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C Di Bello

Publications and source records attributed to C Di Bello.

At least 37 records · Page 2Linked to original sources
PubMed1995

Minimal sequence requirements for synthetic peptides derived from the V3 loop of the human immunodeficiency virus type 1 (HIV-1) to enhance HIV-1 binding to cells and infection.

We previously demonstrated that a 23-mer peptide (DB3) derived from the V3 loop of the surface glycoprotein of HIV-1 MN strain was able to bind to soluble CD4 and enhance HIV-1 infection. The mechanism and structural features required for these biological activities were studied by using shortened DB3 derivatives and DB3 analogs carrying single amino acid substitutions. We found that peptides in which the aromatic amino acid in position 15 or 16 had been replaced by an uncharged hydrophobic residue (DB3-I15 and DB3-I16), analogs in which positively charged amino acids were replaced by corresponding D-enantiomers, and shortened DB3-derivatives lost both enhancing activity and ability to bind to soluble CD4. Other peptide variants in which a positively charged amino acid was replaced by asparagine at positions 3 (DB3-N3), 6 (DB3-N6), and 19 (DB3-N19), respectively, retained both enhancing and binding activities, although with different efficiencies. The CD4 binder peptides DB3 and DB3-N19, but none of the CD4 nonbinder peptides, enhanced CD4 expression on peptide-treated cells as well as gp120 binding to both CD4+ cells and soluble CD4. These findings strongly suggest that the peptide/CD4 interaction induced an increase in both CD4 expression and CD4/gp120 binding affinity, which in turn mediated the enhancement of viral infection. A model of the structural conformation of DB3 peptide required for its biological activities is discussed.

Amino Acid Sequence↗
PubMed1993

Structural studies on synthetic peptides from the principal neutralizing domain of HIV-1 gp120 that bind to CD4 and enhance HIV-1 infection.

In previous studies we have demonstrated that synthetic peptides, corresponding to sequences in the (307-330) region of the gp120 principal neutralizing domain of different HIV-1 isolates are specifically recognized by a site distinct from the high affinity gp120-binding site of CD4. Interestingly, a peptide designed from the HIV-1 MN strain is able to enhance viral infection, while a HTLV-IIIB derived analogue is at least ten-fold less efficient and no effect is shown by other tested peptides. This enhancing effect occurs in the early step of infection and it is not strain restricted. A correlation between structure and biological functions evidenced by CD, FT-IR, and preliminary mono and bidimensional NMR is presented in this paper. The experimental data are compared to the predictions obtained by theoretical calculations.

Amino Acid Sequence↗
PubMed1993

Total synthesis of horse heart cytochrome c. Preparation and characterization of the (1-66)apofragment.

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

Amino Acid Sequence↗
PubMed1992

Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.

A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.

Amino Acid Sequence↗
PubMed1992

Total synthesis of horse heart cytochrome C.

A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced in this important molecule. The present result is an example of how solid phase peptide synthesis of large protein fragments in conjunction with the availability of a specific recognition process may extend the potentiality of the chemical approach to the synthesis of an entire protein.

Amino Acid Sequence↗
PubMed1992

Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a beta-turn in solution; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Conformational studies on bombesin antagonists: CD and NMR characterization of [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14).

The conformational flexibility of the [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14) nonapeptide has been studied by CD and one- and two-dimensional (1D and 2D) nmr techniques. The CD and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for the parent bombesin (BN) and [D-Phe12, Leu14]BN. A preliminary investigation on spantide is also reported. In particular, the results obtained from CD measurements indicate that there is a shift from random coil structures, in aqueous solutions, toward folded structures in apolar media (2,2,2-trifluoroethanol) and in a membrane-mimetic environment (40 mM SDS) for all three peptides, namely BN, [D-Phe12, Leu14]BN, and [Thr6, Leu13 psi(CH2NH) Met14]BN (6-14). Spantide, which also possesses some inhibitory activity against BN but very little sequence similarity, even in water, shows an ordered conformation. Nuclear magnetic resonance parameters such as backbone NH-alpha CH coupling constant values, amidic temperature coefficients, and the presence of only sequential nuclear Overhauser effects have not provided, so far, any clear evidence for a preferential ordered structure in the peptides studied, and this may be due to rapid exchange among different conformers in the nmr time scale.

Amino Acid Sequence↗
PubMed1991

Fluorescence, CD, and NMR studies on spantide, a bombesin and substance P antagonist.

The conformational properties of spantide [(D-Arg, D-Trp, Leu) substance P] have been studied by fluorescence, CD, and nmr techniques. The fluorescence, CD, and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for bombesin. A preliminary investigation on [D-Pro] spantide is also reported.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1990

Evidence for beta-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.

Amino Acid Sequence↗
PubMed1989

Conformation studies on bombesin receptor antagonists: 500 MHz NMR and CD characterization of synthetic (D-Phe12, Leu14)-bombesin.

The conformation flexibility of the tetradecapeptide hormone bombesin and its synthetic antagonist (DPhe12, Leu14)-bombesin has been studied using nuclear magnetic resonance and circular dichroism techniques. The spectral features observed indicate that the ordered structure present in the C-terminal pentapeptide moiety of native BBS is lost in the (DPhe12, Leu14) analog.

Bombesin↗
PubMed1989

Mono and two-dimensional 500-MHz characterization of synthetic bombesin and related peptides in DMSO and DMSO-water.

The proton nmr characterization of bombesin (BBS) and of two peptide fragments corresponding to the (1-6) and (6-14) sequences has been carried out at 500 MHz in dimethyl sulfoxide (DMSO-d6) using two-dimensional (2D) homo and 1H-13C heterocorrelated techniques. All resonances in the nmr spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants are quite similar and around 7.8-8.2 Hz, pointing to an unfolded structure in DMSO-d6. The possibility of secondary structures in highly viscous mixtures of DMSO-d6-water was investigated. The existence of sequential nuclear Overhauser enhancement (NOE) effects in the C-terminal nonapeptide section may indicate a preferential site for secondary structuring.

Bombesin↗
PubMed1989

Solid-phase synthesis of bombesin by continuous flow procedure using Fmoc-amino acids.

Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.

Amino Acids↗
PubMed1988

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques.

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO-d6 using two-dimensional homo- and 1H-13C hetero-correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO-d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193-199] are discussed.

Amino Acid Sequence↗
PubMed1984

Semisynthetic studies on bovine pancreatic ribonuclease.

The S-peptide of the enzyme bovine pancreatic ribonuclease has been used as a model for covalent semisynthesis. Methods for side-chain protection, enzymatic cleavage of the peptide chain at the level of the single arginine-10 and for selective deprotection of the alpha-carboxyl function of this residue, have been examined. The partially protected [1-10] sequence has been coupled to a solid-phase generated [11-15] sequence attached to the polymer. After deblocking from the solid-support, the [1-15] semisynthetic peptide was complexed with native S-protein to give a complex with high biological activity.

Amino Acid Sequence↗
PubMed1980

Formation of an iso-1-cytochrome c-like species containing a covalently bonded heme group from the apoprotein by a yeast cell-free system in the presence of hemin.

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.

Apoproteins↗