Search

Tropoelastin is the dominant building block of elastic fibers, which form a major component of the extracellular matrix, providing structural support to tissues and imbuing them with elasticity and resilience. Recently, the atomistic structure of human tropoelastin is described, obtained through accelerated sampling via replica exchange molecular dynamics simulations. Here, principal component analysis is used to consider the ensemble of structures accessible to tropoelastin at body temperature (37 °C) at which tropoelastin naturally self‐assembles into aggregated coacervates. These coacervates are relevant because they are an essential intermediate assembly stage, where tropoelastin molecules are then cross‐linked at lysine residues and integrated into growing elastic fibers. It is found that the ensemble preserves the canonical tropoelastin structure with an extended molecular body flanked by two protruding legs, and identifies variations in specific domain positioning within this global shape. Furthermore, it is found that lysine residues show a large variation in their location on the tropoelastin molecule compared with other residues. It is hypothesized that this perturbation of the lysines increases their accessibility and enhances cross‐linking. Finally, the principal component modes are extracted to describe the range of tropoelastin's conformational fluctuation to validate tropoelastin's scissor‐twist motion that was predicted earlier.

Publicado em "European Cells and Materials. ISSN 1473-2262. Vol. 33, Suppl. 2, 2017 (0006)", The natural polymer tropoelastin is a structural protein of ECM of tissues requiring elasticity as part of their function, including ligaments and tendons. Tropoelastin has an innate capacity of self-assembly into high-order structures, and together with elastic resilience, structural stability and bioactivity bring forth pleasant singularities in adopting it as a building block to fabricate hydrogels. Moreover, easy tailoring of properties can be attained via incorporation of specific components into the polymeric network, including magnetic nanoparticles (MNPs), which are beneficial for on-demand therapies. Thus, the main goal of this work consisted in developing a magnetically responsive tropoelastin (MagTro) hydrogel as a platform to study the response of tendon cells to a mechanical stimulus induced by application of an external magnetic field (EMF). For this purpose, to first produce hydrogels, a solution of recombinant human tropoelastin was first freeze-dried overnight inside a mould and then chemically cross-linked inside an open desiccator via vapour glutaraldehyde. Thereafter, MagTro hydrogels were obtained through in situ precipitation of MNPs by immersing tropoelastin hydrogels in FeCl2 and FeCl3 solution overnight and secondly by soaking them in NaOH. Hydrogels were then analysed morphologically by Scanning Electron Microscopy (SEM and Cryo-SEM). Enzyme-triggered degradation was studied after 72h at 37oC in a human neutrophil elastase solution. Hydrogels exhibited a quick magnetic responsiveness to an EMF (Fig.1). Interestingly, MagTro hydrogels exhibited smaller pores as observed by Cryo-SEM. This feature can be tuned according to different soft tissue requirements by controlling different parameters of the fabrication process. Additionally, the release of tropoelastin into solution decreased, which suggests the formation of a surface coating of MNPs on tropoelastin network, protecting the hydrogel from a faster degradation. Preliminary results also indicate that cultured cells are viable and spread at the surface of the hydrogel. The application of an EMF to cell-laden MagTro hydrogels will be further investigated. Overall, the streamlined fabrication of MagTro hydrogels was successfully attained and the hydrogel formulation represents a promising potential platform for soft tissue regeneration., The authors acknowledge to BEAM-Master Joint Mobility Project an EU Australian cooperation in Biomedical Engineering Grant Agreement, 2014-1843/001 001-CPT EU-ICI-ECP and to FCT–Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, RC-A PhD grant SFRH/BD/96593/2013 and MEG grant IF/00685/2012., info:eu-repo/semantics/publishedVersion

Interpretation of XPS of untreated and 800 s PIII treated PES.

Background: Negative residues are rare in human tropoelastin, and their contributions to protein structure and function are unknown. Results: Mutating aspartate 72 reduces coacervation, cross-linking, and elastogenesis and alters the N-terminal conformation. Conclusion: Aspartate 72 residue stabilizes the N-terminal structure for functional assembly into elastic fibers. Significance: This work provides the first direct evidence of the importance of the tropoelastin N-terminal structure in elastic fiber assembly., Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.

Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and functionally atypical hydrogels, and markedly impaired and abnormal elastic fiber formation. Our work indicates the crucial significance of both E345 and E414 residues in modulating specific local structure and higher-order assembly of human tropoelastin.