br Results and discussion To check the hypothesis that
Results and discussion To check the AT7519 that the charge of α-synuclein can be altered due to the glycation, we measured zeta potential of native α-synuclein and α-synuclein glycated by two different glycating agents, methylglyoxal (MG) and glyceraldehyde-3-phosphate (GA-3-P). The value for native α-synuclein was −15.9 ± 5.1 mV indicating negative net charge at pH 7.0, which agrees well with the data on isoelectric point of α-synuclein (4.7). The value for α-synuclein glycated by MG was almost the same whereas glycation by GA-3-P resulted in significant increase of zeta potential in absolute value up to −21.5 ± 2.7 mV (Fig. 1). In other words, glycation by GA-3-P caused increase of the α-synuclein net charge to more negative values suggesting modification of positively charged groups of lysine to neutral or negatively charged groups. According to measuring free amino groups, 5 and 8 lysine residues were modified MG and GA-3-P respectively (data not shown). Then we performed molecular modeling of the interaction between glycated α-synuclein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using molecular dynamics simulations. Unfortunately, modeling of the modifications of unfolded protein consisting in cross-linking such as formation of methylglyoxal lysine dimers is too complicated because of many different variations of cross-linked pairs, both inter- and intramolecular. Furthermore, according to the data of SDS PAGE, α-synuclein preparations glycated by MG or GA-3-P yielded 18-kDa band corresponding to the monomer of α-synuclein indicating the absence of inter-molecular cross-linking . Therefore we selected non-cross-linking modification, i.e. formation of N-ε-carboxymethyl lysine (CML, see Fig. 2C for the structure), which is one of the most common advanced glycation product [, , ]. Noteworthy, this modification is associated with change of the protein charge to more negative value, which occurs in the case of α-synuclein glycation by GA-3-P according to zeta potential measurement (Fig. 1). Two different forms of glycated α-synuclein were analyzed: with two modified lysine residues (96 and 102), which undergo SUMOylation in vivo , and with nine modified lysine residues (6, 10, 12, 21, 23, 32, 34, 43, and 96), which undergo ubiquitination in vivo [43,44] (denoted as g-α-synuclein and g2-α-synuclein, respectively). The choice of modification was based on the idea that they cannot be utilized by protein quality control system in contrast to α-synuclein molecules glycated at other sites, and therefore can exhibit a high impact on the cell. Noteworthy, these two models allow estimating influence of different levels of glycation: low (two modified residues) and high (nine modified residues) glycation level. For both forms (and the native α-synuclein as well) 10 independent simulations with different initial position of α-synuclein were performed. The stoichiometry 1:1 was selected for simulations because of two reasons: a small stoichiometry value was expected since the size of the complex of GAPDH and unmodified α-synuclein is similar to the size of free GAPDH , and introducing additional protein molecules would result in significant increase of the box size and, consequently, computational cost since both molecules are large. As it was recently found , native α-synuclein interacts with anion-binding groove between O and R (or P and Q) subunits of GAPDH which comprises NAD+-binding pocket and active site (Fig. 2A). In all 10 simulations, α-synuclein bound to this region of GAPDH (Fig. 2D, E). The following GAPDH residues have been predicted to serve as a binding site: Phe37, Gln78, Arg80, Asp81, Lys84, Glu106, Lys107, Gln113, Pro124, Ala126, Asp127, Gly193, Lys194, Ser210, Thr211, Gly212, Lys215, Lys219 (the most important residues are shown in bold). All these residues are located on the edge of the anion-binding groove (Fig. 2B). Therefore we expected effect of glycation on the binding since glycation of unfolded proteins can influence on their interaction with other charged macromolecules .