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Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability.

Sode K, Ochiai S, Kobayashi N, Usuzaka E - Int. J. Biol. Sci. (2006)

Bottom Line: The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein.The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate.These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-2-4-16, Nakacho, Koganei, Tokyo 184-8588, Japan. sode@cc.tuat.ac.jp

ABSTRACT
The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.

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Effect of amino acid substitution on amyloid fibril formation of α-synuclein. Time course of fibril formation of α-synuclein and its mutants as determined by TfT fluorescence assay analysis: WT (black circles), Rep6 (blue triangles), Rep7 (red squares), Rep67 (green diamonds).
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Figure 3: Effect of amino acid substitution on amyloid fibril formation of α-synuclein. Time course of fibril formation of α-synuclein and its mutants as determined by TfT fluorescence assay analysis: WT (black circles), Rep6 (blue triangles), Rep7 (red squares), Rep67 (green diamonds).

Mentions: The prepared wild type and mutant α-synuclein samples were investigated for their aggregation and fibril formation abilities using the fluorescent dye TfT. Because TfT appears to embed itself within the clearance of pleats of β-sheets, it interacts quite specifically with fibrils but not with most native proteins or amorphous aggregates. Binding of TfT to protein fibrils is accompanied by a characteristic increase in the fluorescence intensity in the vicinity of 482 nm 15. Comparison of the fibrillation patterns of wild type and mutant α-synucleins (Figure 3) shows that Rep7 has a similar fibrillation rate and higher maximal fluorescence intensity than wild type. Therefore, reparation of the 7th repeat sequence, mutating it to contain the characteristic KTKEGV hexameric motif, did not affect the propensity of fibril formation. In contrast, Rep6 and Rep67 had much lower fibril formation rates than both wild type and Rep7, with maximal fibril formation values less than 10% of the wild-type value. Therefore, both Rep6 and Rep67 achieved great reductions in fibril formation ability by reparation of the 6th repeat sequence to contain the characteristic hexamer motif.


Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability.

Sode K, Ochiai S, Kobayashi N, Usuzaka E - Int. J. Biol. Sci. (2006)

Effect of amino acid substitution on amyloid fibril formation of α-synuclein. Time course of fibril formation of α-synuclein and its mutants as determined by TfT fluorescence assay analysis: WT (black circles), Rep6 (blue triangles), Rep7 (red squares), Rep67 (green diamonds).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1622890&req=5

Figure 3: Effect of amino acid substitution on amyloid fibril formation of α-synuclein. Time course of fibril formation of α-synuclein and its mutants as determined by TfT fluorescence assay analysis: WT (black circles), Rep6 (blue triangles), Rep7 (red squares), Rep67 (green diamonds).
Mentions: The prepared wild type and mutant α-synuclein samples were investigated for their aggregation and fibril formation abilities using the fluorescent dye TfT. Because TfT appears to embed itself within the clearance of pleats of β-sheets, it interacts quite specifically with fibrils but not with most native proteins or amorphous aggregates. Binding of TfT to protein fibrils is accompanied by a characteristic increase in the fluorescence intensity in the vicinity of 482 nm 15. Comparison of the fibrillation patterns of wild type and mutant α-synucleins (Figure 3) shows that Rep7 has a similar fibrillation rate and higher maximal fluorescence intensity than wild type. Therefore, reparation of the 7th repeat sequence, mutating it to contain the characteristic KTKEGV hexameric motif, did not affect the propensity of fibril formation. In contrast, Rep6 and Rep67 had much lower fibril formation rates than both wild type and Rep7, with maximal fibril formation values less than 10% of the wild-type value. Therefore, both Rep6 and Rep67 achieved great reductions in fibril formation ability by reparation of the 6th repeat sequence to contain the characteristic hexamer motif.

Bottom Line: The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein.The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate.These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-2-4-16, Nakacho, Koganei, Tokyo 184-8588, Japan. sode@cc.tuat.ac.jp

ABSTRACT
The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.

Show MeSH
Related in: MedlinePlus