Radioimmunological identification of tropoelastin.
Ontology highlight
ABSTRACT:
SUBMITTER: Christner P
PROVIDER: S-EPMC1163887 | biostudies-other | 1976 Aug
REPOSITORIES: biostudies-other
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Project description:Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 μg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 μg of native TE mRNA with a me1 Ψ modification or 10 and 30 μg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 μg of a codon-optimized TE mRNA variant with the me1 Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 μg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.
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Project description:A structural protein blend system based on silkworm silk fibroin and recombinant human tropoelastin is described. Silk fibroin, a semicrystalline fibrous protein with beta-sheet crystals provides mechanical strength and controllable biodegradation, while tropoelastin, a noncrystallizable elastic protein provides elasticity. Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) indicated that silk becomes miscible with tropoelastin at different blend ratios, without macrophase separation. Fourier transform infrared spectroscopy (FTIR) revealed secondary structural changes of the blend system (beta-sheet content) before and after methanol treatment. Atomic Force Microscopy (AFM) nano-indentation demonstrated that blending silk and tropoelastin at different ratios resulted in modification of mechanical features, with resilience from approximately 68%- approximately 97%, and elastic modulus between 2 and 9 Mpa, depending on the ratio of the two polymers. Some of these values are close to those of native aortic elastin or elastin-like polypeptides. Significantly, during blending and drying silk-tropoelastin form micro- and nano-scale porous morphologies which promote human mesenchymal stem cell attachment and proliferation. These blends offer a new protein biomaterial system for cell support and tailored biomaterial properties to match mechanical needs.
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Project description: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.
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Project description:Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk-tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly-d-lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 μm groove width, 0.5 μm groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk-tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs.
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Project description:The folding and trafficking of tropoelastin is thought to be mediated by intracellular chaperones, although the identity and role of any tropoelastin chaperone remain to be determined. To identify proteins that are associated with tropoelastin intracellularly, bifunctional chemical cross-linkers were used to covalently stabilize interactions between tropoelastin and associated proteins in the secretory pathway in intact fetal bovine auricular chondrocytes. Immunoprecipitation of tropoelastin from cell lysates after cross-linking and analysis by SDS-PAGE showed the presence of two proteins of approximately 74 kD (p74) and 78 kD (p78) that coimmunoprecipitated with tropoelastin. Microsequencing of peptide fragments from a cyanogen bromide digest of p78 identified this protein as BiP and sequence analysis identified p74 as the peptidyl-prolyl cis-trans isomerase, FKPB65. The appearance of BiP and FKBP65 in the immunoprecipitations could be enhanced by the addition of brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) to the culture medium for the final 4 h of labeling. Tropoelastin accumulates in the fused ER/Golgi compartment in the presence of BFA if its degradation is inhibited by ALLN (Davis, E.C., and R.P. Mecham. 1996. J. Biol. Chem. 271:3787-3794). The use of BFA and other secretion-disrupting agents suggests that the association of tropoelastin with FKBP65 occurs in the ER. Results from this study provide the first identification of a ligand for an FKBP in the secretory pathway and suggest that the prolyl cis-trans isomerase activity of FKBP65 may be important for the proper folding of the proline-rich tropoelastin molecule before secretion.
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Project description:This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells.Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin.Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W.Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.
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Project description:Background:Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques. We developed gadolinium-labeled tropoelastin-specific magnetic resonance contrast agents (Gd-TESMAs) for tropoelastin imaging in animal models. Methods and Results:Two peptides, VVGSPSAQDEASPLS and YPDHVQYTHY were selected to target tropoelastin. In vitro binding, relaxivity, and biodistribution experiments enabled characterization of the probes and selecting the best candidate for in vivo MRI. MRI was performed in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice and New Zealand white rabbits with stable and rupture-prone plaques using Gd-TESMA. Additionally, human carotid endarterectomy specimens were imaged ex vivo. The VVGSPSAQDEASPLS-based probe discriminated between tropoelastin and cross-linked elastin (64±7% vs 1±2%, P=0.001), had high in vitro relaxivity in solution (r1-free=11.7±0.6mM-1s-1, r1-bound to tropoelastin = 44±1mM-1s-1) and favorable pharmacokinetics. In vivo mice vascular enhancement (4wks=0.13±0.007mm2, 8wks=0.22±0.01mm2, 12wks=0.33±0.01mm2, P<0.001) and R1 relaxation rate (4wks=0.90±0.01 s-1, 8wks=1.40±0.03 s-1, 12wks=1.87±0.04s-1, P<0.001) increased with atherosclerosis progression after Gd-TESMA injection. Conversely, statin-treated (0.13±0.01mm2, R1 =1.37±0.03s-1) and control (0.10±0.005mm2, R1 =0.87±0.05s-1) mice showed less enhancement. Rupture-prone rabbit plaques had higher R1 relaxation rate compared with stale plaques (R1=2.26±0.1s-1vs R1=1.43±0.02s-1, P=0.001), after administration of Gd-TESMA that allowed detection of rupture-prone plaques with high sensitivity (84.4%) and specificity (92.3%). Increased vascular R1 relaxation rate was observed in carotid endarterectomy plaques after soaking (R1pre= 1.1±0.26 s-1 vs R1post= 3.0±0.1s-1, P=0.01). Ex vivo analyses confirmed the MRI findings and showed uptake of the contrast agent to be specific for tropoelastin. Conclusions:MRI of tropoelastin provides a novel biomarker for atherosclerotic plaque progression and instability.
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