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Protein carbamylation links inflammation, smoking, uremia and atherogenesis

Abstract

Post-translational modification and functional impairment of proteins through carbamylation is thought to promote vascular dysfunction during end-stage renal disease. Cyanate, a reactive species in equilibrium with urea, carbamylates protein lysine residues to form ε-carbamyllysine (homocitrulline), altering protein structure and function. We now report the discovery of an alternative and quantitatively dominant mechanism for cyanate formation and protein carbamylation at sites of inflammation and atherosclerotic plaque: myeloperoxidase-catalyzed oxidation of thiocyanate, an anion abundant in blood whose levels are elevated in smokers. We also show that myeloperoxidase-catalyzed lipoprotein carbamylation facilitates multiple pro-atherosclerotic activities, including conversion of low-density lipoprotein into a ligand for macrophage scavenger receptor A1 recognition, cholesterol accumulation and foam-cell formation. In two separate clinical studies (combined n = 1,000 subjects), plasma levels of protein-bound homocitrulline independently predicted increased risk of coronary artery disease, future myocardial infarction, stroke and death. We propose that protein carbamylation is a mechanism linking inflammation, smoking, uremia and coronary artery disease pathogenesis.

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Figure 1: Schematic illustration of pathways for promoting protein carbamylation and their link to atherosclerosis.
Figure 2: Production of OCN and homocitrulline (HCit) in the reaction of proteins with MPO/SCN/H2O2.
Figure 3: MPO is a catalytic source for carbamylation at sites of inflammation and within human atherosclerotic plaque.
Figure 4: Potential pro-atherogenic effects of MPO-catalyzed protein carbamylation.
Figure 5: Case/control study examining the relationship between plasma concentrations of protein-bound HCit and the prevalence of atherosclerotic CVD.
Figure 6: Case/control study examining the relationship between plasma abundance of protein-bound HCit and prospective risk for major adverse cardiac event (MACE; one or more of the following conditions: nonfatal MI, stroke, need for revascularization (Revasc.) or death).

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Acknowledgements

Supported by US National Institutes of Health grants HL70621, P01 HL076491 and P01 HL077107 and by the Cleveland Clinic General Clinical Research Center (M01 RR018390).

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Contributions

Z.W. performed the biochemical, cellular, animal model and mass spectrometry studies, as well as assisting in drafting the manuscript. S.J.N. assisted with clinical studies design and statistical analysis. E.R.R. performed histology studies on human atherosclerotic plaque. O.K. and S.H. generated the monoclonal antibody to carbamylated LDL. J.B. assisted with clinical trial design and statistical analyses. W.F.R. collaborated in MPO transgenic mouse studies. E.J.T. participated in acquisition of clinical data and materials. J.A.D. participated in biochemical and cell biological characterization studies of carbamylated proteins. S.L.H. conceived the idea for the study, designed experiments, drafted manuscript and provided all funding. All authors provided critical review and comments on the manuscript.

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Correspondence to Stanley L Hazen.

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Competing interests

S.L.H. is named as co-inventor on pending patents filed by the Cleveland Clinic that relate to the use of biomarkers to inflammatory and cardiovascular diseases. He is also the scientific founder and a consultant to PrognostiX Inc., and has received honoraria and consulting fees related to cardiovascular biomarkers from Abbott, BioSite, Lilly, Merck, Pfizer, Wyeth and Biophysical.

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Wang, Z., Nicholls, S., Rodriguez, E. et al. Protein carbamylation links inflammation, smoking, uremia and atherogenesis. Nat Med 13, 1176–1184 (2007). https://doi.org/10.1038/nm1637

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