以自旋標定和電子自旋共振光譜分析全長小鼠普立昂蛋白類澱粉纖維之結構

Abstract

普利昂疾病是一種可傳播的致命神經退化性疾病，並且可以在人類和哺乳動物之間傳播。其病原體是一種錯誤折疊的、抗蛋白酶的、且富含 β-折疊的普利昂蛋白聚集體，簡稱為 PrPSc。 PrPSc 從其天然形式的普利昂蛋白（稱為 PrPC）經結構轉化而來，並且可以自我堆疊形成感染性澱粉樣纖維。由於普利昂疾病的關鍵是澱粉樣纖維的形成，因此研究和識別蛋白質的哪些片段發生了結構轉變並參與澱粉樣纖維核心生成可以提供有關普利昂疾病的重要資訊。已知在部分變性條件下普利昂蛋白也可以自發形成非感染性澱粉樣原纖維。在這項研究中，我們利用定點突變、定點自旋標記、引晶實驗和電子自旋共振光譜的方法來研究試管內製備的全長小鼠普利昂蛋白的纖維結構核心。結果得知普利昂澱粉樣纖維的形成與 α到 β 結構轉換有關，主要涉及的區域為序列160-224，而該片斷原為普利昂蛋白中的第二條折板、第二條螺旋和第三條螺旋。我們的方法未來可用於研究感染性普利昂纖維或其他澱粉樣纖維的結構。此外，我們利用不同片段的普利昂蛋白或胜肽所形成的澱粉樣纖維作為種晶來檢查普利昂蛋白中片段108-144在形成澱粉樣纖維時扮演的角色。其結果顯示自發形成的纖維或由全長普利昂（23-230）纖維或縮短的普利昂蛋白（90-230）纖維誘發所形成的纖維，其片段108-144都不在澱粉樣纖維核心中，然而利用普利昂胜肽（108-144）纖維當作晶種時，在129與138這兩個位置上其電子自旋共振光譜有顯著的不同，這表明形成的纖維結構受普利昂胜肽纖維影響。

Prion diseases are a group of transmissible fatal neurodegenerative disorders spreading between humans and other mammals. The pathogenic agent, prion, is a misfolded, protease-resistant, β-sheet-rich protein aggregate called scrapie prion (PrPSc). PrPSc is converted from its native form of the cellular prion protein, called PrPC, and PrPSc can self-associate to form the infectious amyloid. Since the key hallmark in prion disease is amyloid formation, studying and recognizing which segments taking structural transformation and involved in the amyloid core can provide molecular details about prion diseases. It has been known that non-infectious amyloid fibrils could also be formed by prion protein under partially denaturing conditions. In this study, a combination of site-directed mutagenesis, site-directed spin-labeling, seeding assay, and electron spin resonance spectroscopy was employed to recognize the fibril structure of the in vitro prepared full-length mouse prion protein. Our results show that the amyloid core is formed from segment 160224, which includes the original strand 2, helix 2, and helix 3 in the prion protein. Our method can be applied to study the structure of infectious prions or other amyloid fibrils in the future. In addition, different lengths of PrP fibrils were used as seeds to examine the role of segment 108-144 in the amyloidogenesis of full-length prion protein. Our data show that segment 108-144 is not in the amyloid core in the spontaneously formed full-length PrP fibrils or the fibrils seeded by full-length PrP fibrils or PrP (90-230) fibrils. However, the ESR spectra of the fibrils with a spin-labeled at 129 or 138 have peak broadening, suggesting that the resulting fibril structure was altered by the peptide seed.