Papers by Pasquale Palladino
Protein & Peptide Letters, 2003
We have synthesised the CXC chemokine receptor 4 (29-39) peptide, CXCR4{29-39}. This peptide is l... more We have synthesised the CXC chemokine receptor 4 (29-39) peptide, CXCR4{29-39}. This peptide is located in the N-terminal region of the receptor and is likely to be involved in the docking step of the receptor interaction with its natural ligand stromal cell-derived factor-1 chemokine, SDF-1. Preliminary experiments, performed in the presence of micellar detergents to model a membrane-like environment, show that the (1-17) segment of SDF-1 binds to CXCR4{29-39}.
Journal of Peptide Science, 2008
The 173-195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate t... more 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][181][182][183][184][185][186][187][188][189][190][191][192][193][194][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 PrP C 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 hPrP C 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.
Journal of Peptide Science, 2011
Bacteria employ the SecA motor protein to push unfolded proteins across the cytoplasmic membrane ... more Bacteria employ the SecA motor protein to push unfolded proteins across the cytoplasmic membrane through the SecY protein-conducting channel complex. The crystal structure of the SecA-SecY complex shows that the intramolecular regulator of ATPase1 (IRA1) SecA domain, made up of two helices and the loop between them, is partly inserted into the SecY conducting channel, with the loop between the helices as the main functional region. A computational analysis suggested that the entire IRA1 domain is structurally autonomous, and was the basis to synthesize peptide analogs of the SecA IRA1 loop region, to the aim of investigating its conformational preferences. Our study indicates that the loop region populates a predominantly flexible state, even in the presence of structuring agent. This provides indirect evidence that the SecA loop-SecY receptor docking involves loop-mediated opening of the SecY channel.
Relations structure/activité dans les systèmes protéiques : Chémokines et Méthionine Sulfoxyde Réductases
Http Www Theses Fr, 2004
Surface plasmon resonance for the label-free detection of Alzheimer’s β-amyloid peptide aggregation
Analytical and Bioanalytical Chemistry, 2015
Amyloid peptide oligomers and fibrils are studied as targets for therapy and diagnosis of Alzheim... more Amyloid peptide oligomers and fibrils are studied as targets for therapy and diagnosis of Alzheimer's disease. They are usually detected by amyloid incubation, but such method is necessarily associated with Aβ1-42 depletion and dye binding or conjugation, which have a complex influence on fibril growth, provide information about fibril elongation over long time periods only, and might lead to false-positive results in amyloid inhibition assay. Surface plasmon resonance (SPR) is used to study with no labelling and in real time the aggregation of Aβ1-42 amyloid on specific antibodies. SPR data show, for the first time by using SPR, a multi-phase association behavior for Aβ1-42 oligomers accounting for a sigmoidal growth of amyloid as a function of time, with two antibody-dependent aggregation patterns. The new method represents an advantageous alternative to traditional procedures for investigating amyloid self-assembly and inhibition from early-stage oligomer association, on the time scale of seconds to minutes, to long-term polymerization, on the time scale of hours to days.
Functional and Structural Aspects of Poplar Cytosolic and Plastidial Type A Methionine Sulfoxide Reductases
J Biol Chem, 2006
The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, bot... more The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.
Peptide Materials Obtained by Aggregation of Polyphenylalanine Conjugates as Gadolinium-Based Magnetic Resonance Imaging Contrast Agents
Advanced Functional Materials, 2015
Redox activity of melanin from the ink sac of Sepia officinalis by means of colorimetric oxidative assay
Natural Product Research, 2015
The redox properties of natural extract from cuttlefish ink sac (Sepia officinalis) and synthetic... more The redox properties of natural extract from cuttlefish ink sac (Sepia officinalis) and synthetic melanin used as a biomimetic in melanin structural investigation were determined by comparison of this phenol-based heterogeneous pigment with gallic acid used as a standard in Folin-Ciocalteu colorimetric assay widely employed for characterisation of oxidative properties of biomaterials. Reactivity of sepia melanin reported here is much higher than previously indicated and this protocol should allow the redox characterisation of all melanins irrespective of their origin and composition.
High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells
Protein Expression and Purification, 2007
PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activatin... more PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.
New TFA-Free Cleavage and Final Deprotection in Fmoc Solid-Phase Peptide Synthesis: Dilute HCl in Fluoro Alcohol
Organic Letters, 2012
A novel method for cleaving from resin and removing acid-labile protecting groups for the Fmoc so... more A novel method for cleaving from resin and removing acid-labile protecting groups for the Fmoc solid-phase peptide synthesis is described. 0.1 N HCl in hexafluoroisopropanol or trifluoroethanol cleanly and rapidly removes the tert-butyl ester and ether, Boc, trityl, and Pbf groups and cleaves the common resin linkers: Wang, HMPA, Rink amide, and PAL. Addition of just 5-10% of a hydrogen-bonding solvent considerably retards or even fully inhibits the reaction. However, a non-hydrogen-bonding solvent is tolerated.
Molecular BioSystems, 2011
Supramolecular aggregates obtained by self-aggregation of five new cationic amphiphilic CCK8 pept... more Supramolecular aggregates obtained by self-aggregation of five new cationic amphiphilic CCK8 peptides have been obtained in water solution and characterized for: (i) aggregate structure and stability; (ii) CCK8 peptide conformation and bioavailability on the external aggregate surface; and (iii) for their cell binding properties. The cationic amphiphilic CCK8 peptides self-aggregate giving a combination of liposomal and micelle structures, with radii ranging between B60 nm and B90 nm, and between B5 and B10 nm, respectively. The presence of CCK8 peptide well-exposed on the aggregate surface is demonstrated by fluorescence measurements. Peptide conformation changes in the five supramolecular aggregates: the CCK8 conformational behaviour is probably induced by the presence of three charged lysine residues close to the bioactive peptide sequence. Only aggregates in which the CCK8 peptide presents a structural arrangement similar to that found for the same peptide in DPC micelles give promising binding properties to CCK2-R receptors overexpressed by transfected A431 cells. Chemical modifications on the CCK8 N-terminus seem to play an important role in stabilizing the peptide active conformation, either when the peptide derivative is in monomeric or in aggregate form. For their easy preparation procedures and their binding properties, supramolecular aggregates based on cationic peptide amphiphiles can be considered as promising candidates for target selective drug carriers on cancer cells.
C α -Methyl, C α - n -Propylglycine Homo-oligomers
Macromolecules, 2003
ABSTRACT A series of Nα-protected, monodispersed homo-oligopeptide esters to the octamer level fr... more ABSTRACT A series of Nα-protected, monodispersed homo-oligopeptide esters to the octamer level from l-Cα-methyl, Cα-n-propylglycine [or Cα-methylnorvaline, (αMe)Nva] has been synthesized by solution methods and fully characterized. The preferred conformation of these homo-oligomers in solution has been assessed by FT-IR absorption and 1H NMR techniques. Moreover, the molecular structures of the homotrimer and homotetramer have been determined in the crystal state by X-ray diffraction. The obtained results strongly support the view that right-handed, single or multiple, and consecutive β bends are preferentially adopted by the conformationally restricted l-(αMe)Nva homo-oligomers. In particular, 310 helices are formed by the longest homo-oligomers. It is our contention that the [(αMe)Nva]n peptides represent the best available choice among Cα-tetrasubstituted α-amino acid-based homo-oligomers for the construction of relatively easy to make, rigid foldamers with a well-defined screw-sense bias.
Conformation and Self-Association of Peptide Amphiphiles Based on the KTTKS Collagen Sequence
Langmuir, 2012
Studying peptide amphiphiles (PAs), we investigate the influence of alkyl chain length on the agg... more Studying peptide amphiphiles (PAs), we investigate the influence of alkyl chain length on the aggregation behavior of the collagen-derived peptide KTTKS with applications ranging from antiwrinkle cosmetic creams to potential uses in regenerative medicine. We have studied synthetic peptides amphiphiles C(14)-KTTKS (myristoyl-Lys-Thr-Thr-Lys-Ser) and C(18)-KTTKS (stearoyl-Lys-Thr-Thr-Lys-Ser) to investigate in detail their physicochemical properties. It is presumed that the hydrophobic chain in these self-assembling peptide amphiphiles enhances peptide permeation across the skin compared to KTTKS alone. Subsequently C(n)-KTTKS should act as a prodrug and release the peptide by enzymatic cleavage. Our results should be useful in the further development of molecules with collagen-stimulating activity.
Ionic Strength Effects on the Critical Micellar Concentration of Ionic and Nonionic Surfactants: The Binding Model
Langmuir, 2011
We have recently investigated the aggregation behavior of zwitterionic n-dodecyl phosphocholine i... more 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.
Journal of Peptide Science, 2004
Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical s... more Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique β,γ -dichlorinated proline [Pro(Cl) 2 ], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the β,γ -dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear.
Neuroglobin-prion protein interaction: what's the function?
Journal of Peptide Science, 2011
Neuroglobin and cellular prion protein (PrP(C) ) are expressed in the nervous system and co-local... more Neuroglobin and cellular prion protein (PrP(C) ) 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 PrP(C) 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 PrP(C) 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 PrP(C) positively charged regions in thisprotein-protein association.
Functional and Structural Aspects of Poplar Cytosolic and Plastidial Type A Methionine Sulfoxide Reductases
Journal of Biological Chemistry, 2007
The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, bot... more The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.
Reversible thermal transition of polydiacetylene based on KTTKS collagen sequence
Chemical Communications, 2012
Here we explore the physico-chemical properties of a peptide amphiphile obtained by chemical conj... more Here we explore the physico-chemical properties of a peptide amphiphile obtained by chemical conjugation of the collagen-stimulating peptide KTTKS with 10,12-pentacosadiynoic acid which photopolymerizes as a stable and extended polydiacetylene. We investigate the self-assembly of this new polymer and rationalize its peculiar behavior in terms of a thermal conformational transition. Surprisingly, this polymer shows a thermal transition associated with a non-cooperative increase in β-sheet content at high temperature.
Carcinogenesis, 2005
Astins, a family of cyclopentapeptides, isolated from the roots of a medicinal plant Aster tatari... more Astins, a family of cyclopentapeptides, isolated from the roots of a medicinal plant Aster tataricus (Compositae), show antitumour activity. Their chemical structures consist of a 16-membered ring system containing a unique b,g-dichlorinated proline [Pro(Cl 2 )], other non-coded amino acid residues, and a cis conformation in one of the peptide bonds. The b,g-dichlorinated proline residue is considered to play an important role in their antineoplastic activities in vitro on nasopharynx carcinoma (KB) cells and in vivo on sarcoma 180 ascites and P388 lymphocytic leukaemia in mice. The acyclic astins without Pro(Cl 2 ) do not show antitumour activity against S-180 ascites in vivo, suggesting that the cyclic nature of astins plays an important role in their antitumour activities. We synthesized new astin-related cyclopeptides differing from the natural product for the presence of some non-proteinogenic amino acid residues: Aib, Abu, -S(b 3 )-hPhe and a peptide bond surrogate (--SO 2 --NH--) and we tested for their antitumour effect. We observed cytotoxic effects of the newly synthesized cyclic astins, but not with the acyclic analogue astins. We also observed that the cyclic astin induced apoptosis in a human papillary thyroid carcinoma cell line (NPA cell line) and that apoptotis was associated with activation of caspases. The caspase family inhibitor, Z--Val--Asp--(OMe)--FMK, protected NPA cells from cyclic analogue astin-induced apoptosis. To determine which caspase was specifically activated, we assayed caspase activity in astin-treated cells in the presence of specific caspase and 8, 9 or 3 inhibitors, i.e. Z--IETD--FMK, Z--LEHD--FMK Z--DEVD--FMK, which inhibit caspases 8, 9 and 3, respectively. The data presented here show selective antineoplastic properties of the newly synthesized cyclic astins, and suggest, for the first time, a mechanism for their antineoplastic action through the activation of apoptotic pathway.
Chemical Biology & Drug Design, 2012
CXCL9, CXCL10, and CXCL11 are members of a family of small (8-10 kDa) proteins, named chemokines ... more CXCL9, CXCL10, and CXCL11 are members of a family of small (8-10 kDa) proteins, named chemokines (or chemoattractant cytokines), that play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes and have important pro-inflammatory and immune modulatory functions (1). They are CXC chemokines having four conserved cysteines and are distinguished by the presence of one amino acid between the first and second cysteine. These three chemokines bind and activate the same receptor CXCR3 [chemokine (C-X-C motif) receptor 3] (2,3), which is mainly expressed on activated T and natural killer (NK) cells (4), and also appear to mediate distinct biological phenomena in vivo. In particular, the differential activation of CXCR3 by CXCL9, CXCL10, and CXCL11 may induce different biological functions and activate several signaling pathways, triggering the recruitment of inflammatory cells (5). In fact, CXCL11 has significantly higher receptor binding affinity and is a more potent chemo-attractant than CXCL9 or CXCL10 (2).
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Papers by Pasquale Palladino