Claudia Scarinci

Definition of the α2 region of HLA-DR molecules involved in CD4 binding

Abstract HLA class II molecules present antigenic peptides to the T cell receptor of CD4+ T lymphocytes and interact with CD4 during the antigen recognition process. A major CD4 binding site encompassing amino acids (aa) 134–148 in the β2 domain of HLA-DR has been previously identified and residues located within the α2 subunit of murine MHC class II I-A d molecules have been shown to contribute to CD4-class II interaction. To characterize the α2 region of HLA-DR molecules involved in the binding of CD4, we have synthesized overlapping linear and cyclic peptides derived from a region encompassing aa 121–143. We demonstrate that two linear peptides (aa 124–138 and 130–143) and a cyclic one (aa 121–138) specifically bind to CD4-sepharose affinity columns. Although cyclic analogues exhibit more ordered populations as detected by circular dichroism measurements, cyclization did not improve the activity of some peptides. Peptide sequence positioning in HLA-DR1 dimer model indicates that α2 residues 124 to 136 form a solvent-exposed loop which faces the β2 loop delimited by residues 134–148. These data suggest that one CD4 molecule contacts both α2 and β2 loops of the HLA-DR homodimer.

Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site

The selective proteolytic activation of the HIV-1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508-Glu-Lys-Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500-Ala-Lys-Arg503 (site 2). We report on the solution structural analysis of a 19-residue synthetic peptide, p498, which spans the two gp160-processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N-terminal side presents a 9-residue helical segment, enclosing the processing site 2; the C-terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site-directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160-PCs recognition.

Biological and conformational studies on analogues of a synthetic peptide enhancing HIV‐1 infection

We have previously demonstrated that a 23‐amino acid peptide derived from the V3 loop of the surface glycoprotein of the HIV‐1 strain MN is able to bind CD4 and to enhance HIV‐1 infection. Further studies have suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. This paper describes the biological and physico‐chemical characterization of three analogues of reduced sequence that have been designed in order to identify the minimum active sequence of this peptide corresponding to the MN‐HIV‐1 principal neutralizing domain. Biological studies indicate that the entire sequence is required for biological activity and that the sequence 1–18 presents an inhibitory activity. CD and FT‐IR absorption data are discussed here in order to identify possible structure‐function correlations.

Structural Investigation of the HIV‐1 Envelope Glycoprotein gp160 Cleavage Site, 2: Relevance of an N‐Terminal Helix

Proteolytic activation of the HIV‐1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C‐terminus of the sequence R508–E–K–R511 (site 1), in spite of the presence of another consensus sequence, Lys500–Ala–Lys–Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498–Gly516, we previously suggested a possible role of an N‐terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23‐residue peptide h‐REKR, designed to exhibit a large N‐terminal helix, followed by the gp160 native sequence, Arg508–Gly516. h‐REKR is digested by furin with high efficiency, comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98 % trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N‐terminal segment. Such a long helix does not seem to affect the loop conformation of the C‐terminal site 1‐containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

Synthetic Peptides Derived from the Angiostatin K4 Domain Inhibit Endothelial Cell Migration

Angiogenesis, the process by which new capillaries are formed from preexisting blood vessels, represents a vital function for the growth of normal tissues during embryogenesis as well as for the pathological growth of tumors. Experimental evidence has shown that both primary and metastatic tumors need to recruit angiogenic vessels for their growth, and form their own circulatory system by up-regulating angiogenic stimulators and down-regulating angiogenesis inhibitors. Blocking of positive regulators or utilization of negative regulators to suppress angiogenesis results in a delay or regression of induced tumors. In particular, negative regulators of angiogenesis, such as angiostatin, endostatin, and antagonists for integrin v 3, displayed profound antitumor activities in vivo. Angiostatin is a 38 kDa internal fragment of plasminogen, produced by proteolytic cleavage, and consists of four triplydisulfide-bridged kringle structures. In mice, angiostatin inhibits primary tumor growth as well as angiogenesis-dependent growth of metastases. In vitro, angiostatin inhibits endothelial cell migration and reduces endothelial cell growth in proliferation assays. Furthermore, it has been shown that angiostatin induces endothelial cell-specific apoptosis in vitro. Despite intense research efforts, the exact mechanisms by which angiostatin down-regulates neovascularization remain to be determined. Little is also known about the nature of receptors mediating inhibitory effects on endothelial cells, although several receptors for angiostatin have been identified. ATP synthase has been described as a high-affinity angiostatinbinding protein, and antibodies to ATP synthase have been reported to inhibit the antiproliferative activity of angiostatin on endothelial cells. Plasminogen itself has been shown to bind to other cell-surface proteins such as annexin II and alpha enolase. Recently, angiomotin has been identified in a yeast two-hybrid screen as a functional angiostatin-binding protein that mediates the inhibition of endothelial cell motility and tube formation in vitro. A fundamental step for deciphering the biology of angiostatin is to understand the role of the kringle functional domains in the structure/function of angiostatin. Each kringle is composed of approximately 80 amino acids in a double-loop conformation, held together by three disulfide bonds. Angiostatin was originally described as consisting of kringles 1 ± 4, although the COOH terminus was identified later on by Soff and co-workers, who described an angiostatin consisting of kringles 1 ± 4, plus approximately 85% of plasminogen kringle 5. Early attempts to generate recombinant angiostatin indicated the importance of the disulfide-bonded kringle in the inhibition of angiogenesis. Several other findings suggested that the kringle structure itself confers a large part, if not all, of the antiangiogenic activity. While sharing structural similarity and sequence homology, kringle domains appear to have distinct inhibitory profiles on angiogenesis-associated endothelial cell activities. Specifically, the in vitro activity of angiostatin in endothelial-cell proliferation assays resides in kringles 1 ± 3, with kringle 1 being the most potent inhibitor, while kringle 4 is relatively ineffective. In contrast, K4 is the smaller fragment with the most potent antimigratory activity. Finally, Cao and co-workers demonstrated that K5 of plasminogen presents the most potent activity against endothelial-cell proliferation. The reasons why some of the kringle domains are significantly more potent than others and the mechanisms explaining the dissociation of antiproliferative and antimigratory activity attributed to K4, compared with other kringle domains, are still unknown. However, comparisons of different angiostatin-related proteins require careful scrutiny since differences in activity between different kringle domains, or different angiostatin isoforms with varying kringle compositions, may reflect artifacts of the recombinant technology, and not necessarily an inherent difference in the kringle composition. To investigate the different contribution of K4 regions to the antimigratory activity, three linear peptides that mapped the entire K4 sequence were designed (Figure 1), synthesized, and tested for biological activity, the quantification of the biological activity being regarded as the first step in the process of identification of the minimum active sequence in the design of peptides of reduced length. All linear peptides present Nand C-terminal Cys residues protected by Acm or tButhio groups. In [19] Another receptor, Gua-Glu(OBnz)-Trp-Glu(OBnz)-NH2, which is one of the weak binding receptors according to the on-bead assay (Kass 90M ), did not give any reasonable data. The binding constant was too low to be measured accurately in free solution by UV titration, which is again in good agreement with the rather poor binding affinity of this specific receptor observed in the on-bead assay. [20] M. Conza, H. Wennmers, Chem. Commun. 2002, 866 ± 867. [21] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson, W. C. Still, J. Comput. Chem. 1990, 11, 440 ± 467. [22] Because of the limited solubilities both of the free peptide and of the receptors at concentrations 10 mM in aqueous solvents, the NMR studies had to be performed in DMSO. Although the relative importance of H-bonds/ion-pairing versus hydrophobic interactions should be shifted towards the hydrophobic interactions in water, the observation of the H-bonded pattern required for a sheet in DMSO is also a necessary prerequisite for its occurrence in water.

Synthetic peptides for study of human immunodeficiency virus infection

The formation of a complex among gp120, CD4, and CCR5/CXCR4 represents a key step in human immunodeficiency virus (HIV) infection. The use of synthetic peptides reproducing sequences of these surface proteins has increased knowledge about the interactions that determine the penetration of HIV viruses into target cells. The final aim of such investigations is the design of molecules able to inhibit the initial step of infection and the development of high-sensitivity in vitro assays for detection of HIV. In particular, the studies presented herein concern the role of the gp120 V3 loop in the CD4 binding, the importance of the N-terminal sequence of HIV-coreceptor CCR5, the sequences patterned on CXCR4 natural ligand (stromal-derived factor 1 [SDF-1]) as inhibitory peptides, and the importance of substrate secondary structure in determining the enzymatic processing of gp120 precursor (gp160).