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.

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.