Luana Di Leandro

Cystamine restores GSTA3 levels in Vanin-1 null mice.

Free cysteamine levels in mouse tissues have been strictly correlated to the presence of membrane-bound pantetheinase activity encoded by Vanin-1. Vanin-1 is involved in many biological processes in mouse, from thymus homing to sexual development. Vanin-1 -/- mice are fertile and grow and develop normally; they better control inflammation and most of the knockout effects were rescued by cystamine treatment. Gene structure analysis showed the presence of an oxidative stimuli-responsive ARE-like sequence in the promoter. In this paper we investigate antioxidant-detoxifying enzymatic activities at the tissue level, comparing Vanin-1 -/- and wild-type mice. In Vanin-1 null animals we pointed out a decrease in the Se-independent glutathione peroxidase activity. The decrease in enzymatic activity appeared to be correlated to an impairment of GST isoenzyme levels. In particular a significant drop in GSTA3 together with a minor decrement in GSTM1 and an increase in GSTP1 levels was detected in Vanin-1 -/- livers. Cystamine administration to Vanin-1 -/- mice restored specifically GSTA3 levels and the corresponding enzymatic activity without influencing protein expression. A possible role of cystamine on protein stability/folding can be postulated.

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: Is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease‐associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease‐associated strain a 9‐residues insertion was found to be located close to the type I Cu‐site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate‐derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.

Metal-induced self-assembly of peroxiredoxin as a tool for sorting ultrasmall gold nanoparticles into one-dimensional clusters†

Nanomanipulation of matter to create responsive, ordered materials still remains extremely challenging. Supramolecular chemistry has inspired new strategies by which such nanomaterials can be synthesized step by step by exploiting the self-recognition properties of molecules. In this work, the ring-shaped architecture of the 2-Cys peroxiredoxin I protein from Schistosoma mansoni, engineered to have metal ion-binding sites, is used as a template to build up 1D nanoscopic structures through metal-induced self-assembly. Chromatographic and microscopic analyses demonstrate the ability of the protein rings to stack directionally upon interaction with divalent metal ions and form well-defined nanotubes by exploiting the intrinsic recognition properties of the ring surfaces. Taking advantage of such behavior, the rings are then used to capture colloidal Ni(2+)-functionalized ultrasmall gold nanoparticles and arrange them into 1D arrays through stacking into peapod-like complexes. Finally, as the formation of such nano-peapods strictly depends on nanoparticle dimensions, the peroxiredoxin template is used as a colloidal cut-off device to sort by size the encapsulated nanoparticles. These results open up possibilities in developing Prx-based methods to synthesize new advanced functional materials.

Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems

BackgroundAntibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.ResultsAn anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range.ConclusionsWe undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.

Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes

BackgroundChromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level.ResultsWe report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei.ConclusionsAtomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.

Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants

A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.

Ricin and Saporin: Plant Enzymes for the Research and the Clinics

Many plants produce enzymes with N-glycosidase activity, also known as Ribosome Inactivating Proteins. These proteins remove a specific adenine residue from the ribosomal RNA (28S in eukaryotes) inducing the block of pro- tein synthesis by inhibiting the binding of the Elongation Factor 2. Both eukaryotic and prokaryotic ribosomes (with dif- ferent sensitivity) can irreversibly be damaged by the action of these enzymes, suggesting their use as cytotoxic drugs. In fact several applications of targeted N-glycosidases have been developed (i.e. immunotoxins) for the treatment of human diseases such as leukaemia, but biotechnological development has furthermore suggested new applications of targeted N- glycosidases (i.e. Ig192-saporin) that are now used as powerful tools for cell biology research. The high number of en- zymes available and the possibility to express these proteins as recombinant products, allow to predict new formulations and applications discussed in this paper starting from the example of the model toxins ricin and saporin.

Targeted therapy of human glioblastoma via delivery of a toxin through a peptide directed to cell surface nucleolin

Targeted anticancer therapies demand discovery of new cellular targets to be exploited for the delivery of toxic molecules and drugs. In this perspective, in the last few years, nucleolin has been identified as an interesting surface marker to be used for the therapy of glioblastoma. In this study, we investigated whether a synthetic antagonist of cell‐surface nucleolin known as N6L, previously reported to decrease both tumor growth and tumor angiogenesis in several cancer cell lines, including glioblastoma cells, as well as endothelial cells proliferation, could be exploited to deliver a protein toxin (saporin) to glioblastoma cells. The pseudopeptide N6L cross‐linked to saporin‐S6 induced internalization of the toxin inside glioblastoma cancer cells. Our results in vitro demonstrated the effectiveness of this conjugate in inducing cell death, with an ID50 four orders of magnitude lower than that observed for free N6L. Furthermore, the preliminary in vivo study demonstrated efficiency in reducing the tumor mass in an orthotopic mouse model of glioblastoma.

Conformational Changes at the Active Site of Pantetheine Hydrolase During Denaturation by Guanidine Hydrochloride

Conformational changes at the active site of pantetheine hydrolase (EC3.5.1.-) during guanidine hydrochloride (GndHCl) denaturation were investigated by UV and circular dichroism spectroscopy and by electron spin resonance spectroscopy, following the spectral behaviour of the nitroxide radicals (N- (1- oxyl - 2,2,5,5, -tetramethyl-3-pyrrolidinyl) iodacetamide) covalently linked to the two active site cysteine residues. At low denaturant concentrations (0.2 M) no conformational changes may be observed, whereas the catalytic activity, is strongly affected. The results indicate that the active site of pantetheine hydrolase is labile and unfolds under conditions in which no global tertiary struscture modifications can be observed.