Characterization of the aggregation propertis of amyloid core in the TDP-43 C-terminal fragments

過去人們已經認知到，在澱粉樣纖維的形成過程中，一些被酵素切斷的蛋白及胜肽有著核心片段可提供分子間的辨識以及自我組裝的驅動力。在2010年，TDP-43 蛋白中C端的部分已被發現其中的胜肽片段D1 (287-322) 在磷酸鹽緩衝溶液中可以形成澱粉樣纖維。為了更進一步去探討這個片段形成澱粉樣纖維的核心部分，我們依序的合成縮短的胜肽，並且經過圓二色光譜，穿隧式電子顯微鏡，拉曼光譜，以及Thioflavin T (ThT)的測試來確認澱粉樣纖維的形成與否。我們發現，只有包含307-322這段序列的胜肽片段能夠產生構型的變化，而這個構型的變化主要為random coil過渡到β-sheet為主的結構。然而，比307-322更短的胜肽片段：312-322以及307-317無法產生這種構型變化。 並且，我們透過疏水性分析、分子彈性(flexibility)以及β摺版結構的預測來確認這些胜肽片段的生物物理特性。根據CD光譜的結果，我們仔細的分析能形成澱粉樣纖維的胜肽在β摺板吸收強度上升的不同。核心序列相較於較長的胜肽有較快的上升程度。為了更進一步的了解類澱粉樣纖維核心序列的特性，我們把核心胜肽片段TDP307-322中的三個甘胺酸(Glycine)換成三個脯胺酸(Proline)。由於文獻中指出，脯胺酸並不利於β-sheet結構的形成，我們期待胜肽TDP307-322 G3308P3無法有效形成β-sheet的結構，並間接地阻止了澱粉樣纖維的形成。令我們驚訝的是，我們引入了這三個脯胺酸似乎使得這個胜肽在圓二色光譜中產生一個很強的PPⅡ螺旋結構，而我們也進一步的發現其實原本的TDP307-322中也有這樣一個結構出現在構型轉變的過程中。因此，我們推測在307-312這段序列中所包含的胺基酸對於蛋白質聚集以及澱粉樣纖維的形成有顯著的影響。在這裡，我們在核心序列中換了一個以及兩個脯胺酸來看看對於澱粉樣纖維的形成有甚麼樣不同程度的擾動。我們發現，替換不同數量的脯胺酸呈現出精巧調控澱粉樣纖維形成的行為。這個PPⅡ螺旋結構或許在形成澱粉樣纖維的過程中，扮演著形成β摺板的過渡態，並間接在動力學上影響形成速率。若是我們能夠根據可能的過渡狀態發現重新導向蛋白聚集的途徑及控制在蛋白聚集的過程中澱粉樣纖維的特性，我們或許可以扭轉關於寡聚體毒性，細胞膜穿孔或是其他類澱粉樣纖維疾病所能造成的問題。It has been known that the core sequence of cleaved peptides or proteins provides molecular recognition as well as self-assembly driving force during amyloid fibrils formation. In 2010, the peptide fragment D1 (287-322) from the C-terminal domain of TDP-43 has been demonstrated to form amyloid fibrils in phosphate buffer. To further characterize the amyloid core in this fragment, we sequentially synthesize the truncated peptides and confirm the amyloid fibrils formation by Circular Dichroism (CD), Transmission Electronic Microscopy (TEM), Raman spectroscopy and Thioflavin T (ThT) assay. We had found that only the peptides containing the sequence (307-322) may undergo a conformational change from the random coil to a β-sheet rich structure when compared to the shorter peptides (312-322, 307-317). Moreover, the hydrophobicity, average flexibility and beta-sheet predictions have been applied to confirm the biophysical properties of these peptides. According to CD results, amyloid peptides having increasing intensity in β-sheet have been carefully compared. The core sequence shows higher increasing rate compared to the longer amyloid peptides. In order to gain insight into the properties of the amyloid core sequence, three glycines in the peptide TDP307-322 has been replaced to proline, TDP307-322 G3308P3, as proline had been reported to block the β-sheet structure. We expect to see the proline can block the β-sheet structure formation and indirectly inhibit amyloid fibrils formation. To our surprise, the triple-proline we introduced somehow make the peptide to generate a strong poly(L-proline) Ⅱ (PPⅡ)-helical conformation in CD spectra, and as a matter of fact, the wild-type TDP307-322 was observed to contain PPⅡconformation during the conformational change as well. Therefore, we postulate the residues in 307-312 may have significant impact of aggregation and fibril formation. Here, we replace one and two proline substitutions in between zero and three in the core sequence to see the perturbation of amyloid fibrils formation in different extent. What we have found is that the different amounts of proline substitutions demonstrate delicate behavior in tuning the amyloid fibrils formation. The PPⅡ helix conformation might play an important role in kinetically controlling the intermediate state toward β-sheet structure during amyloid fibril formation. If we can find a way to redirect the aggregation pathway based on the possible intermediate state and control the amyloid properties during aggregation, it is possible to reverse the situation which related to oligomer toxicity, membrane pore formation, or some other defect in amyloidogenesis