It was demonstrated that the addition of fold excess
It was demonstrated that the addition of 10-fold excess of α-synuclein without modifications (with respect to the molar concentration of tetrameric GAPDH) leads to partial inactivation of GAPDH after 1-h incubation by 20% (Fig. 4, curve 2) in AG-1024 to insignificant decrease of the specific activity of free GAPDH (Fig. 4, curve 1), which was likely due to the oxidation of the essential cysteine residues of the enzyme. Noteworthy, the structure of GAPDH is affected by the bound α-synuclein molecule, but it is not denatured . Addition of α-synuclein glycated by 1 mM MG or 1 mM GA-3-P leads to the inactivation of the enzyme by 35% and 40%, respectively (Fig. 4, curves 3 and 4, respectively).
Summarizing aforementioned results, we can conclude that α-synuclein glycation results in strengthening of its binding with GAPDH. The binding has been shown to be driven by electrostatic interactions: negatively charged region of α-synuclein interacts with anion-binding groove of GAPDH. Glycation of α-synuclein can lead to the substitution of positively charged residues to negatively charged ones, which consequently enhances the binding. If the glycation level is high, behavior of α-synuclein can be significantly altered because of appearance of new extended anionic regions. As a result, highly glycated α-synuclein can interact not only with anion-binding grooves, but also with other positively charged regions on GAPDH surface. In other words, glycation can result in the increase of α-synuclein reactivity and might cause non-specific interactions with other proteins similarly to linear polyanions, which interact with positively charged regions of the enzyme even if its net charge is negative [46,47].
We did not performed MD simulations of the GAPDH interaction with α-synuclein modified by glyceraldehyde-3-phosphate since the products are unclear , and phosphate group might split off because of lability of the connection bond. Carboxymethyl lysine was chosen since it is considered to be the most widespread advanced glycation end product and can be formed due to glycation by different agents [39,48]. Anyway, change in the charge of the α-synuclein molecule due to glycation should occur in most cases of modifications since reactive positively charged epsilon amino groups of lysine disappear, and therefore most of modified residues have neutral or negative charge instead of positive one of lysine . The significant change of α-synuclein zeta potential due to glycation by glyceraldehyde-3-phosphate has been found in experimental study. Glycation of α-synuclein has been shown to enhance its inactivating action towards GAPDH. The aforementioned results indicate the GAPDH inhibition by glycated α-synuclein and the participation of GAPDH substrate, i.e. glyceraldehyde-3-phosphate, in this process. We suppose that α-synuclein glycation can be one of the factors involving in synucleinopathies development associated with diabetes, and inhibition of glycolysis is a main mechanism of energy metabolism disorders, which appears in these diseases. In addition, since both active and fibrillar forms of GAPDH prevent α-synuclein amyloidogenic transformation [28,30], glycation of GAPDH should alter the α-synuclein oligomerization and fibrillization process.
Materials and methods
Acknowledgements This work was supported by the Russian Science Foundation (project No. 16-14-10027). The molecular dynamics simulations were performed in the Supercomputing Center of Lomonosov Moscow State University .
Introduction Quinoprotein is a general term used for enzymes containing an ortho-quinone or para-quinone as a prosthetic group. Pyrroloquinoline quinone (PQQ; Figure 1) has been referred to as the third redox cofactor [1,2] following nicotinamide pyridine nucleotide (NAD(P)+) and flavin (FAD, FMN). Next to PQQ, other quinone prosthetic groups include topa quinone (TPQ) , tryptophan tryptophylquinone (TTQ) , lysine tyrosylquinone (LTQ) , and cysteine tryptophylquinone (CTQ) [6,7]. PQQ is tightly, but not covalently, bound to the enzyme, primarily via electrostatic interactions, whereas other quinones are formed by posttranslational modifications of amino acid residues and are thus covalently attached to the enzyme . PQQ quinoproteins are known from prokaryotes where they primarily catalyze the dehydrogenation of the primary or secondary hydroxyl group of alcohols or sugars [9, 10, 11]. Some of these PQQ-dependent enzymes also contain one or more heme groups and are called quinohemoproteins [12,13]. A few bacterial species such as methylotrophic bacteria are able to synthesize PQQ, whereas non-PQQ-synthesizing bacteria such as Escherichia coli rely on the environment for its supply .