Mechanisms of Functional Amyloid Formation

功能性淀粉样蛋白形成机制

• 批准号: 8344842
• 负责人: Jennifer Lee
• 金额: $ 24.88万
• 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/8344842
• 关键词: Address; Adopted; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Benign; Biological; Biological Models; Carboxylic Acids; Cells; DNA Sequence Rearrangement; Deposition; Disease; Employee Strikes; Endosomes; Exhibits; Eye; Filament; Fluorescent Probes; Goals; Human; Kinetics; Lysosomes; Measures; Melanins; Melanosomes; Molecular; Molecular Conformation; Morphology; Nature; Organelles; Parkinson Disease; Pigmentation physiologic function; Process; Property; Proteins; Role; Side; Site; Skin; Solutions; Solvents; Staging; Structure; Thioflavin T; Titrations; Transmission Electron Microscopy; Tryptophan; Work; amyloid formation; amyloid structure; beta pleated sheet; cytotoxic; human disease; melanocyte; monomer; polypeptide; protein protein interaction; protonation; scaffold

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. We have begun to study the repeat domain (RPT, residues 315-444), and essential luminal polypeptide region of Pmel17, as a model system of conformational change from soluble and unstructured monomer to aggregated, beta-sheet-containing fibrils. To mimic the changing acidic pH conditions of the maturing melanosome, we measured RPT amyloid formation kinetics as a function of solution pH. Since tryptophan emission is highly sensitive to solvent polarity, local conformational changes, and protein-protein interactions, we exploited the only intrinsic tryptophan (Trp423) as a site-specific fluorescent probe of amyloid structure and aggregation kinetics. We find that Trp423 is exquisitely sensitive to soluble and fibrillar RPT conformation with spectral properties (intensity and mean wavelength) exhibiting distinct temporal changes under the various solution conditions examined. Particularly, spectroscopic differences highlight distinct amyloid morphologies as visualized by TEM and thioflavin T activity. Furthermore, fibril formation kinetics are highly pH dependent and we identified a critical range of solution pH (4.5 to 5.5) for RPT aggregation suggesting that protonation of specific carboxylic acid side chains is critical in facilitating the structural rearrangement necessary for amyloid formation. Upon titration to neutral pH, these fibrils dissolve, supporting a regulatory mechanism whereby if released and exposed to neutral cytosolic pH, RPT would adopt a non-toxic, soluble form. Our current efforts are focused on understanding at the molecular level, which residue(s) contribute to the critical pH regime of RPT amyloid formation.

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。 我们已经开始研究Pmel 17的重复结构域（RPT，残基315-444）和必需的管腔多肽区域，作为从可溶性和非结构化单体到聚集的、含有β折叠的原纤维的构象变化的模型系统。为了模拟不断变化的酸性pH条件下的成熟黑素体，我们测量RPT淀粉样蛋白形成动力学作为溶液pH值的函数。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色氨酸（Trp 423）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。 我们发现，Trp 423是非常敏感的可溶性和纤维状RPT构象与光谱特性（强度和平均波长）表现出不同的时间变化下的各种解决方案的条件下检查。特别地，光谱差异突出了不同的淀粉样蛋白形态，如通过TEM和硫磺素T活性可视化的。 此外，原纤维形成动力学是高度pH依赖性的，我们确定了RPT聚集的溶液pH的临界范围（4.5至5.5），表明特定羧酸侧链的质子化在促进淀粉样蛋白形成所需的结构重排中是至关重要的。在滴定至中性pH时，这些原纤维溶解，支持调节机制，由此如果释放并暴露于中性细胞溶质pH，RPT将采用无毒的可溶形式。 我们目前的努力集中在分子水平上的理解，残基（S）有助于RPT淀粉样蛋白形成的临界pH制度。