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.

突触核蛋白（α -syn）是一种丰富的140残基蛋白，功能不明确，富集于突触前神经元末梢。值得注意的是，大脑中聚集或淀粉样α -syn的存在是帕金森病的一个标志，其构象和聚集动力学与膜密切相关。虽然大量的研究工作已经转向对膜相互作用下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结合如何影响磷脂双层结构和性质。