Mechanisms of Functional Amyloid Formation

Pmel17原纤维作为人体皮肤和眼睛黑色素沉积所需的结构支架。黑色素是在黑素体中合成的，黑素体是与内体和溶酶体相关的细胞器，并储存在负责色素沉着的黑素细胞中。虽然黑素体成熟过程涉及四个不同的阶段，并通过透射电子显微镜（TEM）在超微结构水平上进行了详细的表征，但在每个阶段中，腔内Pmel17原纤维的分子性质尚不清楚。此外，哪些多肽结构域单独或部分构成Pmel17细丝的淀粉样蛋白核心仍有待确定。

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.