Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Akhtar, Kamal; Broekelmann, Thomas J.; Miao, Ming; Keeley, Fred W.; Starcher, Barry; Pierce, Richard A.; Mecham, Robert P.; Adair-Kirk, Tracy L.

doi:10.1074/jbc.m110.126789

Cited by 43 publications

(34 citation statements)

References 63 publications

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“…The peptide corresponding to the C-terminal 25 amino acids of TE (CT-25; FGGALGALGFPGGACLGKSCGRKRK) and a scrambled CT-25 peptide (KACSPARGLCKGFPGRGLGKLPGKG) were synthesized using standard FastMoc chemistry on an ABI-431A synthesizer, purified with reverse phase (C18) HPLC, and the sequence was confirmed by mass spectrometry as described previously (2). ONOO Ϫ Exposure of TE and CT-25 Peptides-Full-length TE, CT-25, and scrambled CT-25 peptides were treated with ONOO Ϫ (Millipore Corp., Bedford, MA) as described previously (17). Briefly, immediately prior to each assay, the concentration of ONOO ؊ was determined spectrophotometrically at 302 nm (⑀ M ϭ 1670 M Ϫ1 cm Ϫ1 ) and diluted in 0.01 N NaOH.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant TE and CT-25 Peptides-Full-length bovine TE was expressed as His 6 fusion protein and purified using nickelnitrilotriacetic acid-agarose beads (Qiagen Inc., Valencia, CA) followed by HPLC as described previously (2,17). The peptide corresponding to the C-terminal 25 amino acids of TE (CT-25; FGGALGALGFPGGACLGKSCGRKRK) and a scrambled CT-25 peptide (KACSPARGLCKGFPGRGLGKLPGKG) were synthesized using standard FastMoc chemistry on an ABI-431A synthesizer, purified with reverse phase (C18) HPLC, and the sequence was confirmed by mass spectrometry as described previously (2).…”

Section: Methodsmentioning

confidence: 99%

“…These conditions are associated with oxidative stress. Recently, we showed that ONOO Ϫ , an oxidant released by activated inflammatory cells, modifies TE and that these modifications prevent the assembly of TE into elastic fibers in vitro (17). Because the interaction of TE with cells plays a role in elastic fiber assembly, we sought to determine whether oxidative modifications of TE by ONOO Ϫ alter the interactions with cells.…”

Section: Smc and Wi-38 Cell Binding Is Mediated Through The C-terminamentioning

confidence: 99%

“…These free radicals can modify proteins, resulting in an alteration of protein structure and function (15, 16). We recently reported that oxidative/nitrosative modifications to TE prevent elastic fiber assembly in vitro, at least in part by inhibiting interactions of TE with other components of elastic fibers (17).In this study, we investigated whether oxidative modification of TE affects its binding to cell surface receptors. We found that SMCs and fetal lung fibroblasts bind to TE via different mechanisms.…”

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Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Akhtar¹,

Broekelmann

Song³

et al. 2011

Journal of Biological Chemistry

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Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin ␣ v ␤ 3 and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO ؊ ) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO ؊ treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO ؊ treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO ؊ prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.Elastic fibers are key extracellular matrix structures that provide the stretch and recoil properties of tissues such as arteries, lungs, and skin. Elastic fibers consist of two major components, elastin and microfibrils. Elastin is the predominant component of elastic fibers, comprising Ͼ90% of the total mass. Tropoelastin (TE), 2 the soluble precursor of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, endothelial cells, and smooth muscle cells (SMCs). Assembly of monomeric TE into elastic fibers is a multistep process. Upon secretion from cells, TE monomers organize into aggregates on the cell surface. These aggregates are then deposited onto pre-existing microfibrils. Microfibrillar components align the TE aggregates, which undergo cross-linking to form mature elastic fibers. Cell surface molecules, such as the 67-kDa elastin-binding protein (EBP), ␣ v ␤ 3 integrin, and glycosaminoglycans (GAGs), have been proposed to be involved in elastic fiber assembly either by promoting aggregation of TE monomers or by keeping the microfibrils close to the cell membrane through interactions with microfibrillar components (1-5).TE is mainly synthesized during late fetal and early postnatal stages of development. Synthesis of TE in normal adult tissues is negligible; however, in several cardiovascular and pulmonary diseases, such as atherosclerosis and emphysema/chronic obstr...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Smc and Wi-38 Cell Binding Is Mediated Through The C-terminamentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Akhtar¹,

Broekelmann

Song³

et al. 2011

Journal of Biological Chemistry

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Anetoderma Associated With a Succinate Dehydrogenase Gene Mutation

et al. 2019

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Mechanical Deformation Accelerates Protein Ageing

et al. 2017

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A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week‐long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

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Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Cited by 43 publications

References 63 publications

Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Oxidative Modifications of the C-terminal Domain of Tropoelastin Prevent Cell Binding

Anetoderma Associated With a Succinate Dehydrogenase Gene Mutation

Mechanical Deformation Accelerates Protein Ageing

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