Carlo Di Bello

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.

Synthetic peptides from the principal neutralizing domain of human immunodeficiency virus type 1 (HIV-1) enhance HIV-1 infection through a CD4-dependent mechanism

The principal neutralizing domain (PND) of Human Immunodeficiency Virus type 1 (HIV-1) is mapped to a 24-amino acid sequence located in the hypervariable V3 region of the viral envelope protein. The PND of HIV-1 isolates from infected individuals corresponds mostly to that of the HIV-1 MN strain. We found that a peptide designed from the PND of HIV-1 MN virus greatly enhanced viral infection, while a peptide-derived PND of HTLV-IIIB virus showed at least 10-fold less efficient activity; no such effect was exhibited by the other peptides tested, including one designed from the PND of HIV-1 RF strain. The observed enhancing effect occurred in the early steps of viral infection and was not strain-restricted as both MN- and IIIB-derived peptides increased heterologous virus expression, including that of the RF strain. The MN- and, to a lesser extent, IIIB-derived peptides also increased CD4 expression on the cell membrane and differentially inhibited CD4 down-regulation induced by the phorbol ester TPA and/or by the monosialoganglioside GM1; the peptides showing no viral infection enhancement had no such effects. These findings demonstrate that the viral enhancement observed took place through a CD4-dependent mechanism and suggest that the PND is involved in HIV-1 infection and spread.

Covalent surface modification of titanium oxide with different adhesive peptides: surface characterization and osteoblast-like cell adhesion.

A fundamental goal in the field of implantology is the design of innovative devices suitable for promoting implant-to-tissue integration. This result can be achieved by means of surface modifications aimed at optimizing tissue regeneration. In the framework of oral and orthopedic implantology, surface modifications concern both the optimization of titanium/titanium alloy surface roughness and the attachment of biochemical factors able to guide cellular adhesion and/or growth. This article focuses on the covalent attachment of two different adhesive peptides to rough titanium disks. The capability of biomimetic surfaces to increase osteoblast adhesion and the specificity of their biological activity due to the presence of cell adhesion signal-motif have also been investigated. In addition, surface analyses by profilometry, X-ray photoelectron spectroscopy, and time of flight-secondary ion mass spectrometry have been carried out to investigate the effects and modifications induced by grafting procedures.

Assessment of novel chemical strategies for covalent attachment of adhesive peptides to rough titanium surfaces: XPS analysis and biological evaluation

Bioactive molecules have been proposed to promote beneficial interactions at bone-implant interfaces for enhancing integration. The main objective of this study was to develop novel methods to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive peptides, through selective reaction involving single functional groups. In the first protocol, an aminoalkylsilane was covalently linked to the Ti oxide layer, followed by covalent binding of glutaric anhydride to the free NH(2) groups. The carboxylic group of glutaric anhydride was used to condense the free N-terminal group of the side-chain protected peptide sequence. Finally, the surface was treated with trifluoroacetic acid to deprotect side-chain groups. In the second protocol, the peptide was directly anchored to the Ti oxide surface via UV activation of an arylazide peptide analogue. X-ray photoelectron spectroscopy analyses confirmed that modifications induced onto surface composition were in agreement with the reactions performed. The peptide density of each biomimetic surface was determined on the basis of radiolabeling and XPS derived reaction yields. The in vitro cellular response of the biomimetic surfaces was evaluated using a primary human osteoblast cell model. Cell adhesion, proliferation, differentiation, and mineralization were examined at initial-, short-, and long-time periods. In was shown that the biomimetic surface obtained through photoprobe-marked analogue that combines an easily-performed modification provides a favorable surface for an enhanced cellular response.

BINDING TO CD4 OF SYNTHETIC PEPTIDES PATTERNED ON THE PRINCIPAL NEUTRALIZING DOMAIN OF THE HIV-1 ENVELOPE PROTEIN

The interaction between the viral envelope protein gp120 and the cellular surface antigen CD4 is a key event in HIV-1 infection. Reciprocal high affinity binding sites have been located in the first domain of CD4 and in the carboxy-terminal region of gp120, respectively. Upon infection, the membranes of the target cells fuse; sites of CD4 and gp120, distinct from their high affinity binding sites, play a role in the post-binding events leading to syncytia formation. We have studied the interactions of CD4 with gp120 and gp120-derived peptides using an in vitro assay based on immobilized recombinant soluble CD4 (sCD4). In this system CD4 binds to recombinant soluble gp120 and to anti-receptor peptides derived from the high affinity CD4-binding site of gp120, as well as to peptides corresponding to the principal neutralizing domain (PND) of the envelope protein, i.e., to the domain required for HIV-1-mediated syncytium formation. Competition experiments performed using epitope-specific mAbs and a variety of peptides indicated that PND-derived peptides are specifically recognized by a CD4 site adjacent to, but distinct from, the high affinity gp120-binding site of CD4. Synthetic peptides patterned on the PND of different viral isolates were retained onto sCD4-based affinity columns at different extent; some of the structural requirements for binding were analyzed. Studies performed on CD4+ T-cells showed that PND-derived peptides also interact with CD4 in its native membrane-bound conformation. These results indicate that a direct contact takes place between CD4 and the gp120 domain participating in HIV-induced syncytia formation.

C-terminal amidation of neuropeptides. Gly-Lys-Arg extension an efficient precursor of C-terminal amide.

Biosynthesis of the C‐terminal carboxamide group of peptide hormones was studied using comparatively pGlu‐His‐Pro‐Gly and Glu‐His‐Pro‐Gly‐Lys‐Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C‐terminal dibasic extension favored a 10‐fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C‐terminal amidation.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)4K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)4K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)4K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)4K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright

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.

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.

THIN-LAYER CHROMATOGRAPHY OF DINITROPYRIDYL- AND NITROPYRIMIDYL-AMINO ACIDS.

Abstract The separation of dinitropyridyl-amino acids and of nitropyrimidyl-amino acids by thin-layer chromatography on silica gel is described. The advantages of this method as compared with conventional procedures are discussed.