錯誤折疊並聚集的促澱粉樣變蛋白和多肽分子通常以β折疊含量豐富的纖維狀澱粉態存在，這種纖維狀澱粉態被認為與多種神經退行性疾病的發病有關，例如老年癡呆症，多聚穀氨醯胺症以及傳染性海綿狀腦病。澱粉態沉積物作為多種神經退行性疾病的顯著標誌，促澱粉樣變蛋白和多肽發生錯誤折疊並聚集進而導致神經毒性的機理仍未被闡明。在當前的研究中，我們選擇酵母感染性蛋白Sup35作為探索促澱粉樣變蛋白聚集機理的模型。Sup35是一種存在於釀酒酵母細胞中的感染性蛋白，作為一種翻譯終止因子，它可以通過改變自身構象，進而形成不溶的纖維狀澱粉態沉澱。根據位置和功能的不同，Sup35可被劃分為3個結構域，即N，M和C。作為控制其感染性的區域，Sup35-NM被廣泛接受為一種用於研究促澱粉樣變蛋白的模型。研究人員已經針對Sup35的聚集機理開展了很多研究，其中最為廣泛接受的是Lindquist 等人提出的β螺旋模型。在這個模型中，相鄰的氨基酸片段形成了一種頭對頭和尾對尾的構象。我們的研究目的就是要探究這種聚集機理模型是否正確。如果不正確，我們將對聚集機理提出一種新的假設。 / 作為探索促澱粉樣變蛋白聚集過程的重要前提，研究人員必須首先製備出只含有單獨的蛋白單體的樣品溶液。否則，相關的動力學過程研究將被干擾。我們通過動態激光光散射研究發現，使用現有的多種用於溶解促澱粉樣變蛋白和多肽的實驗方法並不能製備出真正的蛋白溶液，得到的樣品中總含有微量的、尺寸大約為10-10² nm的聚集體。這些聚集體會極大地影響聚集的動力學過程。這也可以在一定程度上解釋為什麼在不同的文獻報導中，同一種蛋白在相同的環境中卻表現出差異巨大的動力學過程。在當前的研究中，我們將傳統方法與我們實驗室新進開發的超濾法相結合，發展出了一套可以用於製備真正的、不含有聚集體的促澱粉樣變蛋白或多肽溶液的方法。製備出的溶液可以保持其中的蛋白或多肽處於單體狀態至少一個星期，這為研究在生理條件下蛋白的聚集過程提供了重要的基礎。 / 為了研究Sup35不同亞基之間的相互作用，我們分別在其N結構域的頭，腰和尾做了半胱氨酸點突變，並用兩種相互獨立的方法研究亞基之間的相互作用。第一種方法是在突變位點引入空間位阻，從而減弱所謂的頭對頭尾對尾的相互作用。我們的想法很直接，如果Lindquist等人提出的機理是正確的，那麼突變後的蛋白將無法形成纖維狀澱粉態沉澱。第二種方法是通過形成二硫鍵在不同蛋白的半胱氨酸突變位點之間引入連接分子，共有兩種連接分子，一種長約2 Å，另一種長約11 Å。選擇這兩種連接分子的原因是，聚集體中兩條Sup35蛋白鏈之間的距離通常約4.7 Å，連接分子長於或短于這個距離應會對聚集產生不同影響，從而反映出聚集體的結構資訊。 / 在這篇博士論文中，首先，我將介紹促澱粉樣變蛋白研究的背景和激光光散射測量的原理以及研究中用到的主要實驗方法。然後，我將闡述如何將傳統方法和我們實驗室新進發展出的超濾法相結合，從而製備出真正的、不含聚集體的蛋白溶液。接下來，我還將證明通過動態和靜態激光光散射相結合，我們可以得到更多關於促澱粉樣變蛋白的微觀參數，包括分子量，蛋白單體和聚集體的流體力學半徑等。最後，我將針對不同Sup35突變體的聚集動力學過程來研究其亞基之間的相互作用並提出Sup35的聚集模型。 / Misfolding and aggregation of amyloidogenic protein/peptide are frequently found in a β-sheet-rich fibrillar protein conformation known as amyloids, which are related to the onset of neurodegenerative diseases, ranging from Alzheimer and polyglutamine diseases to transmissible spongiform encephalopathies. While amyloid deposits are hallmarks of many neurodegenerative diseases, the mechanism by which these proteins/peptides gain their neurotoxic function upon misfolding and aggregation remains unclear. In the current study, we choose the yeast prion Sup35 as a model system to investigate the aggregation mechanism of amyloidogenic protein. The Sup35 protein is a yeast (Saccharomyces cerevisiae) prion protein, a translation termination factor that can convert into insoluble amyloid fibril. The structure of Sup35 protein can be divided into three regions; namely, N, M, and C based on their positions and different functions. Being the prion-determining region, Sup35-NM has been widely accepted as a model to study the amyloidogenic proteins. Many studies have been focused on the aggregation mechanism. The β-helix model proposed by Lindquist and her coworkers is mostly accepted. In such a model, Sup35-NM is folded to form a “head and a “tail region in the N region and different Sup35-NM chains aggregate together via a cooperative Head-to-Head and Tail-to-Tail stacking. The aim of our current study is to check whether this proposed mechanism is valid. / To gain insight into the mechanism of aggregation process, one must start with a solution that contains only individual (monomeric) protein chains. Otherwise, the kinetic study would be compromised. Our dynamic laser light scattering (LLS) study shows that the existing protocols of dissolving amyloidogenic protein/peptide do not result in a true solution; namely, there always exist a trace amount of interchain aggregates with an average size of ~10-10² nm, which greatly affect the association kinetics, partially explaining why different kinetics were reported even for a solution with identical protein and solvent. In this thesis study, by using a combination of the conventional dissolution procedure and our newly developed ultra-filtration method, we have developed a novel protocol to prepare a true solution of amyloidogenic protein/peptide without any interchain aggregates. The resultant solutions remain in their monomeric state for more than one week, which is vitally important for further study of the interchain association under the physiological conditions / To investigate the inter-subunit relationship, cysteine variants mutated at “Head, Waist, Tail" of the N region have been constructed. Two independent assessments have been proposed to study the inter-subunit interaction. One is to provide steric hindrance to the mutated sites so that the so called “Head-to-Head and Tail-to-Tail" interaction will be attenuated. Our strategy is quite straightforward, if the mechanism proposed by Lindquist and her coworkers is valid, the modified protein should lose its ability to form amyloid fibrils. The other strategy is to introduce disulfide cross-linkage between different mutation sites. Two types of disulfide cross-linkage have been chosen, one with a bond length of ~2 Å and the other, ~11 Å. The reason for such choices is that Sup35-NM has a characteristic inter-strand distance of ~4.7 Å. The disulfide bond length shorter or longer than this distance is supposed to play a different role in the protein aggregation, shedding light on the structural information. / In this Ph.D. thesis, we first introduce the background of amyloidogenic protein research and present the principle and instrumentation of laser light scattering, the main technique applied in our study. Next we show how to obtain a true solution of amyloidogenic protein with no oligomeric aggregates by combining a conventional dissolution procedure and our newly developed ultra-filtration method. We also show how to combine static and dynamic laser light-scattering measurements in the study of protein solutions, which leads to more microscopic parameters, such as the molar mass and the hydrodynamic sizes of individual protein chains and their aggregates. Our focus is on the aggregation kinetics of modified Sup35-NM variants and on the investigation of the inter-subunit interaction. Finally, we propose a model for the aggregation of Sup35-NM prion protein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Diao, Shu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-101). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT (in Chinese) --- p.i / ABSTRACT --- p.iii / Table of content --- p.v / Acknowledgement --- p.viii / Chapter Chapter 1 --- Introduction and background --- p.1 / Chapter 1.1 --- The biological role of amyloidogenic protein --- p.1 / Chapter 1.1.1 --- The role of amyloidogenic protein in human disease --- p.1 / Chapter 1.1.2 --- The functional role of amyloidogenic protein in living system --- p.2 / Chapter 1.1.3 --- The role of amyloidogenic protein asnonchromosomal genetic elements --- p.3 / Chapter 1.2 --- The structure of amyloid fibrils --- p.4 / Chapter 1.2.1 --- Macromolecular structure of amyloid fibrils --- p.5 / Chapter 1.2.2 --- Structure models for protofilament --- p.6 / Chapter 1.2.3 --- The polymorphism of amyloid fibrils --- p.9 / Chapter 1.3 --- Aggregation mechanism of amyloidogenic protein --- p.10 / Chapter 1.3.1 --- The nucleated polymerization mechanism --- p.11 / Chapter 1.3.2 --- Multiple conformations adopted by amyloidogenic protein chains --- p.13 / Chapter 1.3.3 --- Sequence effect on amyloid formation --- p.15 / Chapter 1.4 --- The pathogenesis of amyloid diseases --- p.16 / Chapter 1.4.1 --- Prefibrillar aggregates may be the real culprits --- p.16 / Chapter 1.4.2 --- Strategies to prevent amyloid diseases --- p.17 / Chapter 1.5 --- References and Notes --- p.19 / Chapter Chapter 2 --- Principle of Laser Light Scattering and Instrumentation --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Static Laser Light Scattering --- p.28 / Chapter 2.2.1 --- Scattering by a small particle --- p.28 / Chapter 2.2.2 --- Scattering by many small-particle system --- p.30 / Chapter 2.2.3 --- Scattering by real systems --- p.31 / Chapter 2.3 --- Dynamic Laser Light Scattering --- p.37 / Chapter 2.3.1 --- Power spectrum of scattered light --- p.37 / Chapter 2.3.2 --- Siegert relation --- p.39 / Chapter 2.3.3 --- Translational diffusions --- p.40 / Chapter 2.3.4 --- Analysis of the correlation function profile --- p.42 / Chapter 2.4 --- Combination of Static and Dynamic Light Scattering --- p.44 / Chapter 2.5 --- Practice of Laser Light Scattering --- p.45 / Chapter 2.5.1 --- Light source --- p.45 / Chapter 2.5.2 --- Optics and cell design --- p.46 / Chapter 2.5.3 --- Detector --- p.47 / Chapter 2.5.4 --- Sample Preparation --- p.47 / Chapter 2.5.5 --- Differential refractometer --- p.48 / Chapter 2.6 --- References and Notes --- p.49 / Chapter Chapter 3 --- How to obtain a true solution of amyloidogenic protein/peptide with no oligomeric aggregates --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Experimental section --- p.53 / Chapter 3.3 --- Results and discussion --- p.59 / Chapter 3.4 --- Conclusion --- p.68 / Chapter 3.5 --- References and Notes --- p.71 / Chapter Chapter 4 --- Aggregation mechanism investigation of the Yeast prion protein Sup35-NM --- p.73 / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Experimental section --- p.75 / Chapter 4.3 --- Results and discussion --- p.82 / Chapter 4.3.1 --- Aggregation kinetics of Sup35-NM protein initiated from monomeric state --- p.82 / Chapter 4.3.2 --- Does Sup35-NM protein aggregate in a head-to-head and tail-to-tail fashion? --- p.87 / Chapter 4.3.2.1 --- The effect of dimerization on Sup35-NM aggregation --- p.88 / Chapter 4.3.2.2 --- Inter-subunit investigation by Pyrene excimer fluorescence assay --- p.92 / Chapter 4.3.2.3 --- The effect of PEGylation on Sup35-NM aggregation --- p.94 / Chapter 4.4 --- Conclusion --- p.98 / Chapter 4.5 --- References --- p.100 / Publications --- p.102
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328144 |
| Date | January 2012 |
| Contributors | Diao, Shu., Chinese University of Hong Kong Graduate School. Division of Chemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (viii, 102 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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