From the Cover: Protein carbamylation is a hallmark of aging

Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.Aging is a complex process resulting from the combination of a large number of genetic and acquired factors leading to a decline of organism functions. Cellular senescence, telomere shortening, decreased proliferative capacity, mitochondrial DNA single mutations, and inflammation influence the aging process (1–3). Protein aging is also actively involved in tissue aging. During their biological life, proteins are exposed to various alterations caused by nonenzymatic posttranslational modifications (NEPTMs), like glycation, oxidation, carbonylation, or carbamylation, that contribute to functional and structural alterations of their properties (4).Among them, glycation has generally been recognized to significantly contribute to aging processes. Glycation refers to the binding of sugar carbonyl groups to protein amino groups, resulting in the formation of a Schiff base, which rapidly undergoes a molecular rearrangement to form an Amadori product. These products can be further exposed to irreversible oxidative processes, which lead to the generation of a variety of complex compounds called advanced glycation end products (AGEs). Because glycation and oxidative reactions are closely linked, it is more suitable to name the whole pathway “glycoxidation.” It has been recognized that AGEs accumulated in organisms in an age-dependent manner. N^ε-carboxymethyl-lysine (CML) and pentosidine, two major AGEs, were found in several tissues, like kidney, bone, eye, skeletal muscle, cartilage, arterial wall, or brain, and shown to be correlated to the risk of adverse aging-related outcomes (5–10). Because AGE formation is cumulative and irreversible, glycoxidation particularly affects extracellular matrix (ECM) proteins because of their long biological life. Indeed, glycation promotes collagen cross-linking involved in stiffness and decreased elasticity of skin (11) but also, modifies matrix proteins of other tissues, contributing, for example, to the development of vessel rigidity. This phenomenon is associated with the higher prevalence of cardiovascular diseases and could predict adverse cardiovascular events in both healthy subjects and high-risk patients (12). Moreover, it has been recently shown that exogenous AGEs brought by diet contributed to endothelial dysfunction, arterial stiffness, and aging (13).The justified interest expressed for glycoxidation may, however, have distracted attention from other important pathophysiological mechanisms of aging. Indeed, it has been shown that another NEPTM, carbamylation, participated in protein molecular aging. This reaction corresponds to the binding of isocyanic acid to free amino groups and preferentially occurs on the ε-NH₂ of lysine residues generating homocitrulline (HCit) (Fig. 1), the most characteristic carbamylation-derived product (CDP) (14, 15) that can be specifically quantified (16). Isocyanic acid is mainly formed by the spontaneous dissociation of urea into cyanate and ammonia (15) but may also derive from thiocyanate through myeloperoxidase action (17, 18). Isocyanic acid generated from biomass burning, biofuel use, or tobacco use has been described as a minor environmental source (19).Open in a separate windowFig. 1.Carbamylation reaction.The occurrence of in vivo carbamylation has been known since 1960, when Stark et al. (20) reported that cyanate was able to react with amino acids and proteins. The first deleterious effects of carbamylation in vivo were evidenced in the 1970s. At that time, patients with sickle cell disease were treated with urea or cyanate to promote the carbamylation of HbS, increasing its affinity for oxygen and decreasing its capacity of aggregation. However, these patients developed cataract, which was attributed to the carbamylation of lens proteins (21). Apart from this context, other pathological implications of carbamylation have been discussed only recently (22–24). The intensity of this reaction is particularly amplified during chronic kidney disease (CKD) because of hyperuremia (25, 26). High concentrations of carbamylated plasma proteins constitute a significant risk factor for cardiovascular events and mortality in hemodialysis patients (26, 27). Recently, our laboratory has evidenced in a mouse model that tissue proteins, including skin collagen, were more intensely carbamylated during CKD and accumulated in the organism (28). In addition, carbamylation rate is increased in atherosclerotic plaques because of myeloperoxidase release from inflammatory cells, participating in the development of atherosclerosis in patients with CKD or type 2 diabetes, even in the absence of renal impairment (23, 29, 30).However, to date, no data are available on the involvement of carbamylation in aging. Such a finding would be of potential relevance, because previous studies have evidenced that carbamylation induced deleterious effects on biological functions of proteins (23, 31–33). In this paper, we have examined the age-related evolution of carbamylation of skin proteins and the two major matrix proteins, type I collagen and elastin, in three mammalian species (human, murine, and bovine). Our results show a progressive increase of carbamylated proteins in skin and suggest that carbamylation may be considered a general feature of tissue aging at least as important as glycation.