Mechanisms of Functional Amyloid Formation

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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.