Mechanisms of Functional Amyloid Formation

功能性淀粉样蛋白形成机制

• 批准号: 10253830
• 负责人: Jennifer Lee
• 金额: $ 8.91万
• 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/10253830
• 关键词: Address; Adopted; Alternative Splicing; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Architecture; Bacteria; Benign; Bioinformatics; Biological; Deposition; Development; Disease; Event; Fluorescence; Genes; Goals; Human; In Vitro; Individual; Kinetics; Mammals; Melanins; Melanosomes; Microbial Biofilms; Modification; Operon; Organelles; Organism; Parkinson Disease; Pathologic; Phylogenetic Analysis; Pigments; Process; Protein Isoforms; Protein Precursors; Proteins; Rod; Role; SILV gene; Seeds; Series; Structure; Testing; Transmission Electron Microscopy; Tryptophan; Work; amyloid formation; amyloid structure; cytotoxic; human disease; in vivo; insight; member; polypeptide; prevent

In our work, we have studied a crucial fibril forming domain termed the repeat domain (RPT, residues 315-444) derived from the human functional amyloid, Pmel17, to gain insights into what may differentiate functional from pathological amyloid. Pmel17 is a transmembrane precursor protein that is proteolytically processed to form intralumenal fibrils in melanosomes upon which melanin is deposited. Pmel17 is highly regulated in vivo, undergoing a series of post-translational and proteolytic modifications whereby the timing and sequence of these events permit amyloid formation. RPT is essential for the amyloid structures observed in melanosomes. Fibrils are formed during the early stages of melanosome development and once formed are responsible for the deposition of the pigment melanin. Since melanin precursors are cytotoxic, sequestering their synthesis on fibrils prevents potential detriment to the organelle. During this review period, we investigated the interplay between two biologically-relevant isoforms of the RPT domain. We sought to understand how the two isoforms might influence each other during amyloid formation, and specifically test whether the less abundant isoform (sRPT) serves to nucleate amyloid formation of the more abundant isoform (lRPT). Using intrinsic tryptophan fluorescence, we show that sRPT fibrils cross-seed and accelerated the rate of lRPT aggregation in vitro. Moreover, the macroscopic structure of sRPT fibrils (twisted protofilaments) is propagated to lRPT, which in the absence of sRPT seeds adopts a rod-like architecture as determined by transmission electron microscopy. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a nucleator of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

在我们的工作中，我们研究了源自人类功能性淀粉样蛋白 Pmel17 的一个关键原纤维形成结构域，称为重复结构域（RPT，残基 315-444），以深入了解功能性淀粉样蛋白与病理性淀粉样蛋白的区别。 Pmel17 是一种跨膜前体蛋白，经过蛋白水解加工，在黑素体中形成腔内原纤维，黑色素沉积在其上。 Pmel17 在体内受到高度调控，经历一系列翻译后和蛋白水解修饰，这些事件的时间和顺序允许淀粉样蛋白的形成。 RPT 对于在黑素体中观察到的淀粉样蛋白结构至关重要。原纤维在黑素体发育的早期阶段形成，一旦形成就负责黑色素的沉积。由于黑色素前体具有细胞毒性，将其合成隔离在原纤维上可以防止对细胞器的潜在损害。 在此审查期间，我们研究了 RPT 结构域的两种生物学相关亚型之间的相互作用。我们试图了解这两种同工型在淀粉样蛋白形成过程中如何相互影响，并专门测试较不丰富的同工型 (sRPT) 是否有助于使较丰富的同工型 (lRPT) 的淀粉样蛋白形成成核。利用内在色氨酸荧光，我们发现 sRPT 原纤维交叉种子并加速了体外 lRPT 聚集的速度。此外，sRPT原纤维（扭曲的原丝）的宏观结构被传播到lRPT，在没有sRPT种子的情况下，lRPT采用由透射电子显微镜确定的棒状结构。有趣的是，对其他哺乳动物的 PMEL17 同源物的生物信息学分析发现，长和短 RPT 亚型在该系统发育组的成员中是保守的。总的来说，我们的结果表明，RPT 的短亚型是 PMEL17 功能性淀粉样蛋白形成的成核剂，反映了细菌功能性淀粉样蛋白在生物膜形成过程中如何组装。虽然细菌通过使用同一操纵子内的单个基因来调节淀粉样蛋白的组装，但我们认为高等生物体中功能性淀粉样蛋白形成的调节可以通过选择性剪接来完成。