Accumulation of Carbamylation-Derived Products in Aneurysmal Aorta.

INTRODUCTION: Carbamylation is a nonenzymatic post-translational modification of proteins characterized by the binding of isocyanic acid to amino groups of proteins, which leads to the alteration of their properties. An increase in serum carbamylation-derived products, including homocitrulline (HCit), has been shown to be associated with the development of cardiovascular diseases. METHODS: HCit was quantified by LC-MS/MS within extracts of aneurysmal and control human aortas. A mouse model of aortic aneurysm (ApoE-/- mice perfused with angiotensin II and fed with sodium cyanate) was used to evaluate the role of carbamylation in aneurysm development. RESULTS: HCit quantification showed a greater heterogeneity of values in aneurysmal aortas in comparison with control ones. At the maximum diameter of dilation, HCit values were significantly higher (+94%, p < 0.05) compared with less dilated areas. No differences were observed according to aneurysm size or when comparing ruptured and unruptured aneurysms. No significant effect of carbamylation on aneurysm development was observed using the animal model. CONCLUSIONS: These results evidenced the accumulation of HCit within aneurysmal aortas but do not allow concluding about the exact participation of protein carbamylation in the development of human abdominal aortic aneurysms.

Measurement of Homocitrulline, A Carbamylation-derived Product, in Serum and Tissues by LC-MS/MS.

Carbamylation corresponds to the nonenzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest in evaluating the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaching to the ε-NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This article describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on preanalytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample pretreatment for the quantification of homocitrulline by LC-MS/MS Alternate Protocol: Preanalytical steps for the quantification of homocitrulline in tissue samples Basic Protocol 2: LC-MS/MS quantification of homocitrulline Basic Protocol 3: LC-MS/MS quantification of lysine in hydrolysates.

Carbamylation of elastic fibers is a molecular substratum of aortic stiffness.

Because of their long lifespan, matrix proteins of the vascular wall, such as elastin, are subjected to molecular aging characterized by non-enzymatic post-translational modifications, like carbamylation which results from the binding of cyanate (mainly derived from the dissociation of urea) to protein amino groups. While several studies have demonstrated a relationship between increased plasma concentrations of carbamylated proteins and the development of cardiovascular diseases, molecular mechanisms explaining the involvement of protein carbamylation in these pathological contexts remain to be fully elucidated. The aim of this work was to determine whether vascular elastic fibers could be carbamylated, and if so, what impact this phenomenon would have on the mechanical properties of the vascular wall. Our experiments showed that vascular elastin was carbamylated in vivo. Fiber morphology was unchanged after in vitro carbamylation, as well as its sensitivity to elastase degradation. In mice fed with cyanate-supplemented water in order to increase protein carbamylation within the aortic wall, an increased stiffness in elastic fibers was evidenced by atomic force microscopy, whereas no fragmentation of elastic fiber was observed. In addition, this increased stiffness was also associated with an increase in aortic pulse wave velocity in ApoE-/- mice. These results provide evidence for the carbamylation of elastic fibers which results in an increase in their stiffness at the molecular level. These alterations of vessel wall mechanical properties may contribute to aortic stiffness, suggesting a new role for carbamylation in cardiovascular diseases.

Proteasome-dependent degradation of intracellular carbamylated proteins.

Carbamylation, which corresponds to the binding of isocyanic acid to the amino groups of proteins, is a nonenzymatic post-translational modification responsible for alterations of protein structural and functional properties. Tissue accumulation of carbamylation-derived products and their role in pathological processes such as atherosclerosis or chronic renal failure have been previously documented. However, few studies have focused on the carbamylation of intracellular proteins and their subsequent role in cellular aging. This study aimed to determine the extent of intracellular protein carbamylation, its impact on cell functions and the ability of cells to degrade these modified proteins. Fibroblasts were incubated with cyanate or urea and the carbamylation level was evaluated by immunostaining and homocitrulline quantification. The results showed that carbamylated proteins accumulated intracellularly and that all proteins were susceptible. The presence of intracellular carbamylated proteins did not modify cell proliferation or type I collagen synthesis nor did it induce cell senescence, but it significantly decreased cell motility. Fibroblasts were able to degrade carbamylated proteins through the ubiquitin-proteasome system. In conclusion, intracellular proteins are susceptible to carbamylation but their accumulation does not seem to deeply affect cell function, owing largely to their elimination by the ubiquitin-proteasome system.

Receptor for advanced glycation end products: a key molecule in the genesis of chronic kidney disease vascular calcification and a potential modulator of sodium phosphate co-transporter PIT-1 expression.

BACKGROUND: Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, frequent vascular calcification (VC) and accumulation of uraemic toxins. Advanced glycation end products and S100 proteins interact with the receptor for advanced glycation end products (RAGE). In the present work, we aimed to investigate the role(s) of RAGE in the CKD-VC process. METHODS: Apoe-/- or Apoe-/-Ager (RAGE)-/- male mice were assigned to CKD or sham-operated groups. A high-phosphate diet was given to a subgroup of Apoe-/-and Apoe-/-Ager-/- CKD mice. Primary cultures of Ager+/+ and Ager-/- vascular smooth muscle cells (VSMCs) were established and stimulated with either vehicle, inorganic phosphate (Pi) or RAGE ligands (S100A12; 20 µM). RESULTS: After 12 weeks of CKD we observed a significant increase in RAGE ligand (AGE and S100 proteins) concentrations in the serum of CKD Apoe-/- mice. Ager messenger RNA (mRNA) levels were 4-fold higher in CKD vessels of Apoe-/- mice. CKD Apoe-/- but not CKD Apoe-/- or Ager-/- mice displayed a marked increase in the VC surface area. Similar trends were found in the high-phosphate diet condition. mRNA levels of Runx2 significantly increased in the Apoe-/- CKD group. In vitro, stimulation of Ager+/+VSMCs with Pi or S100A12 induced mineralization and osteoblast transformation, and this was inhibited by phosphonoformic acid (Pi co-transporters inhibitor) and Ager deletion. In vivo and in vitro RAGE was necessary for regulation of the expression of Pit-1, at least in part through production of reactive oxygen species. CONCLUSION: RAGE, through the modulation of Pit-1 expression, is a key molecule in the genesis of VC.

Measurement of Homocitrulline, A Carbamylation-derived Product, in Serum and Tissues by LC-MS/MS.

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the ε-NH2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This unit describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on pre-analytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2018 by John Wiley & Sons, Inc.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvß3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvß3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3ß activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.