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.

PMEL17原纤维是人类皮肤和眼睛中黑色素沉积所需的结构脚手架。黑色素是在黑色素体，与内体和溶酶体相关的细胞器中合成的，并储存在黑色素细胞中，这些细胞是负责色素沉着的细胞。 尽管已显示黑色素体的成熟过程涉及四个不同的阶段，这些阶段在超微结构水平上通过透射电子显微镜（TEM）在超微结构水平上进行了详细表征，但在每个阶段中，在这些阶段中赤叶内PMEL17原纤维的分子特性尚不清楚。 此外，哪个多肽结构域仅或部分构成PMEL17丝的淀粉样蛋白核心也尚待定义。 1。RPT淀粉样蛋白形成是pH依赖性的 我们已经研究了重复域（RPT，残基315-444），作为一种模型系统，是从可溶性和非结构化单体到含有beta的原纤维的构象变化系统。 RPT初级氨基酸序列由10个不完美的残基重复序列组成，这些重复量富含Pro，Ser，Thr和Glu。 RPT包含16个羧酸盐，强调其经历pH诱导构象变化的倾向。 由于pH和蛋白质结构在体内链接，因此我们研究了局部和宏观RPT构象作为pH的详细函数。 由于TRP发射对溶剂极性，局部构象变化和蛋白质 - 蛋白质相互作用高度敏感，因此我们将唯一的固有W423作为淀粉样蛋白结构和聚集动力学的位点特异性荧光探针。 确定了临界pH状态（4.5至5.5），用于原纤维形成，这表明至少三个羧酸参与了聚集所需的结构重排。 RPT聚集期间W423的高响应能力表明，纤维组装中C末端区域的关键作用。 为了研究黑色素pH的变化与RPT原纤维形成之间的直接相关性，进行了pH滴定测定法。 将预制的RPT聚集体（pH 4.0）滴定为pH 7.0。 在pH 5.0时，小的高度弯曲的聚集体变成长条纹的原纤维，让人联想到I和II期黑素体中观察到的原纤维转变。 进一步的中和pH 7.0导致RPT原纤维完全拆卸。这种独特的聚集/分解过程与与疾病相关的淀粉样蛋白相反，淀粉样蛋白因抵抗最严厉的治疗而臭名昭著。 我们在高度酸性的黑色素体（I期）中提出，蛋白质聚集是在纤维伸长率伸长后才发生的，仅在隔室溶液达到优化的pH 5（II期）之后才发生。 在特定GLU残基的质子化后，多肽链中的静电电荷排斥会减少，从而导致形成紧凑的结构，这些结构促进了原纤维形成所需的关键相互作用。 此外，我们的观察结果可以在优化的pH（5.0）下容易骨料（大约2 microm）（约2 microm）（5.0），这可能表明只有纤维稳定而代替潜在的有毒低聚物。 虽然我们的数据表明，原纤维会溶解在第三阶段和IV黑色素体中发现的近中性条件下，但尚不清楚黑色素沉积是否可以使聚合物材料隔离纤维免受溶液的影响，因此可以保护它们免受溶解。 然而，如果释放并暴露于黑色素体以外的中性环境中，原纤维将很容易分解，从而保持其良性本质。 2。探测RPT Fibril拆卸 为了研究原纤维拆卸，我们分别采用了原子力显微镜（AFM）和核磁共振（NMR）光谱法作为超微结构和分子探针。具体而言，我们询问在溶解期间是否规避与疾病相关淀粉样蛋白相关的中间体。 为了监测原纤维拆卸，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被AFM可视化。 在pH 5.0时，观察到长，直和无支柱的原纤维，让人联想到TEM看到的原纤维。 用pH 6.5缓冲液洗涤这些原纤维后，原纤维开始溶解。实时监测显示，大型原纤维的碎片，然后在几分钟的时间内完全消失了较小的碎片。 为了获得残基特异性见解，为NMR光谱制备了同位素标记的纤维rpt。 在pH 5.0时，没有观察到残基378-444的骨架酰胺共振，这表明该区域包含淀粉样蛋白生成核。 这与我们断言C末端区域对原纤维形成很重要。 溶解动力学和光谱数据没有显示稳定中间体的证据。中间体的不存在也通过尺寸排除色谱法验证。 此外，单个GLU主链酰胺共振表现出相似的动力学趋势，这表明原纤维展开是涉及许多去质子化事件的全球事件。