)) in an equilibrium mixture of folded and unfolded ones as a function of temperature. The simplicity of RNA Foster suggests that it can easily be scaled up for high-throughput studies of RNA folding stability both in basic and applied research.

Investigation of de novo totally random biosequences, Part IV: Folding Properties of de novo, totally random RNAs.

This work lies within the framework of a broader project aimed at exploring the realm of all possible folded polypeptides; the main question addressed here is whether the corresponding RNAs also assume a folded conformation. We present an investigation on the structural properties of de novo, totally random RNAs by means of the 'RNA Foster' assay. Experimental results show that all RNAs studied are folded at 37 degrees , so that fold seems to be a common feature of RNAs. Random RNAs' fold shows a surprising thermal stability with an average T(m) value at ca. 50 degrees which prompts the idea that thermo-stable structures might not be as rare as they are commonly thought to be. The results are discussed within the general framework of random RNA properties such as those that might have been produced in a prebiotic scenario.

From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

Preparation and evaluation of the in vitro erythroid differentiation induction properties of some esters of methyl 3,4-O-isopropylidene-beta-D-galactopyranoside and 2,3-O-isopropylidene-D-mannofuranose.

Two series of glycide esters of short fatty acids, designed for avoiding intramolecular transesterification, were prepared and tested for in vitro erythroid differentiation induction activities using the K562 cell line as experimental system. The 6-O-isobutiryl and pivaloyl derivatives of methyl 3,4-O-isopropylidene-beta-D-galactopyranosides as well the same 1-O-esters of 2,3-O-isopropylidene-alpha- and beta-D-mannofuranose exhibit biological activities much higher that the corresponding acids and could be proposed as possible agents to modulate production of embryo-fetal hemoglobins by human erythroid cells.