Mechanisms of Functional Amyloid Formation

PMel17是人类皮肤和眼睛中的一种色素沉积所必需的蛋白质，它被运输到黑素小体并在其中进行蛋白质分解处理，在那里它形成纤维条纹，黑色素沉积在那里。黑色素是在黑素小体中合成的，黑素小体是与内小体和溶酶体相关的细胞器，黑色素储存在黑素细胞中，黑素细胞负责着色。尽管黑素小体的成熟过程包括四个不同的阶段，并在超微结构水平上用透射电子显微镜(TEM)进行了详细的表征，但在每个阶段中，腔内PMel17纤维的分子性质尚不清楚。此外，PMel17细丝的淀粉样蛋白核心是由哪个多肽结构域单独或部分构成的，目前还不清楚。 1.RPT淀粉样蛋白形成过程中谷氨酸残基的鉴定 一些研究表明，其中一个被蛋白质降解的PMel17片段被称为重复结构域(RPT，残基315-444)，对于观察到的黑素小体中的纤维结构是必不可少的。RPT主要氨基酸序列由10个不完全的13个残基重复组成，富含Pro、Ser、Thr和Glu。RPT含有16种羧酸，强调了它容易经历pH诱导的构象变化。由于黑素小体成熟与体内pH密切相关，我们详细研究了局部和宏观RPT构象随pH的变化。 在以前的工作中，使用唯一的固有的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，确定了纤维形成的关键pH区间(4.5-5.5)。W423在RPT聚集过程中的高响应性表明C-末端区域在纤维组装中起关键作用，这表明四个C-末端中至少有一个或多个参与了聚集所需的结构重排。为了描述特定的Glu残基的作用，我们正在研究Ala-和Gln-突变体的聚集动力学，以定位必要的质子化位置，并评估氢键在这一高度依赖于pH的淀粉样蛋白过程中的作用。利用硫代黄素T(THT)荧光建立了高通量分析方法。用W423和THT荧光法同时监测聚集动力学。用圆二色谱和透射电子显微镜分别验证了纤维二级结构和纤维形态的变化。我们的数据显示，E422是这一过程中的关键残基。基于这些结果的工作淀粉样蛋白模型正在研究中。 2.血脂对RPT聚集的影响 我们研究了脂质对RPT聚集的影响，以探索黑素小体内小泡和/或黑素小体膜是否可以启动和促进纤维形成。使用了由磷脂和溶质脂形成的膜模拟物、胶束和囊泡。我们发现溶脂的存在强烈地影响和加速了RPT的聚集，特别是溶血磷脂酰甘油(LPG)和溶血磷脂酰胆碱(LPC)，而磷脂的影响很小。当单体以最佳的LPG与蛋白质比例15-30刺激RPT淀粉样蛋白的形成时，LPG胶束稳定形成小的、蠕虫样的和硫代黄素T不活跃的聚集体。相反，LPC在所有考察的条件下都刺激RPT聚集。随着圆二色谱，一个二级结构的报告者，我们利用含有单一色氨酸的变体来获得蛋白质-脂类相互作用的特定位点信息。我们的数据表明，LPG选择性地结合到C-末端区域，导致部分α-螺旋结构。我们认为，这种由淀粉样蛋白核心残基形成的螺旋中间体是LPG调节RPT聚集的分子基础。有趣的是，LPC和RPT之间没有明显的相互作用，但脂质创造了促进淀粉样蛋白形成的溶液环境。虽然我们的数据不排除其他因素的参与，如膜上的蛋白质-蛋白质相互作用，我们认为特定的蛋白质-脂肪相互作用可以调节体内淀粉样蛋白的形成，因为黑素糖体富含溶质脂。

Pmel17, an essential protein for pigmentation in human skin and eyes, is trafficked to and proteolytic processed in the melanosome, where it forms fibrous striations to which melanin is deposited. 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 ill-defined. 1. Identification of Critical Glutamic Acid Residues in RPT Amyloid Formation Several studies have shown that one of these proteolyzed Pmel17 fragments, termed the repeat domain (RPT, residues 315-444) is essential for the fibrillar structures observed in melanosomes. 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 melanosome maturation is intimately linked to pH in vivo, we have studied the local and macroscopic RPT conformation as a function of pH in detail. In prior work, using 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. The high responsiveness of W423 during RPT aggregation points towards a key role for the C-terminal region in fibril assembly, suggesting the involvement of at least one or more of the four C-terminal Glu in the structural rearrangement necessary for aggregation. To delineate the role of specific Glu residues, we are studying the aggregation kinetics of Ala- and Gln-mutants to pinpoint essential protonation sites and to assess the role of hydrogen bonding in this highly pH dependent amyloid process. A high-throughput assay using thioflavin T (ThT) fluorescence has been implemented. Aggregation kinetics are monitored simultaneously by W423 and ThT fluorescence. Secondary structural and fibril morphological changes are verified by circular dichroism spectroscopy and transmission electron microscopy, respectively. Our data reveal that E422 is a critical residue during this process. A working amyloid model based on these results is under investigation. 2. Effect of Lipids on RPT Aggregation We have investigated the effect of lipids on RPT aggregation to explore whether intramelanosomal vesicles and/or melanosomal membrane can initiate and facilitate fibrillogenesis. Membrane mimics, micelles and vesicles, formed from phospholipids and lysolipids were employed. We find that RPT aggregation is strongly influenced and accelerated by the presence of lysolipids, particularly lysophosphatidylglycerol (LPG) and lysophosphatidylcholine (LPC) whereas phospholipids have minimal effects. While monomers stimulate RPT amyloid formation at an optimal LPG-to-protein ratio 15-30, LPG micelles stabilize formation of small, worm-like, and thioflavin T inactive aggregates. In contrast, LPC stimulates RPT aggregation under all conditions examined. Along with circular dichroism spectroscopy, a reporter of secondary structure, we exploited single-Trp containing variants to gain site-specific information on protein-lipid interaction. Our data suggest that LPG selectively binds to the C-terminal region, inducing a partial alpha-helical structure. We propose that this formation of a helical intermediate by its amyloidogenic core residues is the molecular basis for LPG modulated RPT aggregation. Interestingly, there are no apparent interactions between LPC and RPT and yet the lipid creates a solution environment that promotes amyloid formation. Although our data do not preclude the participation of other factors such as protein-protein interactions on the membrane, we suggest that specific protein-lipid interactions can regulate amyloid formation in vivo as melanosomes are enriched in lysolipids.