Nerve growth factor (NGF) is a protein necessary for development and maintenance of the sympathetic and sensory nervous systems. We have previously shown that the NGF N-terminus peptide NGF(1-14) is ...sufficient to activate TrkA signaling pathways essential for neuronal survival and to induce an increase in brain-derived neurotrophic factor (BDNF) expression. Cu
ions played a critical role in the modulation of the biological activity of NGF(1-14). Using computational, spectroscopic, and biochemical techniques, here we report on the ability of a newly synthesized peptide named d-NGF(1-15), which is the dimeric form of NGF(1-14), to interact with TrkA. We found that d-NGF(1-15) interacts with the TrkA-D5 domain and induces the activation of its signaling pathways. Copper binding to d-NGF(1-15) stabilizes the secondary structure of the peptides, suggesting a strengthening of the noncovalent interactions that allow for the molecular recognition of D5 domain of TrkA and the activation of the signaling pathways. Intriguingly, the signaling cascade induced by the NGF peptides ultimately involves cAMP response element-binding protein (CREB) activation and an increase in BDNF protein level, in keeping with our previous result showing an increase of BDNF mRNA. All these promising connections can pave the way for developing interesting novel drugs for neurodegenerative diseases.
PARP-14 (poly-ADP Ribose Polymerase-14), a member of the PARP family, belongs to the group of Bal proteins (B Aggressive Lymphoma). PARP-14 has recently appeared to be involved in the transduction ...pathway mediated by JNKs (c Jun N terminal Kinases), among which JNK2 promotes cancer cell survival. Several pharmacological PARP inhibitors are currently used as antitumor agents, even though they have also proved to be effective in many inflammatory diseases. Cytokine release from immune system cells characterizes many autoimmune inflammatory disorders, including type I diabetes, in which the inflammatory state causes β cell loss. Nevertheless, growing evidence supports a concomitant implication of glucagon secreting α cells in type I diabetes progression. Here, we provide evidence on the activation of a survival pathway, mediated by PARP-14, in pancreatic α cells, following treatment of αTC1.6 glucagonoma and βTC1 insulinoma cell lines with a cytokine cocktail: interleukin 1 beta (IL-1β), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). Through qPCR, western blot and confocal analysis, we demonstrated higher expression levels of PARP-14 in αTC1.6 cells with respect to βTC1 cells under inflammatory stimuli. By cytofluorimetric and caspase-3 assays, we showed the higher resistance of α cells compared to β cells to apoptosis induced by cytokines. Furthermore, the ability of PJ-34 to modulate the expression of the proteins involved in the survival pathway suggests a protective role of PARP-14. These data shed light on a poorly characterized function of PARP-14 in αTC1.6 cells in inflammatory contexts, widening the potential pharmacological applications of PARP inhibitors.
Chitosan membranes were modified with mouse epidermal growth factor (EGF) by a photochemical technique. Photochemical immobilization was performed via a two-step process, in which EGF was first ...reacted with a heterobifunctional cross-linker sulfo-SANPAH (sulfosuccinimidyl 6(4′-azido-2′-nitrophenyl-amino)hexanoate) and then immobilized on the chitosan membrane by UV irradiation. The success of immobilization process was checked by Fourier transform infrared attenuated total reflection spectroscopy and X-ray photoelectron spectroscopy. Atomic force microscopy was used to evaluate the surface topography. The mitogenic effect of the EGF-modified chitosan membrane was investigated using mouse fibroblasts (L929 cell line), and cell proliferation was investigated by MTT and crystal violet assays. The results obtained from cell culture experiments showed that immobilized EGF stimulated fibroblast growth on chitosan membranes, and a considerable difference in cell proliferation was detected on EGF-modified chitosan membranes.
The Spontaneous Symmetry Breaking (SSB) phenomenon is a natural event in which a system changes its symmetric state, apparently reasonless, in an asymmetrical one. Nevertheless, this occurrence could ...be hiding unknown inductive forces. An intriguing investigation pathway uses supramolecular aggregates of suitable achiral porphyrins, useful to mimic the natural light-harvesting systems (as chlorophyll). Using as SSB probe supramolecular aggregates of 5,10,15,20-tetrakisp(ω-methoxypolyethyleneoxy)phenylporphyrin (StarP), a non-ionic achiral PEGylated porphyrin, we explore here its interaction with weak asymmetric thermal gradients fields. The cross-correlation of the experimental data (circular dichroism, confocal microscopy, atomic force microscopy, and cryo-transmission electron microscopy) revealed that the used building blocks aggregate spontaneously, organizing in flag-like structures whose thermally-induced circular dichroism depends on their features. Finally, thermal gradient-induced enantioselectivity of the supramolecular flag-like aggregates has been shown and linked to their size-dependence mesoscopic deformation, which could be visualized as waving flags in the wind.
Engineered graphene-based derivatives are attractive and promising candidates for nanomedicine applications because of their versatility as 2D nanomaterials. However, the safe application of these ...materials needs to solve the still unanswered issue of graphene nanotoxicity. In this work, we investigated the self-assembly of dityrosine peptides driven by graphene oxide (GO) and/or copper ions in the comparison with the more hydrophobic diphenylalanine dipeptide. To scrutinize the peptide aggregation process, in the absence or presence of GO and/or Cu
, we used atomic force microscopy, circular dichroism, UV-visible, fluorescence and electron paramagnetic resonance spectroscopies. The perturbative effect by the hybrid nanomaterials made of peptide-decorated GO nanosheets on model cell membranes of supported lipid bilayers was investigated. In particular, quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching techniques were used to track the changes in the viscoelastic properties and fluidity of the cell membrane, respectively. Also, cellular experiments with two model tumour cell lines at a short time of incubation, evidenced the high potential of this approach to set up versatile nanoplatforms for nanomedicine and theranostic applications.
Neurotrophins are essential proteins for the development and maintenance of neural functions as well as promising drugs in neurodegenerative disorders. Current limits in their effective clinical ...applications can be overwhelmed by the combined use of peptidomimetic and nanomedicine approaches. Indeed, neurotrophin-mimicking peptides may allow minimizing the adverse side effects of the whole protein drug. Moreover, the immobilization of such peptides on nanomaterials may offer additional advantages, including protection against degradation, enhanced permeability of barrier membranes, and intrinsic therapeutic properties of the nanoparticles (e.g., antiangiogenic and plasmonic features of gold nanoparticles (AuNPs)). In the present article, we scrutinize the functionalization of spherical AuNPs of diameter 12 nm by peptides because of the N-terminal domains of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF), NGF1-14 and BDNF1-12, respectively. The hybrid gold–peptide nanobiointerface was investigated, both in the direct physisorption and in the lipid-bilayer-mediated adsorption processes, by a multitechnique study that included UV–vis and X-ray photoelectron spectroscopies, dynamic light scattering, zeta-potential analyses, and atomic force microscopy. Both peptide- and lipid-dependent features were identified, to have a modulation in the peptide coverage of nanoparticles as well as in the cellular uptake of NGF and BDNF peptides, as investigated by confocal microscopy. The promising potentials of the neurotrophins to cross the blood–brain barrier were demonstrated.
•Overview of synthetic fluorescent probes for cellular Cu and Zn detection.•Dynamic concept of metallostasis.•‘Sophisticated’ metal probes to understand metal-mediated cell signaling.•Perspective ...routes for metal coordination compounds toward dynamic metallostasis.
The intracellular tracking of zinc and copper, metals essential for life, is nowadays pivotal to unravel the complex mechanism that involves the physiological or pathological role of such elements. Traditional methods to determine cellular copper and zinc levels, including those based on the use of fluorescent probes, are aimed at scrutinizing the metallome, to identify both the individual species and their concentrations. The metallome, however, is also a nonstatic concept, as it responds to environmental perturbations in biologically relevant pathways, with highly dynamic spatiotemporal changes. Through an overview of improvements and limits of the state of the art of synthetic fluorescent probes for the detection of intracellular zinc and copper, we report here new routes for the design and the synthesis of novel metal coordination compounds able to overcome the present weaknesses for a new concept of dynamic metallostasis.
•Neurotrophins exert activity in the same brain areas where metal ions are released.•Metal ions regulates neurotrophins activity in learning and memory formation.•Metal ions dyshomeostasis affect ...neurotrophins activity inducing neurodegeneration.•Metal complexes with neurotrophin mimicking peptides activate neuronal signaling.
Some metal ions of the d-block are necessary for the proper growth, functioning and maintenance of the central nervous system. In particular, copper and zinc ions have emerged as effective non-structural intracellular mediator of cell signaling. The high zinc and copper levels in the synaptic cleft are associated with the shaping of synaptic plasticity and, thus, with learning and memory formation processes. Recent findings identify neurotrophins (NTs) as potential targets of metal ions in the kinase signaling networks of neuronal tissues. Neurotrophins are a class of proteins crucial for the growth and preservation of the central nervous system as they preside over processes/functions like neuroplasticity, memory and learning. A deficit in the expression and activity of these proteins is associated to many neurodegenerative pathologies as Alzheimer and Parkinson’s diseases.
Knowing the coordination chemistry of metal ions to NTs is an essential step to identify the basis of NTs/metal ions physiology in learning and memory formation as well as the factors that trigger memory impairment in neurodegeneration, NTs conformations, NTs chemical properties and their biological functions. Unfortunately, characterizing the metal ion coordination of the whole protein is hardly feasible if not impossible. Thus, the design and synthesis of peptides able to mimic the binding and functional sites of proteins may be a viable strategy to explore protein activity and conformational features, metal ion binding as well as to design new molecules for neurodegenerative disorders.
The aggregation of amyloid-β (Aβ) on lipid bilayers has been implicated as a mechanism by which Aβ exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during ...both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aβ misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aβ aggregate, protofibril, and fibril formation. Aβ aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aβ at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aβ aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aβ misfolding.
In this study, chitosan membranes prepared by the solvent casting method were modified with the Arg-Gly-Asp-Ser (RGDS) sequence of fibronectin using the photochemical immobilization technique. The ...results obtained from attenuated total reflection-Fourier transform infrared spectra and X-ray photoelectron spectroscopy studies confirmed the successful immobilization of RGDS on chitosan membranes. The immobilized peptide concentration was determined by ninhydrin analysis on the order of 10
−7 mol/cm
2.
In vitro cell culture studies were performed with L929 mouse fibroblasts to investigate the effect of biomodification on fibroblast cell behaviour in serum-free and 10% serum-containing media. The results obtained from cell culture studies pointed out the specific interactions between biosignal RGDS molecules and fibroblast cells. A triggered cell attachment and proliferation were observed on RGDS-modified chitosan membranes that were more distinguishable in serum-free medium. In addition, the photochemical immobilization technique was realized in the presence of a photomask that was used to immobilize the RGDS molecules in a defined micropattern. L929 mouse fibroblasts attached on the RGDS-micropatterned areas indicating integrin-mediated interactions.
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