Gianluca Damonte

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.

Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin–NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.

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.

Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences.

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser87 and Thr56 as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38α knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca2+ concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

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.

Abscisic Acid Released by Human Monocytes Activates Monocytes and Vascular Smooth Muscle Cell Responses Involved in Atherogenesis

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-κB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.

Achievements and perspectives of top-down proteomics

Over the last years, top‐down (TD) MS has gained a remarkable space in proteomics, rapidly trespassing the limit between a promising approach and a solid, established technique. Several research groups worldwide have implemented TD analysis in their routine work on proteomics, deriving structural information on proteins with the level of accuracy that is impossible to achieve with classical bottom‐up approaches. Complete maps of PTMs and assessment of single aminoacid polymorphisms are only a few of the results that can be obtained with this technique. Despite some existing technical and economical limitations, TD analysis is at present the most powerful instrument for MS‐based proteomics and its implementation in routine workflow is a rapidly approaching turning point in proteomics. In this review article, the state‐of‐the‐art of TD approach is described along with its major advantages and drawbacks and the most recent trends in TD analysis are discussed. References for all the covered topics are reported in the text, with the aim to support both newcomers and mass spectrometrists already introduced to TD proteomics.

Contribution of two conserved glycine residues to fibrillogenesis of the 106–126 prion protein fragment. Evidence that a soluble variant of the 106–126 peptide is neurotoxic

The fibrillogenic peptide corresponding to the residues 106–126 of the prion protein sequence (PrP 106–126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106–126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106–126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, β‐structured peptide. Moreover, PrP 106–126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild‐type PrP 106–126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106–126.