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Abstract
Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devise a proteomic strategy to identify sites of carboxymethyllysine modification, one of the most abundant advanced glycation end products. We identify over 1000 sites of protein carboxymethylation in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we find that protein glycation triggers a proteotoxic response and indirectly affects the protein degradation machinery. In primary endothelial cells, we show that glyoxal induces cell cycle perturbation and that carboxymethyllysine modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of carboxymethyllysine modification for cellular function and pinpoint specific protein networks that might become compromised during aging.
Highlights
Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins
Our data reveal that CML accumulation in primary endothelial cells inhibits proliferation, which is due to both altered expression of cell cycle regulators, and glycation of tubulins associated with impaired microtubule dynamics
Aiming to identify sites of CML modification on proteins, we developed a proteomic workflow employing an antibody-based enrichment of CML-modified peptides coupled to mass spectrometry (CMLpepIP)
Summary
Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Glycation is increasingly seen as driver of metabolic disease and aging, and may elicit specific effects by targeting signaling proteins[23,24] In this context, glycation of the ryanodine receptor associated with mitochondrial damage[25], reversible inhibitory glycation of nuclear factor erythroid 2-related factor 2 (Nrf2)[26], and methylglyoxal-induced dimerization of Kelch-like ECH-associated protein 1 (KEAP1) with subsequent activation of the KEAP1/Nrf[2] transcriptional program[27] have been reported. We developed a proteomic workflow based on selective enrichment of CML-modified peptides coupled to mass spectrometry for identification and quantification We applied this approach to two cellular models (mouse embryonic fibroblasts (MEF) and human umbilical vein endothelial cells (HUVEC)), and to organs of young and chronologically aged mice. Our data reveal that CML accumulation in primary endothelial cells inhibits proliferation, which is due to both altered expression of cell cycle regulators, and glycation of tubulins associated with impaired microtubule dynamics
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