• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • Indeed a significant difference was observed


    Indeed, a significant difference was observed for the binding of glycated α-synuclein. Thus, g-α-synuclein bound to other part of GAPDH in 4 of 10 simulations (Fig. 2F, brown, yellow and dark-green chains, and Fig. 2G, brown chain). This additional binding site is located on the interface between O and Q (or P and R) subunits of GAPDH. In other 6 simulations, g-α-synuclein chains are bound to the anion-binding groove (Fig. 2F, G). However, in both binding sites, g-α-synuclein chain is more spread on the surface of GAPDH compared to the native protein without modifications. In the case of highly glycated α-synuclein, g2-α-synuclein, this effect is much more pronounced. In 9-cis-Retinoic Acid reviews to native and mildly glycated α-synuclein, which form a kind of clew, g2-α-synuclein looks like a “rope” (Fig. 2H, I). Furthermore, extended chains of g2-α-synuclein braid the GAPDH molecule and interact with many regions of the GAPDH surface in addition to the anion-binding groove. For example, green chain of g2-α-synuclein in Fig. 2H interacts with both anion-binding grooves, i.e. between subunits O and R and subunits P and Q. As a result, the following residues of GAPDH can be involved in the binding in addition to mentioned above: Lys3, Asn24, Pro36, Asn41, His57, Thr59, Lys61, Glu63, Asn64, Gly65, Asn70, Glu79, Lys86, Thr99, Gly100, Val101, Thr103, Thr104, Met105, Glu278. In other words, highly glycated α-synuclein seems to bind with GAPDH more efficiently. This conclusion has been corroborated with calculation the number of bonds between α-synuclein and GAPDH (Fig. 3A). The numbers of H-bonds, ion pairs, and, as a result, all pairs of closely located atoms, were almost twice higher in the case of g2-α-synuclein comparing with native α-synuclein. The same was true for pairs of nonpolar atoms indicating that hydrophobic interaction also increased due to the glycation, probably because of increase of the binding site (and increase of the part of α-synuclein which is bound to GAPDH). As for mildly glycated α-synuclein, g-α-synuclein, the difference with native form was much less. It formed higher number of H-bonds compared to native α-synuclein, but the number of ion pairs as well as estimated impact of hydrophobic interactions were almost the same (Fig. 3A). Noteworthy, glycation resulted in significant change of α-synuclein region involved in the binding. Thus, native α-synuclein without modifications interacted with GAPDH generally via C-terminal region (Fig. 3B, top panel) enriched with negatively charged residues (see the band below the bar chart, negatively and positively charged residues are shown in pink and blue, respectively). Substitution of two lysine residues to negatively charged CML (colored in red) in g-α-synuclein resulted in enlargement of the region involved in the binding (Fig. 3B, middle panel). Furthermore, substitution of 9 lysine residues to negatively charged CML in g2-α-synuclein resulted in a dramatic change of α-synuclein N-terminus charge: it became strongly acidic. As a result, the modified N-terminal region efficiently interacted with GAPDH (Fig. 3B, bottom panel). Since the main binding site of α-synuclein comprises NAD+-binding pocket and active site of GAPDH, the binding should affect the enzymatic activity of GAPDH. As it was shown in our previous work [28], α-synuclein competes with NAD+ in the binding and the binding of α-synuclein to GAPDH with partially oxidized active site cysteines resulted in the subsequent inactivation of the enzyme. To investigate possible effect of glycated α-synuclein on the enzymatic activity of GAPDH we used preparations of α-synuclein glycated by 1 mM MG or GA-3-P. The experiments were performed using the preparations of GAPDH with partially oxidized sulfhydryl groups of the active site, exhibiting 55–65% of the original activity, since, as it was shown earlier, α-synuclein did not affect the activity of GAPDH with completely reduced sulfhydryl groups.