Protein Misfolding and Aggregation

蛋白质错误折叠和聚集

• 批准号: 8746551
• 负责人: Jennifer Lee
• 金额: $ 40.96万
• 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/8746551
• 关键词: Accounting; Adopted; Amyloid; Binding; Brain; C-terminal; Caliber; Charge; Circular Dichroism Spectroscopy; Complex; Data; Deposition; Disease; Disease Progression; Event; Fluorescence Spectroscopy; Generations; Glycerol; Hydrocarbons; Investigation; Kinetics; Label; Lasers; Ligation; Lipid Bilayers; Lipids; Maps; Mass Spectrum Analysis; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular Conformation; N-terminal; Negative Staining; Nerve Degeneration; Neurons; Neutrons; Parkinson Disease; Penetration; Peptides; Phosphatidic Acid; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Phosphorylcholine; Physiological; Preparation; Property; Protein Region; Proteins; Reaction; Reporting; Research; Role; Sampling; Shapes; Site; Solutions; Solvents; Structure; Tail; Techniques; Thick; Transmission Electron Microscopy; Tube; Tubular formation; Variant; Vesicle; Work; alpha helix; alpha synuclein; amyloid formation; density; membrane model; presynaptic; protein aggregate; protein aggregation; protein misfolding; protein profiling; unilamellar vesicle

alpha-Synuclein (alpha-syn) is an abundant 140 residue protein of ill-defined function enriched in the presynaptic neuronal terminals. Notably, the presence of aggregated or amyloid alpha-syn in the brain is a hallmark of Parkinson's disease and its conformation and aggregation kinetics are intimately tied to membranes. While substantial research efforts have been geared towards the understanding of protein conformational dynamics upon membrane interaction, a central question that remains is how protein association influences phospholipid bilayer structure and properties. 1. Neutron Reflectometry Studies of alpha-Synuclein at the Lipid Membrane Interface In prior work, we have assessed the membrane penetration depth of alpha-syn on the residue-level and from the perspective of the bilayer by using site-specific fluorescence spectroscopy and neutron reflectometry (NR), respectively. While the penetration depths obtained from two different membrane models, unilamellar vesicles and sparsely tethered bilayer lipid membrane (tBLM), were highly consistent, the profile for protein occupancy above the bilayer characterized by NR were somewhat unexpected. Specifically, alpha-syn extends into the hydrocarbon core as well as into the bulk solvent region. It is generally thought that the first 100 residues are membrane interacting, adopting an -helical conformation whereas the acidic C-terminal tail remaining disordered in solution. The thickness of the embedded protein region is around 15 angstroms comparable to that would be expected of an alpha-helix. However, the observed protein density out into the bulk solvent appears to be more extensive than would be expected for the last 40 C-terminal residues. Experimental evidence is needed to determine the specific residues that account for the different density regions. Towards the aim to delineate the involvement of specific protein regions, we have produced a segmental isotopically-labeled alpha-syn variant where the N-terminal (residues 1-86) and C-terminal regions (residues 87-140) are deuterated and protonated, respectively. Having the N-terminal region deuterated, the contrast between the protonated lipid and the peptide would be easily distinguishable. Using mass spectrometry, we estimate the level of deuteration to be at least 90 to 96%. Initial ligation reaction was rapid and efficient (< 30 min). NR data have been successfully collected using protein concentration as low as 10 nM. Ongoing efforts are geared toward optimization of sample preparation and investigation of effects of phospholipid headgroups on alpha-syn structure on the tBLM. 2. Membrane Remodeling by alpha-Synuclein and Effects on Amyloid Formation An emerging view is that alpha-syn can strongly influence the structure and properties of phospholipid bilayers. Recent examples include membrane thinning, membrane curvature generation, as well as formation of tubular structures. Presence of anionic phospholipids, e.g. phosphatidylglycerol (PG), phosphatidylserine (PS), or phosphatidic acid (PA), and folding of alpha-helical structure are thought to be essential for membrane binding and remodeling (deformation) by alpha-syn. Membrane shapes along with bilayer integrity are crucial in cellular activities such as intracellular vesicular transport. Accordingly, it is compelling to hypothesize that alpha-syn bends and remodels membranes as part of its physiological as well as pathological function. Recent reports have found that alpha-syn induces membrane tubulation in vesicles containing anionic lipids, especially POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). Interaction with negatively charged lipids and the formation of alpha-helix structure by alpha-syn are proposed to be important factors. Unexpectedly, we found that POPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine) vesicles of average diameter 100 nm are remodeled rapidly ( seconds) into tube-like structures by alpha-syn as visualized by negative staining transmission electron microscopy (TEM). Even in the presence of low amounts of alpha-syn (hundreds of nanomolar), tubules were clearly observed by TEM under a wide variety of lipid-to-protein ratios. Moreover, tubulation inhibits alpha-syn amyloid formation. These results appear to contradict the current hypothesis for membrane curvature generation mechanism which involves the formation of amphipathic helical structure upon membrane association as secondary structure changes of alpha-syn in the presence of POPC vesicles are undetectable by circular dichroism spectroscopy. Other techniques including fluorescence spectroscopies are currently being employed to map the specific interacting region in order to unravel this apparent paradox.

α-突触核蛋白（α-Syn）是一种丰富的140个残基蛋白，具有富含突触前神经元末端的不确定功能。值得注意的是，大脑中聚集或淀粉样蛋白α-Syn的存在是帕金森氏病的标志，其构象和聚集动力学与膜密切相关。尽管大量的研究工作旨在了解膜相互作用时蛋白质构象动力学的理解，但仍然存在的一个核心问题是蛋白质关联如何影响磷脂双层的结构和特性。 1。脂质膜界面上α-突触核蛋白的中子反射法研究 在先前的工作中，我们分别使用位点特异性的荧光光谱和中子反射测量法（NR）评估了残基水平和双层的膜渗透深度。虽然从两个不同的膜模型获得的渗透深度，但单层囊泡和稀疏的束缚双层脂质膜（TBLM）高度一致，而由NR表征的双层上方的蛋白质占用率很高。具体而言，α-Syn延伸到碳氢化合物核心以及大量溶剂区域。人们普遍认为，前100个残基是膜相互作用的，采用 - 螺旋构象，而酸性C末端尾巴含有溶液中残留的无序。嵌入式蛋白质区域的厚度约为15埃，与α-螺旋相当。然而，观察到的蛋白质密度超过最近40个C末端残基所预期的要广泛。需要实验证据来确定解释不同密度区域的特定残基。 为了描绘特定蛋白质区域的参与，我们产生了一个分段的同位素标记的α-syn变体，其中N端（残基1-86）和C末端区域（残基87-140）分别被剥离和蛋白研究。具有N末端区域的剥离，质子化脂质和肽之间的对比很容易区分。使用质谱法，我们估计剥离水平至少为90％至96％。初始连接反应是快速有效的（<30分钟）。使用低至10 nm的蛋白质浓度成功收集了NR数据。持续的努力旨在优化样品制备，并研究磷脂头组对α-Syn结构TBLM的影响。 2。通过α-核蛋白重塑膜和对淀粉样蛋白形成的影响 一个新的观点是，α-syn可以强烈影响磷脂双层的结构和特性。最近的例子包括膜变薄，膜曲率产生以及管状结构的形成。阴离子磷脂的存在，例如磷脂酰甘油（PG），磷脂酰丝氨酸（PS）或磷脂酸（PA）以及α-螺旋结构的折叠被认为对于α-Syn的膜结合和重塑（变形）是必不可少的。膜形状以及双层完整性在细胞内囊泡转运等细胞活性中至关重要。因此，令人信服的是假设α-Syn弯曲并重塑膜是其生理和病理功能的一部分。 最近的报道发现，α-syn诱导含有阴离子脂质的囊泡，尤其是POPG（1-甲状腺素-2-烯酰基-SN-甘油-3-甘油-3-磷酸 - （1'-rac-甘油））。提议与带负电荷的脂质的相互作用，并被α-Syn形成α-螺旋结构是重要因素。出乎意料的是，我们发现POPC（1-甲米酰基-2螺旋酰Sn-甘油-3-磷酸胆碱）的平均直径为100 nm的囊泡通过α-Syn迅速重塑（秒），并被α-Syn迅速重塑到管状结构中，并通过负模板的变速器传输电子显微镜（TEM）可视化。即使存在较低量的α-syn（数百个纳米极），在多种脂质与蛋白质比下，TEM清楚地观察到了小管。此外，管抑制了α-syn淀粉样蛋白的形成。这些结果似乎与膜曲率产生机制的当前假设相矛盾，膜曲率产生机制涉及膜结合上两亲性螺旋结构的形成，因为在POPC囊泡存在下α-Syn的二级结构变化是无法通过圆形二色谱检测的。目前正在使用包括荧光光谱在内的其他技术来绘制特定的相互作用区域，以揭示这种明显的悖论。