Here, in addition to confirming the effects of C-terminal residue deletion on α-syn fibril formation ( and ), we probed the contributions of electrostatic repulsion directly by constructing substitution mutants where C-terminal Asp and/or Glu residues were altered to Asn residues (). From the results, it was revealed clearly that the acceleration of fibril formation, mainly fibril nucleus formation is due mainly to the elimination of negative charges, but not to decreases in polypeptide length of the C-terminal region. This result also agrees well with the finding that the fibril formation of α-syn is accelerated by charge shielding through addition of NaCl (Yagi et al. 2005
, or spermine (Hoyer et al. 2004
), or by lowering the pH (Hoyer et al. 2004
; Cho et al. 2009
; McClendon et al. 2009
; Wu et al. 2009
). The importance of the C-terminal 40 amino acids for fibril formation is also reported by Horvath et al. (2012
) very recently. They observed an accelerated fibril formation of α-syn in the presence of a dihydro thiazolo ring fused 2-pyridone derivative, and found through NMR experiments that the compound interacts with amino acid residues 1–100, while residues 101–140 remained flexible in solution. This result demonstrates that masking the N-terminal region of the α-syn polypeptide results in conditions favorable for fibril formation, most likely by increasing accessibility to important portions of the C-terminal region.
As the fibril nucleus is stabilized by oligomerization, electrostatic repulsion between molecules may exert an inhibitory effect during fibril nucleus formation. Recently, we determined the fibril nucleus core peptide region of α-syn as the segment corresponding to Ala76–Lys96 (Yagi et al. 2010
). When the 14 negative charges located between positions 104 and 139, which are relatively close to this nucleus core region, are removed by either deletion or mutation, intermolecular interactions of the fibril nucleus peptide regions would be favored, which in turn would accelerate fibril nucleus formation. Even under conditions similar to the physiological salt concentration, the effects of negative charge were observed, as seen in b, suggesting that this electrostatic contribution is significant.
In the C-terminal region of α-syn, we also find tyrosine residues at positions 125, 133, and 136. We have examined the relative contributions of these Tyr residues on fibril formation by mutating them to Ala residue(s) (), and found that Tyr136 was a critical element that promoted fibril formation. Simply changing Tyr136 to Ala was sufficient to significantly suppress both fibril nucleus formation (evidenced by the increased lag time) and fibril extension (seen by a decrease in the rate of fluorescence intensity increase). This Tyr could be replaced by Trp or Phe with minimal effects to the fibril formation mechanism, but not by Ser, Glu, or Leu (). These findings demonstrate that aromatic residues at position 136 are necessary for fibril formation. In other words, a possible way to suppress the fibril formation of α-syn may be to change Tyr136 to other nonaromatic amino acid residues.
Because the two factors that we focused upon in this study were located in the same C-terminal region of the α-syn polypeptide, we combined these two mutants to probe for any synergistic effects on fibril formation. Our results surprisingly pointed toward a very complex nucleation mechanism that dictated synuclein fibrillation. First of all, the relative importance of the tyrosine residue at position 136 was highlighted in our experiments. The results seen with the Syn119-140CF/Y136A mutant was a good example of the dominance of the tyrosine residue in dictating the formation of fibers (). However, if we refrained from neutralizing all of the negative charges in the C-terminal region, removing only the charges between residues 130 and 140, we observe that the absence of Tyr136 may be overcome, leading to fibrillation. This result is in apparent conflict with the dominant effects of tyrosine substitution seen in the other mutants probed in this study.
When we observed the shapes of the fibrils formed in , we found that fibrils formed by Syn130-140CF/Y136A were slightly different from the other samples (). Perhaps another, alternate pathway of fibril formation that is accessible only to this mutant exists. This may be because retaining the negative charges between residues 119 and 129 allows access to a new site that promotes nucleation, perhaps due to differences in the overall secondary structure. In Syn119-140CF/Y136A, removal of all of the negative charges in this region may cause the alternate site to be occluded once more, resulting in the complete suppression of fibril formation brought about by the absence of Tyr 136.
Our results have revealed that there may be many pathways involving multiple factors in the C-terminal region that initiate the formation of α-syn fibrils, and further careful analysis is necessary to completely understand the process of fibril nucleation and extension. In this context, we feel it worthwhile to emphasize another experimental result that was reported by others and confirmed by us; that α-syn also shows an increased tendency to form fibrils when the C-terminal region of interest is completely removed (). A complete understanding of the process of α-syn fibril formation must therefore provide an understanding of all of these diverse facets of the initial steps of fibril formation.
We have attempted to figure out a possible mechanism of α-syn amyloid fibril formation that explains our findings. The schematic model is shown in . α-Syn is intrinsically disordered and the polypeptide may assume an expanded conformation due to the repulsion of negative charges located in the C-terminal region, including other ensemble conformations (Heise et al. 2005
). Also, charge repulsion should suppress various intermolecular interactions important to molecular association (Levitan et al. 2011
). Upon deletion of the C-terminal negative charges or addition of NaCl, the electrostatic repulsion is reduced or shielded and intermolecular interactions centered upon this region is able to occur. Then, intermolecular interactions involving Tyr136 are initiated, probably due to the aromatic hydrophobic (Makin et al. 2005
; Levy et al. 2006
; Yagi et al. 2008
) or π–π ring stacking interaction (Levy et al. 2006
). The commitment of Tyr136 in this step is very important for fibril formation. From this increased intermolecular interaction, the fibril core region (Ala76–Lys96) (Yagi et al. 2010
), which is relatively close to the C-terminal region, now begins to form the fibril nucleus. Once the fibril nucleus forms tightly, fibril extension reaction begins rapidly. During this extension step, Tyr136 also affects the fibril extension rate through aromatic ring interactions. For the C-terminal truncation mutants that lack both negative charges and Tyr136, fibrillation must wait until the hydrophobic characteristics of the fibril core region trigger molecular association. Thus, the negative charges and Tyr136 located in the C-terminal region of α-syn both play critical roles in the mechanism of amyloid fibril formation.
Figure 8 A schematic model of α-syn fibril formation mechanism. Roles of the C-terminal negative charges and Tyr136 on the fibril formation, especially on the fibril nucleus formation step, are shown. The long blue squares represent the fibril core region (more ...)
Finally, these findings in this study may shed light on the gradual and persistent fibrillation mechanism of this intrinsically disordered protein, and also may lead to the development of a medical treatment for Parkinson's disease. In our hands, a mutant α-syn in which the amino acid residues between 119 and 140 have been deleted (Syn118) readily forms fibrils. In contrast, Syn119-140CF/Y136A, where the relevant amino acids in the same sequence region (negatively charged residues, and Tyr136) have been substituted, is unable to form fibrils (). This comparison seems to suggest that the charge-neutralized, tyrosine-deleted C-terminal region of Syn119-140CF/Y136A may be actively inhibiting the fibril formation of α-syn, perhaps through intramolecular or intermolecular interaction with the fibril core sequence (residues Ala76–Lys96; Yagi et al. 2010
). If true, a synthetic peptide corresponding to the C-terminal amino acid sequence of Syn119-140CF/Y136A might conceivably be utilized as an inhibitor of fibrillation, i.e., such peptide administered in vivo may interact with α-syn and prevent intermolecular interactions. Through utilization of this peptide, a new medical treatment for Parkinson's disease may eventually be developed.