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. 1. RPT Amyloid Formation Is pH Dependent We have studied the repeat domain (RPT, residues 315-444) as a model system of conformational change from soluble and unstructured monomer to aggregated, beta-sheet-containing fibrils. 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 pH and protein structure are linked in vivo, we studied the local and macroscopic RPT conformation as a function of pH in detail. Since Trp emission is highly sensitive to solvent polarity, local conformational changes, and protein-protein interactions, we exploited 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 suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. The high responsiveness of W423 during RPT aggregation points towards a key role for the C-terminal region in fibril assembly. To investigate a direct correlation between changes in melanosomal pH and formation of RPT fibrils, a pH titration assay was performed. Preformed RPT aggregates (pH 4.0) were titrated to pH 7.0. At pH 5.0, small, highly curved aggregates change into long striated fibrils, reminiscent of the fibril transition observed in stage I and II melanosomes. Further neutralization to pH 7.0 resulted in complete disassembly of RPT fibrils. This unique aggregation/disaggregation process is in contrast to disease-related amyloids, which are notorious for resisting the harshest treatments. We propose in the highly acidic melanosome (stage I), protein aggregation is initiated with fibril elongation occurring only after the compartment solution reaches an optimized pH 5 (stage II). Upon protonation of specific Glu residues, the electrostatic charge repulsion within the polypeptide chain reduces, thereby leading to formation of compact structures that promote key interactions required for fibril formation. In addition, our observation that RPT will readily aggregate (approx. 2 microM) at the optimized pH (5.0) could suggest that only fibrils are stabilized in lieu of potentially toxic oligomers. While our data show that fibrils would dissolve in the near neutral conditions found in stage III and IV melanosomes, it is unclear whether upon melanin deposition, the polymeric material could sequester the fibrils from solution and hence protect them from dissolution. Nevertheless, if released and exposed to the neutral environments outside the melanosome, fibrils will readily disintegrate and thus maintain their benign nature. 2. Probing RPT Fibril Disassembly To investigate fibril disassembly, we employed atomic force microscopy (AFM) and nuclear magnetic resonance (NMR) spectroscopy as ultrastructural and molecular probes, respectively. Specifically, we asked whether intermediates associated with disease-related amyloids are circumvented during dissolution. To monitor fibril disassembly, preformed RPT amyloid was deposited on mica and visualized by AFM under wet buffer conditions. At pH 5.0, long, straight and unbranched fibrils were observed, reminiscent to those seen by TEM. Upon washing these fibrils with pH 6.5 buffer, the fibrils begin to dissolve. Real-time monitoring reveals fragmentation of large fibrils followed by complete disappearance of smaller fragments on the order of minutes. To obtain residue specific insight, isotopically labeled fibrillar RPT was prepared for NMR spectroscopy. At pH 5.0, no backbone amide resonances were observed for residues 378-444, suggesting this region contains the amyloidogenic core. This is consistent with our assertion that the C-terminal region is important for fibril formation. Dissolution kinetics and spectra data showed no evidence of stable intermediates. The absence of intermediates also was verified by size exclusion chromatography. Furthermore, individual Glu backbone amide resonances exhibited similar kinetic trends, suggesting fibril unfolding is a global event involving many deprotonation events.
| Jiang, Zhiping; Lee, Jennifer C (2014) Lysophospholipid-containing membranes modulate the fibril formation of the repeat domain of a human functional amyloid, pmel17. J Mol Biol 426:4074-4086 |
| McGlinchey, Ryan P; Jiang, Zhiping; Lee, Jennifer C (2014) Molecular origin of pH-dependent fibril formation of a functional amyloid. Chembiochem 15:1569-72 |
| McGlinchey, Ryan P; Yap, Thai Leong; Lee, Jennifer C (2011) The yin and yang of amyloid: insights from ?-synuclein and repeat domain of Pmel17. Phys Chem Chem Phys 13:20066-75 |
| McGlinchey, Ryan P; Gruschus, James M; Nagy, Attila et al. (2011) Probing fibril dissolution of the repeat domain of a functional amyloid, Pmel17, on the microscopic and residue level. Biochemistry 50:10567-9 |
| Pfefferkorn, Candace M; McGlinchey, Ryan P; Lee, Jennifer C (2010) Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17. Proc Natl Acad Sci U S A 107:21447-52 |
