Analyzing structural alterations of mitochondrial intermembrane space superoxide scavengers cytochrome-c and SOD1 after methylglyoxal treatment

Abstract

Mitochondria are quantitatively the most important sources of reactive oxygen species (ROS) which are formed as by-products during cellular respiration. ROS generation occurs when single electrons are transferred to molecular oxygen. This leads to a number of different ROS types, among them superoxide. Although most studies focus on ROS generation in the mitochondrial matrix, the intermembrane space (IMS) is also important in this regard. The main scavengers for the detoxification of superoxide in the IMS are Cu, Zn superoxide dismutase (SOD1) and cytochrome-c. Similar to ROS, certain reactive carbonyl species are known for their high reactivity. The consequences are deleterious modifications to essential components compromising cellular functions and contributing to the etiology of severe pathological conditions like cancer, diabetes and neurodegeneration. In this study, we investigated the susceptibility of SOD1 and cytochrome-c to in vitro glycation by the dicarbonyl methylglyoxal (MGO) and the resulting effects on their structure. We utilized experimental techniques like immunodetection of the MGO-mediated modification 5-hydro-5-methylimidazolone, differential scanning calorimetry, fluorescence emission and circular dichroism measurements. We found that glycation of cytochrome-c leads to monomer aggregation, an altered secondary structure (increase in alpha helical content) and slightly more compact folding. In addition to structural changes, glycated cytochrome-c displays an altered thermal unfolding behavior. Subjecting SOD1 to MGO does not influence its secondary structure. However, similar to cytochrome-c, subunit aggregation is observed under denaturating conditions. Furthermore, the appearance of a second peak in the calorimetry diagram indirectly suggests de-metallation of SOD1 when high MGO levels are used. In conclusion, our data demonstrate that MGO has the potential to alter several structural parameters in important proteins of energy metabolism (cytochrome-c) and antioxidant defense (cytochrome-c, SOD1).

Post-Translational Modifications of Cytochrome c in Cell Life and Disease

“…Whereas C c glycosylation is an enzyme-directed mechanism, glycation is a non-enzymatic chemical process [ 133 ]. The in vitro glycation of Arg91 in horse heart C c induces conformational changes in the protein structure due to an increase in the α-helical content which alters the secondary structure and, therefore, protein folding [ 99 ]. C c glycation leads to an aggregation process through monomer addition, driven by the exposition of new hydrophobic segments to the solvent [ 100 ].…”

“…Figure 3 graphically summarizes all the described modifications of C c that are present in human pathologies. Carbonylation, along with glycation, promote protein aggregation―this includes C c , which is one of the principal features in neurodegenerative diseases [ 99 , 146 ]. On the contrary, C c phosphorylated in positions Ser47 and Tyr97, or nitrosylated has been revealed as a neuroprotective agent against these neuronal disorders [ 49 , 55 , 81 , 123 ].…”

Understanding the Role of Protein Glycation in the Amyloid Aggregation Process

“…Moreover, SOD1 has been shown to be glycated in vivo and glycation sites have been identified and they are six lysine residues (number 3, 9, 30, 36, 122, 128) spread along the protein sequence [ 152 ]. SOD1 glycation has been studied in vitro in the presence of different glycating agents and the effects of glycation in amyloid aggregation has been also evaluated [ 153 , 154 , 155 ]. In particular, SOD1 can be efficiently glycated in vitro by glucose, D-ribose, GO, and MGO and glycation was shown to promote protein unfolding, loss of copper binding and inhibition of the enzymatic activity [ 154 , 156 , 157 , 158 ].…”

“…These results have been confirmed by ex vivo experiments reporting that SOD1 extracted from erythrocytes of diabetic patients was significantly more glycated and has a lower enzymatic activity, with respect to controls [ 159 ]. Although promoting SOD1 unfolding and loss of metal binding, glycation has been shown to inhibit amyloid aggregation in SOD1 and promote the formation of stable cross-linked AGEs [ 153 , 155 ]. Taken together, these results suggest that glycation, besides having protective role in SOD1 amyloid aggregation, could impair the correct maturation of SOD1 in vivo as promoting protein unfolding, demetallation, and loss of enzymatic activity, thus triggering cellular oxidative stress.…”

On the Prediction of In Vitro Arginine Glycation of Short Peptides Using Artificial Neural Networks

“…In proteins, the side chains of lysine and arginine are the main targets of AGE formation [ 10 , 11 ]. One of the most reactive glycating compounds is the reactive carbonyl species (RCS) methylglyoxal (MGO) which is formed as a toxic by–product by metabolic activity, e.g., during glycolysis [ 12 ].…”