Mechanisms of Functional Amyloid Formation

功能性淀粉样蛋白形成机制

• 批准号: 8557989
• 负责人: Jennifer Lee
• 金额: $ 45.93万
• 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/8557989
• 关键词: Address; Alzheimer' s Disease; Amides; Amino Acid Sequence; Amyloid; Amyloid Fibrils; Atomic Force Microscopy; Benign; Biological; Biological Assay; Biological Models; Buffers; C-terminal; Carboxylic Acids; Cells; Charge; DNA Sequence Rearrangement; Data; Deposition; Disease; Electrostatics; Employee Strikes; Endosomes; Environment; Event; Exhibits; Eye; Filament; Fluorescent Probes; Goals; Human; Individual; Kinetics; Label; Link; Lysosomes; Melanins; Melanosomes; Mica; Molecular; Molecular Conformation; Molecular Probes; Molecular Sieve Chromatography; Monitor; NMR Spectroscopy; Nature; Organelles; Parkinson Disease; Pigmentation physiologic function; Process; Proteins; Role; Site; Skin; Solutions; Solvents; Staging; Structure; Time; Titrations; Transmission Electron Microscopy; Vertebral column; Work; amyloid formation; amyloid structure; beta pleated sheet; carboxylate; cytotoxic; deprotonation; human disease; in vivo; insight; melanocyte; monomer; polypeptide; prolyl-serine; protein aggregation; protein protein interaction; protein structure; protonation; scaffold; trend

Pmel17 fibrils serve as the structural scaffolding required for melanin deposition in human skin and eyes. Melanin is synthesized in melanosomes, organelles related to both endosomes and lysosomes, and stored in melanocytes, cells responsible for pigmentation. While the melanosome maturation process has been shown to involve four distinct stages that have been characterized in detail at the ultrastructural level by transmission electron microscopy (TEM), the molecular nature of the intralumenal Pmel17 fibrils during each of these stages is not known. Moreover, which polypeptide domain solely or partly constitutes the amyloid core of the Pmel17 filaments also remains to be defined. 1. RPT Amyloid Formation Is pH Dependent We have studied the repeat domain (RPT, residues 315-444) as a model system of conformational change from soluble and unstructured monomer to aggregated, beta-sheet-containing fibrils. The RPT primary amino acid sequence is comprised of 10 imperfect 13 residue repeats that are rich in Pro, Ser, Thr, and Glu. RPT contains 16 carboxylates underscoring its propensity to undergo pH induced conformational changes. Because pH and protein structure are linked in vivo, we studied the local and macroscopic RPT conformation as a function of pH in detail. Since Trp emission is highly sensitive to solvent polarity, local conformational changes, and protein-protein interactions, we exploited the only intrinsic W423 as a site-specific fluorescent probe of amyloid structure and aggregation kinetics. A critical pH regime (4.5 to 5.5) was identified for fibril formation suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. The high responsiveness of W423 during RPT aggregation points towards a key role for the C-terminal region in fibril assembly. To investigate a direct correlation between changes in melanosomal pH and formation of RPT fibrils, a pH titration assay was performed. Preformed RPT aggregates (pH 4.0) were titrated to pH 7.0. At pH 5.0, small, highly curved aggregates change into long striated fibrils, reminiscent of the fibril transition observed in stage I and II melanosomes. Further neutralization to pH 7.0 resulted in complete disassembly of RPT fibrils. This unique aggregation/disaggregation process is in contrast to disease-related amyloids, which are notorious for resisting the harshest treatments. We propose in the highly acidic melanosome (stage I), protein aggregation is initiated with fibril elongation occurring only after the compartment solution reaches an optimized pH 5 (stage II). Upon protonation of specific Glu residues, the electrostatic charge repulsion within the polypeptide chain reduces, thereby leading to formation of compact structures that promote key interactions required for fibril formation. In addition, our observation that RPT will readily aggregate (approx. 2 microM) at the optimized pH (5.0) could suggest that only fibrils are stabilized in lieu of potentially toxic oligomers. While our data show that fibrils would dissolve in the near neutral conditions found in stage III and IV melanosomes, it is unclear whether upon melanin deposition, the polymeric material could sequester the fibrils from solution and hence protect them from dissolution. Nevertheless, if released and exposed to the neutral environments outside the melanosome, fibrils will readily disintegrate and thus maintain their benign nature. 2. Probing RPT Fibril Disassembly To investigate fibril disassembly, we employed atomic force microscopy (AFM) and nuclear magnetic resonance (NMR) spectroscopy as ultrastructural and molecular probes, respectively. Specifically, we asked whether intermediates associated with disease-related amyloids are circumvented during dissolution. To monitor fibril disassembly, preformed RPT amyloid was deposited on mica and visualized by AFM under wet buffer conditions. At pH 5.0, long, straight and unbranched fibrils were observed, reminiscent to those seen by TEM. Upon washing these fibrils with pH 6.5 buffer, the fibrils begin to dissolve. Real-time monitoring reveals fragmentation of large fibrils followed by complete disappearance of smaller fragments on the order of minutes. To obtain residue specific insight, isotopically labeled fibrillar RPT was prepared for NMR spectroscopy. At pH 5.0, no backbone amide resonances were observed for residues 378-444, suggesting this region contains the amyloidogenic core. This is consistent with our assertion that the C-terminal region is important for fibril formation. Dissolution kinetics and spectra data showed no evidence of stable intermediates. The absence of intermediates also was verified by size exclusion chromatography. Furthermore, individual Glu backbone amide resonances exhibited similar kinetic trends, suggesting fibril unfolding is a global event involving many deprotonation events.

Pmel 17原纤维作为人类皮肤和眼睛中黑色素沉积所需的结构支架。黑色素在黑素体（与内体和溶酶体两者相关的细胞器）中合成，并储存在黑素细胞（负责色素沉着的细胞）中。 虽然黑素体成熟过程已被证明涉及四个不同的阶段，其特征在于在超微结构水平的透射电子显微镜（TEM），在这些阶段中的每一个的腔内Pmel 17原纤维的分子性质是未知的。 此外，哪个多肽结构域单独或部分构成Pmel 17细丝的淀粉样蛋白核心也有待确定。 1. RPT淀粉样蛋白形成具有pH依赖性 我们研究了重复结构域（RPT，残基315-444）作为一个模型系统的构象变化，从可溶性和非结构化的单体聚集，β-折叠含原纤维。 RPT一级氨基酸序列由富含Pro、Ser、Thr和Glu的10个不完全的13残基重复序列组成。 RPT含有16种羧酸盐，强调其倾向于经历pH诱导的构象变化。 由于pH和蛋白质结构在体内是联系在一起的，我们详细研究了局部和宏观RPT构象作为pH的函数。 由于色氨酸发射是高度敏感的溶剂极性，局部构象变化，和蛋白质-蛋白质相互作用，我们利用唯一的内在W 423作为淀粉样蛋白的结构和聚集动力学的位点特异性荧光探针。 一个关键的pH值制度（4.5至5.5）被确定为原纤维的形成，表明参与至少三个羧酸的结构重排所需的聚集。 W 423在RPT聚集过程中的高响应性指向C-末端区域在原纤维组装中的关键作用。 为了研究黑素体pH变化与RPT原纤维形成之间的直接相关性，进行pH滴定测定。 将预形成的RPT聚集体（pH 4.0）滴定至pH 7.0。 在pH 5.0时，小的高度弯曲的聚集体变成长的条纹原纤维，让人想起在阶段I和II黑素体中观察到的原纤维转变。 进一步中和至pH 7.0导致RPT原纤维完全分解。这种独特的聚集/解聚过程与疾病相关的淀粉样蛋白形成对比，后者因抵抗最严厉的治疗而臭名昭着。 我们建议在高度酸性的黑素体（第一阶段），蛋白质聚集开始与原纤维伸长发生后，只有在室溶液达到最佳的pH值5（第二阶段）。 在特定Glu残基质子化后，多肽链内的静电电荷排斥减少，从而导致形成促进原纤维形成所需的关键相互作用的紧凑结构。 此外，我们观察到RPT很容易聚集（约。2 μ M）的低聚物，这表明只有原纤维被稳定化，而不是潜在的毒性低聚物。 虽然我们的数据显示，原纤维会在III期和IV期黑素体中发现的近中性条件下溶解，但尚不清楚在黑色素沉积时，聚合物材料是否可以将原纤维从溶液中隔离出来，从而保护它们免受溶解。 然而，如果释放并暴露于黑素体外的中性环境，原纤维将容易分解，从而保持其良性性质。 2.探测RPT Fibril 为了研究纤维解体，我们采用原子力显微镜（AFM）和核磁共振（NMR）光谱作为超微结构和分子探针，分别。具体而言，我们询问是否与疾病相关的淀粉样蛋白的中间体在溶解过程中规避。 为了监测原纤维分解，将预先形成的RPT淀粉样蛋白沉积在云母上，并在湿缓冲液条件下通过AFM可视化。 在pH 5.0时，观察到长的、直的和不分支的原纤维，使人联想到通过TEM观察到的那些。 在用pH 6.5的缓冲液洗涤这些原纤维时，原纤维开始溶解。实时监测显示大纤维的断裂，随后在几分钟内较小片段完全消失。 为了获得残留物特异性洞察，制备同位素标记的纤维状RPT用于NMR光谱。 在pH 5.0时，未观察到残基378-444的骨架酰胺共振，表明该区域含有淀粉样蛋白生成核心。 这与我们的断言一致，即C-末端区域对于原纤维形成是重要的。 溶出动力学和光谱数据显示没有稳定中间体的证据。还通过尺寸排阻色谱法验证了不存在中间体。 此外，个别Glu骨架酰胺共振表现出类似的动力学趋势，这表明原纤维展开是一个全球性的事件，涉及许多去质子化事件。