Protein Misfolding and Aggregation

蛋白质错误折叠和聚集

• 批准号: 8149474
• 负责人: Jennifer Lee
• 金额: $ 45.54万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149474
• 关键词: protein misfolding

1. Role of Membrane Interactions on the Mechanism of alpha-Synuclein Amyloid Formation Understanding the environmental factors affecting the aggregation of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of alpha-syn are intimately connected to Parkinsons disease (PD) etiology. Membrane interactions are of particular interest because alpha-syn localizes near synaptic vesicles and mitochondrial membranes in vivo. Specifically, the protein undergoes disordered-to-helical structural changes with the addition of membrane mimics such as SDS micelles and upon binding to anionic phospholipid vesicles (SUV) of varying size and composition. To develop a detailed understanding of how membranes influence alpha-syn conformation, site-specific probes of protein conformational heterogeneity and polypeptide-membrane interactions are necessary. Fluorescence spectroscopy is particularly suited for this application because of the availability of environmentally sensitive fluorophores and the ease of performing experiments near physiological temperatures and concentrations even down to a single molecule. In our studies, we have employed anionic SUVs and SDS micelles as membrane mimics to investigate membrane-induced conformational changes by fluorescence as well as circular dichroism (CD) spectroscopy. Tryptophan was substituted at four different aromatic residues (F4W, Y39W, F94W, and Y125W) to report information on local polypeptide environment and conformational heterogeneity between SUV- and SDS-micelles-bound alpha-syn. With this approach, we aim to determine the crucial protein-to-membrane conditions and key sites of interaction that promote protein aggregation and ultimately, monitor membrane-mediated amyloid formation processes. 1.1 Tryptophan Probes at the alpha-Synuclein Membrane Interface Measurements of steady-state and time-resolved fluorescence of single tryptophan-containing alpha-syn variants have revealed distinct phospholipid vesicle and micelle interactions at residues 4, 39, 94, and 125 (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622). Our circular dichroism (CD) data confirm that Trp mutations do not affect alpha-syn membrane binding properties saturating at an estimated lipid-to-protein molar ratio of 380 or approximately 120 proteins covering 7% of the surface area of an 80 nm diameter vesicle. Fluorophores at positions 4 and 94 are the most sensitive to the lipid bilayer with pronounced spectral blue-shifts (W4: lambda max 23 nm; W94: lambda max 10 nm) and quantum yield increases (W4, W94: 3 fold) while W39 and W125 remain primarily water-exposed. Time-resolved fluorescence data show that all sites (except W125) have subpopulations that interact with the membrane. Specifically, the presence of protein conformational heterogeneity in the bilayer, suggest that the both W4 and W94 exhibit high membrane affinity. Notably, both of these sites have not been characterized previously in the vesicle-bound alpha-syn structure. 1.2 Promotion of Inter-alpha-Synuclein Interactions by the Membrane Surface There is a strong relationship between membranes and alpha-syn aggregation behavior, measurements of protein conformation and dynamics on the membrane surface are necessary to gain insight into how this protein converts from a benign to a pathogenic form. We have begun to investigate the specific relationship between amyloid formation and vesicle concentration. Initial data indicate that under low lipid-to-protein ratios (< 140, below the midpoint transition) protein aggregation is stimulated with the lag phase times hasten from protein alone or under saturation conditions. This is striking because using our equilibrium isotherm data (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622), we would estimate a staggering 14,000 proteins-per-vesicle (lipid-to-protein 4); a value significantly higher than the maximum binding sites per vesicle (120). Under these solution conditions, it is not possible for all proteins to adopt known membrane-bound structures. While our data suggest that alpha-syn proteins are not closely packed at saturation, it is plausible that changing the lipid-to-protein ratio could result in conformational rearrangement at the vesicle surface. The local conformational change between the solvent-to-lipid or different lipid-bound states is evidenced in the lipid concentration dependent W94 time-resolved data, indicating that protein-membrane stoichiometry is crucial in modulating membrane interactions in this region of the protein. Because all W4 populations are sensitive to the membrane at all lipid concentrations, while W94 has both lipid and solvent exposed conformers that are dependent on lipid-to-protein ratio, we suggest that W4 could act as a membrane anchor. It is noteworthy that the hydrophobic non-amyloid beta component (NAC, 61-95) region has been proposed to be secluded from intermolecular contacts by the intramolecular N- and C-terminal interactions. Similarly, it is feasible for membrane stimulated aggregation to arise because as the local protein concentration increases at the surface, polypeptide conformational rearrangement ensues leading to exposure of potential protein-protein interaction sites promoting amyloid formation. Currently, experiments using bimolecular quenching and Forster energy transfer measurements are underway to identify and define conformational changes that ensue under these conditions that promote protein aggregation. 2. Fluorescent Probes of alpha-Synuclein Fibril Assembly Upon aggregation, alpha-syn undergoes conformational changes from a disordered monomer to beta-sheet fibrils (amyloid). Importantly, amyloids have been shown as the major constituent in Lewy bodies, a pathological hallmark of PD. A mature fibril is approximately 5&#8722;20 nm in diameter with varying lengths (up to micrometers) and can be comprised of single or multiple filaments (2&#8722;5 nm). These filamentous structures are characterized by a cross-beta fold where each beta-strand is aligned perpendicular to the fibril axis with interstrand hydrogen bonds running along the fibril. Though it is generally thought that the alpha-syn fibril core is composed of residues 30&#8722;100, the molecular details of fibrillar assembly is poorly understood especially pertaining to the N- and C-terminal regions. We have prepared single-Cys mutants derivatized with an environment sensitive dansyl (Dns) fluorophore at specific residues spanning across the alpha-syn sequence (G7C, V26C, V51C, V66C, V77C, L100C, and Y136C) and characterized both soluble and aggregated forms by measurements of steady-state and time-resolved fluorescence. To unravel the role of these individual residues, we utilized these fluorescent variants to monitor the fibril assembly kinetics of the wild-type protein (1.5 Dns-protein: 100 wild-type). Dns fluorescence demonstrates to be generally an effective probe, particularly, changes in both mean emission wavelength and quantum yield are sensitive to amyloid formation.

1. 膜相互作用对 α-突触核蛋白淀粉样蛋白形成机制的作用 了解影响 α-突触核蛋白 (α-syn) 聚集的环境因素非常重要，因为 α-syn 的积累和沉积与帕金森病 (PD) 病因密切相关。膜相互作用特别令人感兴趣，因为 α-syn 在体内定位于突触小泡和线粒体膜附近。具体来说，通过添加膜模拟物（例如 SDS 胶束）以及与不同大小和组成的阴离子磷脂囊泡 (SUV) 结合，该蛋白质会经历无序至螺旋的结构变化。为了详细了解膜如何影响 α-syn 构象，需要对蛋白质构象异质性和多肽-膜相互作用进行位点特异性探针。荧光光谱法特别适合这种应用，因为可以使用环境敏感的荧光团，并且可以轻松地在接近生理温度和浓度（甚至低至单个分子）的条件下进行实验。 在我们的研究中，我们采用阴离子 SUV 和 SDS 胶束作为膜模拟物，通过荧光和圆二色性 (CD) 光谱研究膜诱导的构象变化。色氨酸在四个不同的芳香族残基（F4W、Y39W、F94W 和 Y125W）处被取代，以报告局部多肽环境以及 SUV 和 SDS 胶束结合的 α-syn 之间构象异质性的信息。 通过这种方法，我们的目标是确定促进蛋白质聚集的关键蛋白质与膜条件和相互作用的关键位点，并最终监测膜介导的淀粉样蛋白形成过程。 1.1 α-突触核蛋白膜界面处的色氨酸探针 对含有单个色氨酸的 α-syn 变体的稳态和时间分辨荧光的测量揭示了残基 4、39、94 和 125 处不同的磷脂囊泡和胶束相互作用 (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622)。 我们的圆二色性 (CD) 数据证实，Trp 突变不会影响 α-syn 膜结合特性，估计脂质与蛋白质摩尔比为 380 或约 120 个蛋白质，覆盖 80 nm 直径囊泡表面积的 7% 时达到饱和。 位置 4 和 94 处的荧光团对脂质双层最敏感，具有明显的光谱蓝移（W4：λ max 23 nm；W94：λ max 10 nm）并且量子产率增加（W4、W94：3 倍），而 W39 和 W125 仍然主要暴露于水。 时间分辨荧光数据显示所有位点（W125 除外）都有与膜相互作用的亚群。 具体而言，双层中蛋白质构象异质性的存在表明W4和W94均表现出高膜亲和力。值得注意的是，这两个位点之前都没有在囊泡结合的 α-syn 结构中得到表征。 1.2 膜表面促进α-突触核蛋白间相互作用 膜和 α-syn 聚集行为之间存在密切关系，需要测量膜表面的蛋白质构象和动力学，以深入了解该蛋白质如何从良性形式转化为致病形式。 我们已经开始研究淀粉样蛋白形成和囊泡浓度之间的具体关系。 初始数据表明，在低脂质与蛋白质比率（< 140，低于中点转变）下，蛋白质聚集受到单独蛋白质或在饱和条件下的滞后期时间加速的刺激。这是惊人的，因为使用我们的平衡等温线数据（Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622），我们估计每个囊泡有惊人的 14,000 个蛋白质（脂质到蛋白质 4）；该值明显高于每个囊泡的最大结合位点 (120)。 在这些溶液条件下，不可能所有蛋白质都采用已知的膜结合结构。 虽然我们的数据表明 α-syn 蛋白在饱和状态下并不紧密堆积，但改变脂质与蛋白质的比例可能会导致囊泡表面的构象重排，这似乎是合理的。 溶剂与脂质或不同脂质结合状态之间的局部构象变化在脂质浓度依赖性 W94 时间分辨数据中得到证实，表明蛋白质-膜化学计量对于调节蛋白质该区域的膜相互作用至关重要。 由于所有 W4 群体在所有脂质浓度下都对膜敏感，而 W94 具有依赖于脂质与蛋白质比率的脂质和溶剂暴露构象异构体，因此我们建议 W4 可以充当膜锚定物。 值得注意的是，疏水性非淀粉样β成分（NAC，61-95）区域被认为通过分子内N端和C端相互作用而与分子间接触隔离。 类似地，膜刺激聚集的发生也是可行的，因为随着表面局部蛋白质浓度的增加，多肽构象重排随之发生，导致潜在的蛋白质-蛋白质相互作用位点暴露，促进淀粉样蛋白形成。 目前，正在进行使用双分子猝灭和福斯特能量转移测量的实验，以识别和定义在这些条件下发生的促进蛋白质聚集的构象变化。 2. α-突触核蛋白原纤维组装的荧光探针 聚集后，α-syn 经历从无序单体到 β-片原纤维（淀粉样蛋白）的构象变化。重要的是，淀粉样蛋白已被证明是路易体的主要成分，路易体是帕金森病的病理标志。成熟原纤维的直径约为 5−20 nm，长度各异（可达微米），可由单根或多根细丝 (2−5 nm) 组成。这些丝状结构的特征是交叉β折叠，其中每个β链垂直于原纤维轴排列，链间氢键沿着原纤维延伸。尽管人们普遍认为 α-syn 原纤维核心由残基 30−100 组成，但人们对原纤维组装的分子细节知之甚少，尤其是与 N 端和 C 端区域有关的细节。 我们制备了在跨越 α-syn 序列（G7C、V26C、V51C、V66C、V77C、L100C 和 Y136C）的特定残基处用环境敏感丹酰基 (Dns) 荧光团衍生的单半胱氨酸突变体，并通过测量稳态和时间分辨荧光来表征可溶形式和聚集形式。 为了揭示这些单独残基的作用，我们利用这些荧光变体来监测野生型蛋白（1.5 Dns 蛋白：100 野生型）的原纤维组装动力学。 Dns 荧光通常被证明是一种有效的探针，特别是平均发射波长和量子产率的变化对淀粉样蛋白的形成敏感。