Spectroscopic evidence revealing polyproline II structure in hydrophobic, putatively elastomeric sequences encoded by specific exons of human tropoelastin.

Elastin, the protein responsible for tissue elasticity, is contained in arterial walls, lungs, and skin. Given the cassette like organization of the human tropoelastin gene, giving rise to alternating exons encoding for crosslink domains and elastomeric domains, it is tempting to suggest that polypeptides encoded by different exons could adopt structures independent of the other exons. The results obtained with the polypeptide sequences encoded by exons 3, 7, and 30 of human tropoelastin are described. It is shown that these hydrophobic exons may partly assume the polyproline II (PPII) structure, as found by circular dichroism studies in aqueous solution. Classical Raman spectroscopy evidences a specific sharp band at 1314 cm(-1), which is assigned to the PPII structure adopted by these exons in the solid state. As these sequences are among those putatively responsible for elastomeric properties, these findings are of particular interest in relation to the current models of the elasticity of elastin.

Dissection of human tropoelastin: exon-by-exon chemical synthesis and related conformational studies.

Polypeptide sequences encoding the single exons of human tropoelastin were synthesized and their conformations were studied in different solvents and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of labile conformations such as poly-proline II helix (PPII) and beta-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as alpha-helices, are only present in the poly-alanine cross-linking domains. These findings give a strong experimental basis to the understanding of the molecular mechanism of elasticity of elastin. In particular, they strongly support the description of the native relaxed state of the protein in terms of trans-conformational equilibria between extended and folded structures as previously proposed [Debelle, L., and Tamburro, A. M. (1999) Int. J. Biochem. Cell. Biol. 31, 261-272].

Rational design of tropoelastin peptide-based inhibitors of metalloproteinases.

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.

Las fibras elásticas y el pulmón / The elastic fibers and the lung

La matriz extracelular (ME) del tejido conectivo pulmonar se diferencia en membranas basales y matriz intersticial. Esta última se constituye por un armazón de proteínas colagénicas fibrosas y de fibras elásticas amorfas, que están embebidas dentro de una sustancia basal viscoelástica compuesta por proteoglicanos y glicoproteínas. La ME le proporciona al pulmón sus propiedad mecánicas. Las colágenas proveen tensión, mientras que las fibras elásticas permiten el desarrollo de la expansión elástica, con la subsecuente recuperación característica de este órgano. Las fibras elásticas se constituyen por fibras amorfas de elastina polimérica combinada con ciertas glicoproteínas conocidas como microfibrillas, con proporciones variables según su estadio ontogénico. Este material amorfo constituye toda una malla que comprende un armazón continuo, por todo el pulmón, y en conexión estrecha con las fibras de colágena permite la actividad mecánica del pulmón. La elastina es una molécula altamente hidrofóbica, con una relación intron: exón de 15:1, sintetizada junto con un receptor que guía su polimerización en sitios determinados por las microfibrillas previamente depositadas

Coacervation of sequential polypeptide models of tropoelastin. Synthesis of H-(Val-Ala-Pro-Gly)n-Val-OMe and H-(Val-Pro-Gly-Gly)n-Val-OMe.

Syntheses of two sequential polytetrapeptide models, H-(Val-Ala-Pro-Gly)n-Val-OMe and H-(Val-Pro-Gly-Gly)n-Val-OMe via the p-nitrophenyl ester method are described. The p-nitrophenyl ester method gave high yields (85%--100%) of large molecular weight polymers. H-(Val-Pro-Gly-Gly)n-Val-OMe exhibited the interesting property of coacervation on raising the temperature of aqueous solutions while H-(Val-Ala-Pro-Gly)n-Val-OMe precipitates irreversibly under similar conditions. Whereas non-dialyzed lower molecular weight polymers of H-(Val-Pro-Gly-Gly)n-Val-OMe with n = 8 to 40 did not coacervate, but did show a transition to increased intramolecular order on raising the temperature of aqueous solutions above 50 degrees C, the dialyzed higher molecular weight polymer, n greater than 40, does coacervate beginning at about 50 degrees C. This demonstrates the molecular weight dependence of coacervation and also suggests the importance to coacervation of side chain interactions in the Val-Pro sequence. The increase in intramolecular order, seen as the formation of a 14-atom hydrogen-bonded ring, occurs simultaneously with the Val-Pro hydrophobic side chain association.