Proteasomal degradation of glycated proteins depends on substrate unfolding: Preferred degradation of moderately modified myoglobin.

The Maillard reaction generates protein modifications which can accumulate during hyperglycemia or aging and may have inflammatory consequences. The proteasome is one of the major intracellular systems involved in the proteolytic degradation of modified proteins but its role in the degradation of glycated proteins is scarcely studied. In this study, chemical and structural changes of glycated myoglobin were analyzed and its degradation by 20S proteasome was studied. Myoglobin was incubated with physiological (5-10 mM), moderate (50-100 mM) and severe levels (300 mM) of glucose or methylglyoxal (MGO, 50 mM). Glycation increased myoglobin's fluorescence and surface hydrophobicity. Severe glycation generated crosslinked proteins as shown by gel electrophoresis. The concentration of advanced glycation endproducts (AGEs) N-ε-carboxymethyl lysine (CML), N-ε-carboxyethyl lysine (CEL), methylglyoxal-derived hydroimidazolone-1 (MG-H1), pentosidine and pyrraline was analyzed after enzymatic hydrolysis followed by UPLC-MS/MS. Higher concentrations of glucose increased all analyzed AGEs and incubation with MGO led to a pronounced increase of CEL and MG-H1. The binding of the heme group to apo-myoglobin was decreased with increasing glycation indicating the loss of tertiary protein structure. Proteasomal degradation of modified myoglobin compared to native myoglobin depends on the degree of glycation: physiological conditions decreased proteasomal degradation whereas moderate glycation increased degradation. Severe glycation again decreased proteolytic cleavage which might be due to crosslinking of protein monomers. The activity of the proteasomal subunit β5 is influenced by the presence of glycated myoglobin. In conclusion, the role of the proteasome in the degradation of glycated proteins is highly dependent on the level of glycation and consequent protein unfolding.