A proposed interaction mechanism between elastin-derived peptides and the elastin/laminin receptor-binding domain.

Elastin-derived peptides (EDPs) have been intensively studied in view of their widely diverse biological activities. These are triggered both in normal and tumor cells, through peptide anchoring at the surface of the elastin-binding protein (EBP), a subunit of the elastin/laminin receptor. In this study, we investigated both the structure of the Sgal peptide, representing the elastin-binding domain of EBP, and its interaction with EDPs, through a combination of experimental and theoretical methods. Although the conformation of the Sgal peptide is highly flexible, we detected a type I beta-turn at the QDEA sequence. This represents the best structured motif in the entire Sgal peptide, which might therefore contribute to its binding activity. We further propose a novel three-dimensional model for the interaction between the Sgal peptide and EDPs; folding of the EDPs at the GXXP motif, in a conformation close to a type VIII beta-turn, provides the efficient contact of the protein with the Q residue of the Sgal peptide. This residue is exposed to the peptide surface, because of the beta-turn structure of the QDEA residues in the peptide sequence. We further show that this complex is stabilized by three hydrogen bonds involving EDPs backbone atoms.

Structural and biological properties of Cucumber mosaic virus particles carrying hepatitis C virus-derived epitopes.

The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.

Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study.

Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view.

AFM study of the elastin-like biopolymer poly(ValGlyGlyValGly).

In this paper, we report an AFM study on the supramolecular structures adopted by the synthetic polypentapeptide poly(ValGlyGlyValGly), whose monomeric sequence is an abundant, simple building block of elastin. The polypeptide was analyzed by deposition from both methanolic and aqueous suspensions, showing different behaviors. In methanol, the polypeptide is able to evolve, in a time-dependent way, from layers to ribbons to beaded filaments. When the equilibrium is reached, the formation of well-defined dendritic structures is also observed. This restructuring of the polypentapeptide seems to be reminiscent of a sort of Rayleigh instability. When deposited from aqueous suspensions, the polypeptide self-assembles either in fibrillar networks or in amyloid-like patterns, both of them being found in elastin or elastin-related polypeptides. As a general finding, poly(ValGlyGlyValGly) seems to constitute an excellent mimetic of the supramolecular properties of native elastin.

Elastin-based biopolymers: chemical synthesis and structural characterization of linear and cross-linked poly(OrnGlyGlyOrnGly).

Poly(OrnGlyGlyOrnGly) was synthesized by classical procedures in solution. The monomeric sequence -OrnGlyGlyOrnGly- was chosen as a modification of -ValGlyGlyValGly-, typical of elastin, to impart primary amine functionality, susceptible to cross-linking with appropriate bifunctional reagents. Herein we focus on the cross-linking of poly(OrnGlyGlyOrnGly) with glutaraldehyde. The polymers, both linear and cross-linked, were characterized and investigated for their molecular and supramolecular properties. Circular dichroism studies performed on linear poly(OrnGlyGlyOrnGly) revealed a variety of conformations similar to elastin. At a supramolecular level, different kinds of aggregates were found such as the elastin-like twisted-rope pattern of filaments and fibrils, together with other specific morphologies, similar to those recently identified in some elastin-mimetic polypeptides.

Novel properties of peptides derived from the sequence coded by exon 26A of human elastin.

The exon 26A is a rarely expressed human elastin exon that codes for a hydrophilic and charged amino acid sequence. The functional role of elastin containing this additional sequence is unknown. The present investigation was aimed to determine the effect of synthetic peptides derived from this exon on the vascular tone of rat thoracic aorta. On phenilephrine-preconstricted rat thoracic aortic rings the peptides LSPELREGD and REGD cause dose-dependent relaxation in the concentration range from 10(-9) to 10(-5) M. omega-nitro-L-arginine methyl ester, a known inhibitor of the NO synthase, highly inhibits, although to a different extent, the relaxation induced by these peptides. Removal of endothelium and blocking of ATP-sensitive potassium channels by glibenclamide significantly inhibited the vasorelaxant activity of LSPELREGD but not that of REGD, suggesting a different mechanism of action and possibly a different receptor.

On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences.

The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.

Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer.

Previous studies afforded on peptides and polypeptides containing repetitive sequences of elastin have largely demonstrated that their molecular and supramolecular properties are fully representative of those of tropoelastin, the soluble, linear precursor of elastin itself. In the attempt to synthesize cross-linked elastin-mimetic polypeptides, the repeating sequence VGGVG (V: valine; G: glycine), typical of elastin, was modified to incorporate lysine residues, yielding the polymer poly(KGGVG) (K: lysine). This imparts primary amine functionality susceptible to cross-linking reaction with appropriate bifunctional cross-linking reagents. We report herein the chemical synthesis and cross-linking of poly(KGGVG) with glutaraldehyde (GTA) and with disuccinimidyl glutarate (DSG). In both cases, the characterization of the polymers, both linear and cross-linked, has been carried out by CD spectroscopy and transmission electron microscopy measurements. The obtained results, although not conclusive, demonstrate that poly(KGGVG), both linear and cross-linked, may be considered very similar to tropoelastin and mature elastin, as concerns its molecular and supramolecular properties.

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.

Synthesis and structural characterization of poly(LGGVG), an elastin-like polypeptide.

Poly(LGGVG) a potential elastin-like biomaterial has been synthesized and studied both in solution (by circular dicroism and nuclear magnetic resonance) and in the aggregated state (by transmission electron microscopy). For sake of comparison, also the conformation of the protected (Boc-LGGVG-OEt) and free (H(2)(+)-LGGVG-OH) 'monomers' has been investigated. While in the latter ones the presence has been evidenced of more or less stable type II beta-turns, the polymer showed a conformational ensemble, possibly comprising type II beta-turns, type I beta-turns and open (unordered) structures. At supramolecular level, twisted-rope aggregates were observed by transmission electron microscopy for the polymer. Thus, the title compound has shown to possess, at both molecular and supramolecular level, physico-chemical properties very similar to those of elastin, so to give some confidence that it could really constitute the precursor of an artificial substitute of elastin itself.

Elastin: molecular description and function.

Elastin, the protein responsible for the elastic properties of vertebrate tissues, has been thought to be solely restricted to that role. As a consequence, elastin was conventionally described as an amorphous polymer. Recent results in the biomedical, biochemical and biophysical fields have lead to the conclusion that the presence of elastin in the extracellular space has very complex implications involving many other molecules. The present review describes the current state of knowledge concerning elastin as an elastic macromolecule. First, the genetic, biological, biochemical and biophysical processes leading to a functional polymer are described. Second, the elastic function of elastin is discussed. The controversy on elastin structure and elasticity is discussed and a novel dynamic mechanism of elasticity proposed. Finally, pathologies where the elastin molecule is involved are considered. This updated description of functional elastin provides the required background for the understanding of its pathologies and defines clearly the properties a substance should possess to be qualified as a good elastic biomaterial.

Different period gene repeats take 'turns' at fine-tuning the circadian clock.

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.

Structural studies of cucumber mosaic virus: Fourier transform infrared spectroscopic studies.

The secondary structure of cucumber mosaic virus (CMV) was investigated in solution using Fourier transform infrared (FT-IR) spectroscopy. The amide I region of intact CMV revealed a doublet at 1671 cm-1 and 1653 cm-1, respectively. In order to isolate the IR bands arising from the protein backbone of CMV, the FT-IR spectra of the RNA component, isolated by phenol-SDS treatment of purified CMV and subsequent precipitation by ethanol, was obtained separately and digitally subtracted from the intact CMV spectra. After digital subtraction, the amide I region contained two bands at 1682 cm-1 and 1644 cm-1. The former band was ascribed to beta-sheet structures, while the later band occurs in the region between alpha-helix and "unordered" structures. Resolution enhancement of the finger print amide I region was accomplished using Fourier self-deconvolution of the digitally subtracted FT-IR spectrum of CMV which further confirmed the presence of anti-parallel beta-sheet structure in the protein coat of CMV. Chou-Fasman predictions on the the coat protein also revealed the presence of beta-sheet structure in agreement with FT-IR studies.

The amino acid sequence coded by the rarely expressed exon 26A of human elastin contains a stable beta-turn with chemotactic activity for monocytes.

The structural and biological properties of the amino acid sequence coded by the rarely expressed exon 26A of human elastin were investigated. The C-terminal portion of this sequence, corresponding to residues 600-619 of human tropoelastin, REGDPSSSQHLPSTPSSPRV and three shorter derived peptides, LREGDPSS, SSSQHLPS, and LPSTPSSP, were synthesized and studied. Spectroscopic analyses by CD and NMR have identified a type II beta-turn within the sequence REGD of the octapeptide LREGDPSS. This structural motif was found also in the tetrapeptide REGD in both trifluoroethanol and water. The CD spectrum of the tetrapeptide REGD in trifluoroethanol was consistent with a pure type II beta-turn. A high chemotactic activity for monocytes was exhibited by the structured peptides REGD (CI 0.90 at 10(-)7 M) and LREGDPSS (CI 0.80 at 10(-)11 M), at variance with the unfolded peptides LPSTPSSP and SSSQHLPS, suggesting that this activity is strictly correlated with folded structures. Because the exon 26A of human elastin is expressed in the neointima of hypertensive pulmonary arteries, and macrophages are present in this pathologic tissue [Liptay et al. (1993) J. Clin. Invest. 91, 588-594], the chemotactic activity for human monocytes reported in this paper is consistent with an active role played by the exon 26A in inducing the migration of the monocyte/macrophage cells to the neointima.

Identification of elastin peptides with vasorelaxant activity on rat thoracic aorta.

Elastin peptides obtained in vivo from the enzymatic degradation of elastic fibers are present in the circulating human blood. In order to verify the role that these peptides may have in the regulation of the vascular tone, the activity of several peptides identified in the elastolytic digest of human elastin and some of their structural homologues has been tested. Three of these peptides show a vasorelaxant activity in isolated rat aorta precontracted by phenylephrine. The activity observed is higher in the absence of the endothelium; in these conditions the IC50 for the peptides Val-Gly-Val-Ala-Pro-Gly, Val-Gly-Val-Pro-Gly and Val-Gly-Val-Hyp-Gly was 40 +/- 2, 73 +/- 2 and 10 +/- 1 ng/ml, respectively. They are active in the range of the pathological circulating concentration and their role could be important in the regulation of vascular tone during several elastin degradative diseases.

Quantum molecular modeling of the elastinic tetrapeptide Val-Pro-Gly-Gly.

The free Val-Pro-Gly-Gly tetrapeptide belonging to the Proline-rich sequences of elastin has been studied both theoretically and experimentally. The molecular modelisation was carried out using AM1 and ab initio quantum computations while the conformation in solution was ascertained by circular dichroism spectroscopy performed on the synthesized tetrapeptide. Experimental and theoretical investigations lead to the conclusion that the most probable structure is constituted by a type II beta-turn.

Structure and dynamics of elastin building blocks. Boc-LG-OEt, Boc-VGG-OH.

Short di- and tripeptides such as Boc-LG-OEt, Boc-VG-OEt and Boc-VGG-OH, corresponding to abundant repetitive sequences in elastin, have been extensively studied both in solid state, by X-ray diffraction, and in solution by circular dicroism and nuclear magnetic resonance. Furthermore, theoretical procedures such as simulated annealing and molecular dynamics were also performed on these peptides. In general, the results indicate that no one single structure (be folded or extended) could be representative for these sequences in the protein, but rather that a multiplicity of interconverting conformers, ranging from folded to extended structures, should be considered. In any case, these structures, e.g. beta-turns, polyglycine II and beta-conformations, are those previously suggested to participate to conformational equilibria of elastin.

Circular dichroism studies of CMV-D and CMV-S: two strains of cucumber mosaic cucumovirus with a different biological behaviour.

Cucumber mosaic cucumovirus is a plant virus in which a typical satellite RNA system is present, displaying a dualistic biological behaviour. In fact, it has been shown that satRNA is able either to aggravate or attenuate the viral disease symptomatology with a modulating capability going from death of the host plant to a surprising absence of symptoms. D-satRNA and S-satRNA have been considered the prototype necrogenic and non necrogenic satRNAs respectively. On the basis of circular dichroism spectroscopy, it is suggested that the different biological behaviours can be explained by taking into account the different capabilities exerted by S- and D-satRNAs in inducing structuring effects onto CMV-S and CMV-D genomic RNAs.