Mechanisms of Functional 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性支架。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部分或全部由哪个多肽结构域组成还有待确定。 我们已经开始研究重复结构域(RPT，残基315-444)，它是PMel17的一个重要的管腔多肽区域，作为一个模型系统。为了模拟成熟黑素小体的酸性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，我们利用唯一的内源性色氨酸(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Trp423对可溶的和纤维状的RPT构象非常敏感，在所考察的各种溶液条件下，光谱性质(强度和平均波长)显示出明显的时间变化。另外，还采用了透射电子显微镜和圆二色谱等辅助技术对纤维超微结构和二级结构含量进行了表征。

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), an essential luminal polypeptide region of Pmel17, as a model system. 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. Complementary techniques, TEM and circular dichroism spectroscopy, also are employed to characterize fibrillar ultrastructures and secondary structural content, respectively.