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At least 235 records · Page 13Linked to original sources
PubMed1995

Elastin gene mutations in transgenic mice.

We have constructed several rat tropoelastin minigene recombinants encoding the complete sequence of rat tropoelastin, two isoforms of rat tropoelastin and a truncated tropoelastin lacking the domains encoded by exons 19-31 of the rat gene. Coding and non-coding domains in all these recombinants were placed under the transcriptional control of 3 kb of the promoter domain of the rat tropoelastin gene. These minigenes were used to prepare a total of 28 separate founder lines of transgenic mice. A species-specific reverse-transcriptase polymerase chain reaction (RT-PCR) assay was established to demonstrate the synthesis of rat and mouse tropoelastin mRNA in several tissues obtained from both neonatal and adult transgenic mice. Thermolytic digestion of insoluble elastin isolated from several neonatal mouse tissues revealed the presence of rat tropoelastin peptides in progeny from all those founder mice in which detectable levels of rat tropoelastin mRNA were noted. Phenotypic and histopathological assessment of transgenic and non-transgenic animals revealed the development of two diverse elastic tissue disorders. The progeny of two separate founder lines overexpressing the rat tropoelastin isoform lacking exon 33, developed an emphysematous phenotype in early adulthood. In contrast, transgenic mice, in which expression of the truncated rat tropoelastin minigene lacking exons 19-31 had been observed, died of a ruptured ascending aortic aneurysm. Tropoelastin gene mutations, therefore, will result in heritable disorders of elastic tissue. Moreover, different mutations in the tropoelastin gene will be responsible for very different abnormalities in elastic tissue function.

Animals↗
PubMed2000

Mechanism of abnormal elastin gene expression in the pinguecular part of pterygia.

Elastodysplasia and elastodystrophy are two known manifestations in the conjunctival, ie, pinguecular, part of pterygia. But the mechanisms are still not understood. The purpose of this study is to investigate the mechanism of enhanced elastin gene expression in fibroblasts from the pinguecular part of pterygia, which is related to abnormal elastic fiber expression in the pinguecular part of pterygia. Elastin in surgical specimens of normal conjunctiva and the pinguecular part of pterygia from age-matched patients was detected by immunohistochemical staining. Northern hybridization and quantification of radiolabeled tropoelastin were performed in conjunctival fibroblasts cultured under different doses of ultraviolet (UV) B irradiation, and in cultured pinguecular fibroblasts from pterygia. In vitro translation was also performed to analyze the tropoelastin production in rabbit reticulocyte lysate. The level of tropoelastin in reticulolysates from UV-treated conjunctival and pinguecular fibroblasts of pterygia was higher than in normal conjunctival fibroblasts. The coding sequence and 3'- untranslated region of tropoelastin mRNAs were amplified by reverse transcription-polymerase chain reaction, and mutations were checked by DNA sequencing. Immunohistochemical staining revealed elastin in pinguecular subepithelial connective tissues of pterygia, but not in normal conjunctiva. Tropoelastin mRNA levels were not elevated in cultured pinguecular or conjunctival fibroblasts with or without ultraviolet B irradiation. However, tropoelastin synthesis was enhanced in culture medium of pinguecular and UV-irradiated conjunctival fibroblasts, but not in normal conjunctival fibroblasts. Direct DNA sequencing revealed mutations in the 3'-untranslated region but not in the coding sequence of tropoelastin mRNA, in both pinguecular and UV-irradiated conjunctival fibroblasts. The increased expression of tropoelastin in pinguecular and UV-irradiated fibroblasts is not a result of increased levels of steady-state mRNA, but is a result of posttranscriptional modification of tropoelastin.

3' Untranslated Regions↗
PubMed2001

[A turning point in the knowledge of the structure-function-activity relations of elastin].

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.

Amino Acid Sequence↗