Ming Miao

Functional consequences of homocysteinylation of the elastic fiber proteins fibrillin-1 and tropoelastin.

Homocystinuria caused by cystathionine-β-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains ∼13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-β-synthase-deficient homocystinuria.

Oxidative and nitrosative modifications of tropoelastin prevent elastic fiber assembly in vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO−) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO− and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.

Sequence and domain arrangements influence mechanical properties of elastin-like polymeric elastomers.

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self-assembly that aligns lysine residues for crosslinking. As a result, both the full-length monomer as well as elastin-like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full-length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self-assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full-length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties.

Elastin binding protein and FKBP65 modulate in vitro self-assembly of human tropoelastin.

Elastin is a protein that provides the unusual properties of extensibility and elastic recoil to tissues. Assembly of polymeric elastin into its final architecture in the extracellular matrix involves both self-aggregation properties of its monomeric precursor, tropoelastin, and interactions with several matrix-associated proteins that appear to act by modulating the intrinsic self-assembly of tropoelastin. Because of its highly nonpolar character and propensity to self-aggregate, it has been suggested that mechanisms limiting self-aggregation must also be present during the transit of tropoelastin through the cell prior to secretion. Both the elastin binding protein (EBP) and FKBP65 have been suggested to fulfill that role in the Golgi and endoplasmic reticulum compartments of the cell, respectively. However, details about the nature of the interactions between these proteins as well as about the mechanism by which they may act to limit self-aggregation are lacking. In this study, we demonstrate that both EBP and FKBP65 have strong binding affinities for tropoelastin, with the dissociation constant of EBP approximately 4-fold lower than that of FKBP65. Both proteins also modify the kinetics of self-assembly of tropoelastin in an in vitro system, consistent with a role in attenuating the premature intracellular self-aggregation of tropoelastin through a mechanism that limits the growth and maturation of aggregates. The ability of FKBP65 to modulate the self-assembly of tropoelastin is independent of its enzymatic activity to promote the cis-trans isomerization of proline residues in proteins.

Polymorphisms in the Human Tropoelastin Gene Modify In Vitro Self-Assembly and Mechanical Properties of Elastin-Like Polypeptides

Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease.

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin

Elastin is a self‐assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline‐poor and, as an isolated polypeptide, is susceptible to formation of amyloid‐like structures comprised of stacked β‐sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin‐like polypeptides designed with different numbers of proline‐poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline‐poor hydrophobic sequences to self‐assembly through characterization of phase separation, and to the tensile properties of cross‐linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β‐structure, both affecting polypeptide chain flexibility and cross‐link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence‐structure‐function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties.

Characterization of an unusual tropoelastin with truncated C-terminus in the frog

Tropoelastin is the monomeric form of elastin, a major polymeric protein of the extracellular elastic matrix of vertebrate tissues with properties of extensibility and elastic recoil. Mammalian and avian species contain a single gene for tropoelastin. A tropoelastin gene has also previously been identified in amphibians. In contrast, two tropoelastin genes with different tissue expression patterns have been described in teleosts. While general characteristics of tropoelastins, such as alternating arrangements of hydrophobic and crosslinking domains, are conserved across a wide phylogenetic range, sequences of these domains are highly variable, particularly when amphibian and teleost tropoelastins are included. For this reason exon-to-exon correspondence is not clear, and overall alignment of tropoelastin sequences across all species is not possible. An exception to this is the C-terminal exon, whose coding sequence has been very well-conserved across all species described to date. In mammalians this C-terminal domain has been shown to be important for interactions with cells and other matrix-associated proteins involved in matrix assembly. Here we identify and characterize a second tropoelastin gene in the frog with several unusual characteristics, the most striking of which is truncation of the C-terminal domain, deleting normally conserved sequence motifs. We demonstrate that, in spite of the absence of these motifs, both frog tropoelastin genes are expressed and incorporated into the elastic matrix, although with differential tissue localizations.

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue

Polymeric elastin provides the physiologically essential properties of extensibility and elastic recoil to large arteries, heart valves, lungs, skin and other tissues. Although the detailed relationship between sequence, structure and mechanical properties of elastin remains a matter of investigation, data from both the full‐length monomer, tropoelastin, and smaller elastin‐like polypeptides have demonstrated that variations in protein sequence can affect both polymeric assembly and tensile mechanical properties. Here we model known splice variants of human tropoelastin (hTE), assessing effects on shape, polymeric assembly and mechanical properties. Additionally we investigate effects of known single nucleotide polymorphisms in hTE, some of which have been associated with later‐onset loss of structural integrity of elastic tissues and others predicted to affect material properties of elastin matrices on the basis of their location in evolutionarily conserved sites in amniote tropoelastins. Results of these studies show that such sequence variations can significantly alter both the assembly of tropoelastin monomers into a polymeric network and the tensile mechanical properties of that network. Such variations could provide a temporal‐ or tissue‐specific means to customize material properties of elastic tissues to different functional requirements. Conversely, aberrant splicing inappropriate for a tissue or developmental stage or polymorphisms affecting polymeric assembly could compromise the functionality and durability of elastic tissues. To our knowledge, this is the first example of a study that assesses the consequences of known polymorphisms and domain/splice variants in tropoelastin on assembly and detailed elastomeric properties of polymeric elastin.