Pasquale Palladino

Effects of Lipoic Acid, Caffeic Acid and a Synthesized Lipoyl-Caffeic Conjugate on Human Hepatoma Cell Lines

Hepatocellular carcinoma (HCC) is among the most aggressive and fatal cancers. Its treatment with conventional chemotherapeutic agents is inefficient, due to several side effects linked to impaired organ function typical of liver diseases. Consequently, there exists a decisive requirement to explore possible alternative chemopreventive and therapeutic strategies. The use of dietary antioxidants and micronutrients has been proposed for HCC successful management. The aim of this work was to test in vitro the effects of lipoic acid, caffeic acid and a new synthesized lipoyl-caffeic conjugate on human hepatoma cell lines in order to assess their effect on tumor cell growth. The results of cytotoxicity assays at different times showed that the cell viability was directly proportional to the molecule concentrations and incubation times. Moreover, to evaluate the pro- or anti-inflammatory effects of these molecules, the cytokine concentrations were evaluated in treated and untreated cellular supernatants. The obtained cytokine pattern showed that, at the increasing of three molecules concentrations, three pro-inflammatory cytokines such as IL-1β, IL-8 and TNF-α decreased whereas the anti-inflammatory cytokine such as IL-10 increased.

The human prion protein α2 helix: A thermodynamic study of its conformational preferences

We have synthesized both free and terminally‐blocked peptide corresponding to the second helical region of the globular domain of normal human prion protein, which has recently gained the attention of structural biologists because of a possible role in the nucleation process and fibrillization of prion protein. The profile of the circular dichroism spectrum of the free peptide was that typical of α‐helix, but was converted to that of β‐structure in about 16 h. Instead, below 2.1 × 10−5 M, the spectrum of the blocked peptide exhibited a single band centered at 200 nm, unequivocally associated to random conformations, which did not evolve even after 24 h. Conformational preferences of this last peptide have been investigated as a function of temperature, using trifluoroethanol or low‐concentration sodium dodecyl sulfate as α‐ or β‐structure inducers, respectively. Extrapolation of free energy data to zero concentration of structuring agent highlighted that the peptide prefers α‐helical to β‐type organization, in spite of results from prediction algorithms. However, the free energy difference between the two forms, as obtained by a thermodynamic cycle, is subtle (roughly 5–8 kJ mol−1 at any temperature from 280 K to 350 K), suggesting conformational ambivalence. This result supports the view that, in the prion protein, the structural behavior of the peptide is governed by the cellular microenvironment. Proteins 2005.

Does tetracycline bind helix 2 of prion? An integrated spectroscopical and computational study of the interaction between the antibiotic and α helix 2 human prion protein fragments

We demonstrate here that tetracycline (TC) can strongly interact (KD′ = 189 ± 7 nM) with model peptides derived from the C‐terminal globular domain of the prion protein, hPrP [173‐195], and that interaction concerns residues within the C‐terminal half of the helix 2, a short region previously indicated as endowed with ambivalent conformational behavior and implicated in PrP conversion to the β‐sheet‐rich, infective scrapie variant. Data have been confirmed by binding studies with the N‐terminal truncated 180‐195 variant that displays a dissociation constant of 483 ± 30 nM. Remarkably, TC does not influence the structure of the N‐terminally fluoresceinated peptides that both show α‐helical conformations. Docking calculations and molecular dynamics simulations suggest a direct, strong interaction of the antibiotic with exposed side chain functional groups of threonines 190‐193 on the solvent‐exposed surface of helix 2. Proteins 2007

Ionic strength effects on the critical micellar concentration of ionic and nonionic surfactants: the binding model.

We have recently investigated the aggregation behavior of zwitterionic n-dodecyl phosphocholine in the presence of high salt. As double logarithmic Corrin-Harkins plots of the critical micellar concentration versus the salt concentration were not linear, here we re-examine those data in the context of the binding model of surfactant aggregation, as previously developed by us for ionic surfactants. We have also re-examined plenty of data available in the literature on the salt-dependent aggregation of neutral surfactants. The use of double-logarithmic plots allowed us to show that the binding model is of general applicability. Indeed, it permits unified treatment of ionic and uncharged aggregation without requiring the introduction of linear terms in the salt concentration, as needed in the empirical Corrin-Harkins treatment of nonionic surfactants. The use of this model could be of help in a broad range of surfactant-based applications in the presence of high salt.

The prion protein: structural features and related toxic peptides

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrPC) into an insoluble, partially protease‐resistant abnormal scrapie form (PrPSc). PrPC is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrPC to PrPSc parallels a conformational change of the polypeptide from a predominantly α‐helical to a highly β‐sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure‐activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N‐terminal and globular C‐terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high β‐sheet propensity and tendency to form β‐sheet‐like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.

Conformational Diseases and Structure-Toxicity Relationships: Lessons from Prion-Derived Peptides

The physiological form of the prion protein is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. Available evidence suggests that this protein is essential for neuronal integrity in the brain, possibly with a role in copper metabolism and cellular response to oxidative stress. In prion diseases, the benign cellular form of the protein is converted into an insoluble, protease-resistant abnormal scrapie form. This conversion parallels a conformational change of the polypeptide from a predominantly alpha-helical to a highly beta-sheet secondary structure. The scrapie form accumulates in the central nervous system of affected individuals, and its protease-resistant core aggregates into amyloid fibrils outside the cell. The pathogenesis and molecular basis of the nerve cell loss that accompanies this process are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. A large amount of structure-activity studies is based on the prion fragment approach, but the resulting information is often difficult to untangle. This overview focuses on the most relevant structural and functional aspects of the prion-induced conformational disease linked to peptides derived from the unstructured N-terminal and globular C-terminal domains.

NMR structure and CD titration with metal cations of human prion alpha2-helix-related peptides.

The 173–195 segment corresponding to the helix 2 of the C-globular prion protein domain could be one of several “spots” of intrinsic conformational flexibility. In fact, it possesses chameleon conformational behaviour and gathers several disease-associated point mutations. We have performed spectroscopic studies on the wild-type fragment 173–195 and on its D178N mutant dissolved in trifluoroethanol to mimic the in vivo system, both in the presence and in the absence of metal cations. NMR data showed that the structure of the D178N mutant is characterized by two short helices separated by a kink, whereas the wild-type peptide is fully helical. Both peptides retained these structural organizations, as monitored by CD, in the presence of metal cations. NMR spectra were however not in favour of the formation of definite ion-peptide complexes. This agrees with previous evidence that other regions of the prion protein are likely the natural target of metal cation binding.

Structural characterization of a neurotoxic threonine-rich peptide corresponding to the human prion protein α2-helical 180–195 segment, and comparison with full-length α2-helix-derived peptides†

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrPC full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrPC makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright

Neuroglobin-prion protein interaction: What's the function?

Neuroglobin and cellular prion protein (PrPC) are expressed in the nervous system and co‐localized in the retinal ganglion cell layer. Both proteins do not have an unambiguously assigned function, and it was recently reported that PrPC aggregates rapidly in the presence of neuroglobin, whereas it does not aggregate in the presence of myoglobin, another globin with different tissue specificity. Electrostatic complementarity between the unstructured PrPC N‐terminus and neuroglobin has been proposed to mediate this specific interaction. To verifythis hypothesis experimentally, we have used a combined approach of automated docking and molecular dynamics (MD) studies carried out on short stretches of prion protein (PrP) N‐terminus to identify the minimal electrostatically interacting aminoacidic sequences with neuroglobin. Subsequently, we have performed the synthesis of these peptides by solid phase methods, and we tested their interaction with neuroglobin by surface plasmon resonance (SPR). Preliminary results confirm unequivocally the specific interaction between synthetic PrP peptides and neuroglobin suggesting a crucial role of PrPC positively charged regions in thisprotein–protein association. Copyright

The N-terminal Region of CXCL11 as Structural Template for CXCR3 Molecular Recognition: Synthesis, Conformational Analysis, and Binding Studies

The chemokines and their receptors play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. The membrane receptor CXCR3 binds three chemokines, CXCL9, CXCL10, and CXCL11, and its involvement is recognized in many inflammatory diseases and cancers. Therefore, the inhibition of CXCR3 pathway through interactions with three ligands was indicated as putative therapeutic target for the treatment of these diseases, and some inhibitory compounds have already been described in the literature. Recently, we studied the interaction between CXCR3 and its three natural ligands and showed that three CXCR3 ligands bound the receptor mainly by their N‐terminal regions using aromatic and electrostatic interactions, and, in particular, CXCL11 had the highest affinity for CXCR3. In light of these results, we focused our attention on what structural region(s) of CXCL11 interacted with CXCR3 and what were the structural features. Therefore, we have synthesized three peptides, corresponding to the N‐terminal region of CXCL11, but with different aromatic amino acids, analyzed their conformations by circular dichroism, NMR, and molecular dynamics simulations, simulated their complexes with CXCR3 by docking methods, and validated these data by in vitro studies. The results showed that two peptides were able to bind CXCR3 and to mimic the molecular recognition of CXCL11 and demonstrated that N‐terminal region of CXCL11 can be used as template and starting point to obtain new molecules by de novo design approaches.