Mechanisms of Functional Amyloid Formation

Pmel17 原纤维充当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，这些阶段已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，但每个阶段中管腔内 Pmel17 原纤维的分子性质尚不清楚。 此外，哪个多肽结构域单独或部分构成Pmel17丝的淀粉样蛋白核心也仍有待确定。 我们已经开始研究重复结构域（RPT，残基 315-444），Pmel17 的一个必需的管腔多肽区域，作为模型系统。为了模拟成熟黑素体不断变化的酸性 pH 条件，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，因此我们利用唯一的内在色氨酸（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 对可溶性纤维状 RPT 构象极其敏感，其光谱特性（强度和平均波长）在所检查的各种溶液条件下表现出明显的时间变化。 TEM 和圆二色光谱等补充技术也分别用于表征纤维超微结构和二级结构含量。

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.