Umberto Benatti

Tryptophan-derived Catabolites Are Responsible for Inhibition of T and Natural Killer Cell Proliferation Induced by Indoleamine 2,3-Dioxygenase

Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4+ T lymphocytes, CD8+ T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only l-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by l-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

Pyroglutamate‐modified amyloid β‐peptides – AβN3(pE) – strongly affect cultured neuron and astrocyte survival

N‐terminally truncated amyloid‐β (Aβ) peptides are present in early and diffuse plaques of individuals with Alzheimers disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Downs syndrome (DS). The pyroglutamate‐containing isoforms at position 3 [AβN3(pE)−40/42] represent the prominent form among the N‐truncated species, and may account for more than 50% of Aβ accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Aβ1–40, Aβ1–42, AβN3(pE)−40 and AβN3(pE)−42. Our data show that fibre morphology of Aβ peptides is greatly influenced by the C‐terminus while toxicity, interaction with cell membranes and degradation are influenced by the N‐terminus. AβN3(pE)−40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AβN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AβN3(pE)−40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full‐length peptides resulted partially degraded. These findings suggest that formation of N‐terminally modified peptides may enhance β‐amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

Synthesis of GDP-L-fucose by the Human FX Protein

FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity. Its function has been unrecognized despite partial structural and genetic characterization. Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen. In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing. This sequence revealed a significant homology with many proteins from different organisms. Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E. coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose. Accordingly, a possible role of FX in this metabolism was investigated. The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells. Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.

p38 MAP kinase mediates the cell death induced by prp106-126 in the sh-sy5y neuroblastoma cells

Prion diseases are neurodegenerative pathologies characterized by the accumulation in the brain of a protease-resistant form of the prion protein (PrP(c)), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) maintains many PrP(Sc) characteristics. We investigated the intracellular signaling responsible for the PrP106-126-dependent cell death of SH-SY5Y, a cell line derived from a human neuroblastoma. In this cell line, PrP106-126 induced apoptotic cell death and caused activation of caspase-3, although the blockade of this enzyme did not inhibit cell death. The p38 MAP kinase blockers, SB203580 and PD169316, prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. Taken together, our data suggest that the p38 MAP kinase pathway can mediate the SH-SY5Y cell death induced by PrP106-126.

Identification of an import signal for, and the nuclear localization of, human lactoferrin

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly‐Arg‐Arg‐Arg‐Arg, corresponding to a region of the N‐terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N‐terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 μM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro‐inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 °C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

The metabolism of 6-deoxyhexoses in bacterial and animal cells

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

Expression, purification and characterization of GDP-d-mannose 4,6-dehydratase from Escherichia coli

GDP‐d‐mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP‐d‐mannose to GDP‐l‐fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST‐GMD protein and the thrombin‐cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST‐GMD fusion protein has a K m of 0.22±0.04 mM and a specific activity of 2.3±0.2 μmol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP‐l‐β‐fucose, the end‐product of the pathway, inhibits it specifically. The GST‐GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.

Structural Characterization of Siliceous Spicules from Marine Sponges

Siliceous sponges, one of the few animal groups involved in a biosilicification process, deposit hydrated silica in discrete skeletal elements called spicules. A multidisciplinary analysis of the structural features of the protein axial filaments inside the spicules of a number of marine sponges, belonging to two different classes (Demospongiae and Hexactinellida), is presented, together with a preliminary analysis of the biosilicification process. The study was carried out by a unique combination of techniques: fiber diffraction using synchrotron radiation, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetric (DSC), Fourier transform infrared spectroscopy (FTIR), and molecular modeling. From a phylogenetic point of view, the main result is the structural difference between the dimension and packing of the protein units in the spicule filaments of the Demospongiae and the Hexactinellida species. Models of the protein organization in the spicule axial filaments, consistent with the various experimental evidences, are given. The three different species of demosponges analyzed have similar general structural features, but they differ in the degree of order. The structural information on the spicule axial filaments can help shed some light on the still unknown molecular mechanisms controlling biosilicification.

An improved procedure for rapid isolation or glucose 6-phosphate dehydrogenase from human erythrocytes ☆

Abstract Coupling of N6-(aminohexyl)-adenosine 2′,5′-bisphosphate to BrCN-activated agarose was exploited to develop a simple procedure by which homogeneous glucose 6-phosphate dehydrogenase can be isolated in good yield and in a short time (2 days) from human erythrocytes. The method involves three steps, i.e., chromatography on DEAE-Sephadex, chromatography on phosphocellulose and affinity chromatography on the above ligand-matrix complex. This procedure is applicable for the purification of glucose 6-phosphate dehydrogenase from single donors.

Molecular Cloning of Silicatein Gene from Marine Sponge Petrosia ficiformis (Porifera, Demospongiae) and Development of Primmorphs as a Model for Biosilicification Studies

In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.