Livio Paolillo

The Proprotein Convertase SKI-1/S1P IN VITRO ANALYSIS OF LASSA VIRUS GLYCOPROTEIN-DERIVED SUBSTRATES AND EX VIVO VALIDATION OF IRREVERSIBLE PEPTIDE INHIBITORS

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations ≤100 μm these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

NMR studies of prebiotic polypeptides

Several polypeptides prepared by means of pyrocondensation have been the subject of structural investigations. Attention has been focused on the constitutional characterization of homo-and co-polymers containing Asp and Glu residues, whose role is essential for the formation of the so-called proteinoids. Contrary to the literature data based on chemical degradation, nmr studies show conclusively that in thermal poly-aspartic acid only β-peptide linkages are present. This result casts serious doubt on the role thermal condensation might have played in prebiotic polypeptide syntheses.

Solid State and Solution Conformation of [Ala7]‐Phalloidin: A Synthetic Phallotoxin Analogue

Phallotoxins are toxic compounds produced by poisonous mushroom Amanita phalloides and belong to the class of bicyclic peptides with a transannular thioether bridge. Their intoxication mechanism in the liver involves a specific binding of the toxins to F-actin that, consequently, prevents the depolymerization equilibrium with G-actin. Even though the conformational features of phallotoxins have been worked out in solution, the exact mechanism of interaction with F-actin is still unknown. In this study a toxic phalloidin synthetic derivative, bicyclo(Ala1-D-Thr2-Cys3-cis-4-hydroxy-Pro4-Ala5-2-mercapto-Trp6-Ala7)(S-3-->6) has been synthesized. A substitution at position 7. with an Ala residue replaces the 4,5-dihydroxy-Leu present in the natural phalloidin. This analogue has formed crystals suitable for X-ray analysis, and represents the first case for such a class of compounds. The solid-state structure as well as the solution conformation have been evaluated. NMR techniques have been used to extract interproton distances as restraints in subsequent molecular dynamics calculations. Finally, a direct comparison between structures in solution and in the solid state is presented.

Evidence for β-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.

Structural investigations on the Nodal-Cripto binding: a theoretical and experimental approach.

Nodal, a member of the transforming growth factor-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. Up to date structural information on the interaction of Nodal with its molecular partners are unknown. To deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto-dependent tumor progression, we present here a molecular model of activin receptor-like kinase 4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal, which encompasses residues 43-69 and includes the prehelix loop and the H3 helix. This hypothesis has been subsequently assessed by surface plasmon resonance binding assays between the full-length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition, the binding affinity between the full-length Nodal and Cripto proteins has been evaluated for the first time.

Radical polymerization of methyl methacrylate in the presence of biodegradable poly(L-lactic acid). Preparation of blends, chemical-physical characterization and morphology

Poly(L-lactic) (PLLA)-based containing poly(methyl methacrylate) (PMMA) as dispersed phase are reported in the present paper. The blends are prepared by reactive (NR-type blends) as well as non reactive (NR-type blends) methodologies, according to the preparation of the blends either polymerizing the acrylic monomer in the presence of PLLA or melt mixing the two polymers (PLLA and PMMA) in bulk. Thermal as well as morphological studied performed on both R and NR blends have revealed an intimate dispersion of PLLA and PMMA. Unlikely previous studies carried out on different aliphatic polyesters but following similar chemical approach, mechanical tests on the PLLA-type blends. The results are interpreted on the basis of extensive cross-linking of the system caused by the high sensitivity of PPLA to radical attack. The copolymeric phase formed during the reactive blending is isolated and characterised by NMR technique.

Preparation of poly(β-hydroxybutyrate)/poly(methyl methacrylate) blends by reactive blending and their characterisation

Poly(β.-hydroxybutyrate) (PHB) is a biotechnologically produced polyester, highly crystalline, totally biodegradable with low versatility in mechanical properties. To overcome this problem we performed a reactive blending procedure with a glassy acrylic polymer, poly(methyl methacrylate) (PMMA), via water suspension polymerisation of the acrylic monomer precursor onto poly(β-hydroxybutyrate). It was possible to isolate the co-polymeric phase formed between the two polymers and to characterize it using NMR and differential scanning calorimetry (DSC) techniques. Fracture tests on the blends gave a satisfactory response: in fact the Critical Energy Release Factor (Gc) calculated by Linear Elastic Fracture Mechanism (LEFM) theory of a PHB/PMMA 70/30 wt/wt blend is less than two times larger than that of neat PHB.

NMR conformational studies on a synthetic peptide reproducing the [1‐20] processing domain of the pro‐ocytocin‐neurophysin precursor

The combined use of several nuclear magnetic resonance and restrained molecular dynamics techniques allowed the formulation of a molecular model for the preferred solution conformation of a synthetic peptide reproducing the [1-20] processing domain of the pro-ocytocin-neurophysin precursor. In the model, the conformation of the 20-membered tocin ring, with the two Cys1 and Cys6 residues bridged by a disulphide bond, is very close to that observed for isolated ocytocin in the solid state; in addition, a type II beta-turn is postulated for the 7-10 segment of the acyclic tail containing the Lys11-Arg12 processing site, and connecting ocytocin to the neurophysin domain, while the C-terminal 13-20 segment of the molecule is believed to assume a helical structure. This particular structural organization could be important in participating as the favorable conformation for optimal substrate-enzyme active site recognition and processing by specific endoproteases.

Solvent‐mediated conformational transition in β‐alanine containing cyclic peptides. VIII

In the present paper we describe the solution nmr structural analysis and restrained molecular dynamic simulation of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-beta-Ala-beta-Ala). The conformational analysis carried out in CD3CN and dimethylsulfoxide (DMSO) solutions by nmr spectroscopy was based on interproton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling constants. A restrained molecular dynamic simulation in vacuo was also performed to build refined molecular models. The molecule is present in both solvent systems as two slowly interconverting conformers, characterized by a cis-trans isomerism around the beta-Ala5-Pro1 peptide bond. In CD3CN solution, the conformer with a ci5 peptide bond is quite similar to that observed in the solid state, while the conformer containing all trans peptide bonds is characterized by an intramolecular hydrogen bond stabilizing a C10- and a C13-ring structure. In DMSO solution, the trans isomer is partly similar to that observed in CD3CN solution while the cis isomer is different from that observed in the solid state. The effect of the solvent in stabilizing different conformations was also investigated in DMSO-CD3CN solvent mixtures.

Self-assembling properties of ionic-complementary peptides.

Self‐complementary synthetic peptides, composed by 8 and 16 residues, were analyzed by CD, NMR and small angle neutron scattering (SANS) techniques in order to investigate the relevance of charge and hydrophobic interactions in determining their self‐assembling properties. All the sequences are potentially able to form fibrils and membranes as they share, with the prototype EAK16, a strictly alternating arrangement of polar and nonpolar residues. We find that 16‐mer peptides show higher self‐assembling propensities than the 8‐mer analogs and that the aggregation processes are favored by salts and neutral pH. Peptide hydrophobic character appears as the most relevant factor in determining self‐assembling. Solution conformational analysis, diffusion and SANS measurements all together show that the sequences with a higher self‐assemble propensity are distributed, in mild conditions, between light and heavy forms. For some of the systems, the light form is mostly constituted by monomers in a random conformation, while the heavy one is constituted by β‐aggregates. In our study we also verified that sequences designed to adopt extended conformation, when dissolved in alcohol‐water mixtures, can easily fold in helix structures. In that media, the prototype of the series appears distributed between helical monomers and β‐aggregates. It is worth noticing that the structural conversion from helical monomer to β‐aggregates, mimics β‐amyloid peptide aggregation mechanisms. Copyright