Elastin calcification in in vitro models and its prevention by MGP's N-terminal peptide.

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP3) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP's phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.

The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Direct observation of structure and dynamics during phase separation of an elastomeric protein.

Despite its growing importance in biology and in biomaterials development, liquid-liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient ß-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

Effect of the Ionic Concentration of Simulated Body Fluid on the Minerals Formed on Cross-Linked Elastin-Like Polypeptide Membranes.

Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.

Cross-Linked Elastin-like Polypeptide Membranes as a Model for Medial Arterial Calcification.

Calcium phosphate minerals deposit on the elastin-rich medial layers of arteries in the majority of seniors, diabetic, and chronic kidney disease patients, causing severe cardiovascular complications. There is no cure for medial calcification, and the mechanism of mineral formation on elastin layers is unknown. Here we propose cross-linked elastin-like polypeptide membranes as models to study medial calcification. Calcium phosphates deposit first on fibers and filaments and then spread to globular structures present in the membranes. Mineral phase evolution analyzed by near-edge X-ray spectroscopy matches that previously observed in a mouse model of medial calcification, showing that this simple system captures some of the key in vivo findings. This work shows how minerals form and evolve upon nucleation on elastin and provides an in vitro model that can be tuned to study hypotheses related to arterial calcification mechanisms and test drugs to stop or revert mineralization.

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.

Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers.

Elastin and silk spidroins are fibrous, structural proteins with elastomeric properties of extension and recoil. While elastin is highly extensible and has excellent recovery of elastic energy, silks are particularly strong and tough. This study describes the biophysical characterization of recombinant polypeptides designed by combining spider wrapping silk and elastin-like sequences as a strategy to rationally increase the strength of elastin-based materials while maintaining extensibility. We demonstrate a thermo-responsive phase separation and spontaneous colloid-like droplet formation from silk-elastin block copolymers, and from a 34 residue disordered region of Argiope trifasciata wrapping silk alone, and measure a comprehensive suite of tensile mechanical properties from cross-linked materials. Silk-elastin materials exhibited significantly increased strength, toughness, and stiffness compared to an elastin-only material, while retaining high failure strains and low energy loss upon recoil. These data demonstrate the mechanical tunability of protein polymer biomaterials through modular, chimeric recombination, and provide structural insights into mechanical design. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 693-703, 2016.

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity.

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms ß-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of ß-sheet formation at this location. Our data support a dual role for confined ß-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

Conformational transitions of the cross-linking domains of elastin during self-assembly.

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that ß-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for ß-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.

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 ß-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 ß-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.

Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective.

The extracellular matrix is an integral and dynamic component of all tissues. Macromolecular compositions and structural architectures of the matrix are tissue-specific and typically are strongly influenced by the magnitude and direction of biomechanical forces experienced as part of normal tissue function. Fibrous extracellular networks of collagen and elastin provide the dominant response to tissue mechanical forces. These matrix proteins enable tissues to withstand high tensile and repetitive stresses without plastic deformation or rupture. Here we provide an overview of the hierarchical molecular and supramolecular assembly of collagens and elastic fibers, and review their capacity for mechanical behavior in response to force. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

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.

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.

Development and validation of an automated microfluidic perfusion platform for parallelized screening of compounds in vitro.

Monoamine transporters are of great interest for their role in the physiological activity of the body and their link to mental and behavioural disorders. Currently, static well-plate assays or manual perfusion systems are used to characterize the interaction of psychostimulants, antidepressants and drugs of abuse with the transporters but still suffer from significant drawbacks caused by lack of automation, for example, low reproducibility, non-comparability of results. An automated microfluidic platform was developed to address the need for more standardized procedures for cell-based assays. An automated system was used to control and drive the simultaneous perfusion of 12 channels on a microfluidic chip, establishing a more standardized protocol to perform release assays to study monoamine transporter-mediated substrate efflux. D-Amphetamine, GBR12909 (norepinephrine transporter) and p-chloroamphetamine, paroxetine (serotonin transporter) were used as control compounds to validate the system. The platform was able to produce the expected releasing (D-Amphetamine, p-chloroamphetamine) or inhibiting (GBR12909, paroxetine) profiles for the two transporters. The reduction of manual operation and introduction of automated flow control enabled the implementation of stronger standardized protocols and the possibility of obtaining higher throughput by increasing parallelization.

Proline periodicity modulates the self-assembly properties of elastin-like polypeptides.

Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in ß-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in ß-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized ß-sheet in mediating self-assembly interactions.

Structural disorder and dynamics of elastin.

Elastin is a self-assembling, extracellular-matrix protein that is the major provider of tissue elasticity. Here we review structural studies of elastin from over four decades, and draw together evidence for solution flexibility and conformational disorder that is inherent in all levels of structural organization. The characterization of disorder is consistent with an entropy-driven mechanism of elastic recoil. We conclude that conformational disorder is a constitutive feature of elastin structure and function.

Sequence variants of human tropoelastin affecting assembly, structural characteristics and functional properties of polymeric elastin in health and disease.

Elastin is the polymeric protein responsible for the physiologically important properties of extensibility and elastic recoil of cardiovascular, pulmonary and many other tissues. In spite of significant advances in the understanding how monomeric tropoelastin is assembled into the polymeric elastic matrix, details of this assembly process are still lacking. In particular it is not clear how the various architectures and more subtle elastic properties required by diverse elastic tissues can arise from the protein product of a single gene. While monomeric tropoelastin has the intrinsic ability to self-assemble into fibrillar structures, it is clear that in vivo assembly is guided by interactions with cells and other matrix-associated components. In addition, the multiplicity of reported mRNA isoforms of human tropoelastin, if translated into protein variants, could modulate not only interactions with these matrix-associated components but also self-assembly and functional properties. Critical information identifying such protein isoforms of human tropoelastin is only now emerging from mass spectrometric studies. Increased levels of complexity of the assembly process provide additional opportunities for production of polymeric elastins with aberrant architectures and sub-optimal functional properties that could affect the longer-term structural integrity of elastic matrices. Biophysical techniques, such as SAXS, NMR and molecular dynamics, have provided a means to discern details of the effects of sequence variants, including both alternate splicing isoforms and genetic polymorphisms, on the dynamic flexibility of elastin required for its elastomeric properties. Such approaches promise to provide important new insights into the relationship between sequence, structural characteristics, assembly and functional properties of elastin in both health and disease.

Genipin Cross-Linking of Elastin and Elastin-Based Proteins.

Genipin is a natural plant-derived compound that covalently cross-links biopolymers into lattice networks with good biocompatibility, controllable swelling, and mechanical properties. This protocol describes the genipin cross-linking of elastic proteins, including tropoelastin and elastin-based polypeptides, through steps of elastin phase-separation upon addition of salt and heat, centrifugation to rapidly concentrate the dense protein phase, and incubation. This method is applicable for the fabrication of elastic materials suitable for use as scaffolds for biomedical applications.

Role of Liquid-Liquid Phase Separation in Assembly of Elastin and Other Extracellular Matrix Proteins.

Liquid-liquid phase separation resulting in formation of colloidal droplets has recently attracted attention as a mechanism for rapid and transient assembly of intracellular macromolecules into functional units. Phase separation also appears to be a widespread and evolutionarily ancient mechanism for organization of proteins of the extracellular matrix into fibrillar, polymeric assemblies. Elastin, which provides the physical properties of extensibility and elastic recoil to large arteries, lungs and other tissues, is the best-characterized extracellular matrix protein whose polymeric assembly is initiated by phase separation. Recent studies have provided an atomistic description of the conformational ensemble of elastin-like proteins, and have begun to uncover how the interplay of local secondary structure, hydrophobicity and conformational disorder govern the structure, assembly and function of elastin. Monomeric elastin is a non-polar, glycine-rich, low-complexity, modular protein that remains predominantly disordered even in the crosslinked polymeric state, consistent with its function as an entropic elastomer. Unlike intracellular phase separation, which is reversible, phase separation of elastin and other matrix proteins proceeds to stabilization and clustering of condensed phase droplets and subsequent molecular organization into fibrillar, supramolecular structures. Short ß-sheets appear to mediate the interaction and organization of these phase-separated droplets and modulate the ultimate material properties of the matrix. Whether phase separation is intracellular or extracellular, reversible or network-forming, understanding the sequence determinants of such varied assembly behaviors and differential fates of the colloidal droplets will provide important insights into aberrant assembly with pathological consequences and elucidate fundamental principles for the rational design of biomimetic materials.

Biomechanical Design of Elastic Protein Biomaterials: A Balance of Protein Structure and Conformational Disorder.

Elastic biomaterials are found across biology where they fulfill diverse load-bearing and energy storage and dissipation functions. This class of biomaterials comprises elastic proteins that provide materials with combinations of extensibility, stiffness, tensile strength, toughness, and viscoelastic properties. Differences in mechanical properties are due in large part to variations in the ratio of secondary structure and conformational disorder of constituent protein monomers, arising from differences in amino acid sequence. This natural diversity provides rich inspiration for the design of elastic biomaterials. Here, we review the relationship between sequence, structure, disorder, and mechanical properties of elastic proteins from natural materials ranging from highly extensible and soft, to mechanically strong and tough. We describe molecular strategies as well as recombinant efforts to design materials with tailored mechanical properties, with the ultimate aim of rationally engineering biomaterials for advanced biomedical applications.