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.

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。 我们已经开始研究重复域（RPT，残基315-444）和PMEL17的必需腔多肽区域，作为从可溶性和非结构性单体到聚集的β-折叠纤维的模型变化模型系统。为了模仿成熟黑色素体的酸性pH条件的变化，我们测量了RPT淀粉样蛋白形成动力学作为溶液pH的函数。 由于色氨酸的发射对溶剂极性，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色氨酸（TRP423）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，在所检查的各种溶液条件下，TRP423对具有光谱特性（强度和平均波长）的可溶性和原纤维RPT构型非常敏感。尤其是，光谱差异突出了TEM和硫非类硫素T活性可视化的不同淀粉样形态。 此外，原纤维形成动力学是高度依赖性的，我们确定了RPT聚集的关键溶液pH（4.5至5.5），这表明特定羧酸侧链的质子化对于促进淀粉样蛋白形成所需的结构重排至关重要。滴定到中性pH值后，这些原纤维溶解，支持一种调节机制，如果释放并暴露于中性的胞质pH值，RPT将采用一种无毒的可溶性形式。 我们目前的努力集中在分子水平上的理解上，残基有助于RPT淀粉样蛋白形成的关键pH状态。