The emerging concept of functional amyloids is challenging the way we view amyloids, which have been previously thought as either a cause or consequence of human diseases as in Alzheimers and Parkinsons. In our work, we have studied a crucial fibril forming domain termed the repeat domain (RPT, residues 315444) derived from the human functional amyloid, Pmel17, to gain insights into what may differentiate functional from pathological amyloid. Pmel17 is a transmembrane precursor protein that is proteolytically processed to form intralumenal fibrils in melanosomes upon which melanin is deposited. Pmel17 is highly regulated in vivo, undergoing a series of post-translational and proteolytic modifications whereby the timing and sequence of these events permit amyloid formation. RPT is essential for the amyloid structures observed in melanosomes. Fibrils are formed during the early stages of melanosome development and once formed are responsible for the deposition of the pigment melanin. Since melanin precursors are cytotoxic, sequestering their synthesis on fibrils prevents potential detriment to the organelle. A distinguishing feature that we have discovered is that not only does RPT form amyloid at a mildly acidic, melanosomal pH regime (4.5-5.5) but these fibrils completely dissolve at pH ≥6. This reversible polymerization behavior highly contrasts those exhibited by disease-related amyloids, which only upon the harshest treatments will disassemble, e.g. chemical denaturants and non-physiological pH. A potential biological implication for this observed disaggregation process is that if RPT filaments were to escape from the melanosome, they would dissolve under neutral cytosolic pH, and thus remain benign. While this is a compelling hypothesis, there is no current data supporting fibril dissolution in vivo and other domains may be involved. Nevertheless, our results support the requirement of the acidic melanosome pH for amyloid assembly where protonation of specific carboxylic acids promotes key interactions for RPT fibril formation by reducing either intra- or inter-molecular electrostatic repulsion. We have identified specific carboxylic acids (protonation sites) that are necessary for aggregation and assessed the role of hydrogen bonding in fibril formation by utilizing Ala- and Gln-mutants, respectively. Specifically, effects of mutations at residues, E404, E422, E425 and E430 on RPT aggregation kinetics and pH dependence of amyloid formation were studied. Protonation of the C-terminal glutamic acids is shown to be vital, likely through the inhibition of intra/intermolecular electrostatic repulsion. Particularly, both charge neutralization and hydrogen bonding play key roles at position E422, where the introduction of an amide (-NH2 vs. -OH) sidechain accelerates aggregation via the increase of hydrogen bonding capability. This is in strong agreement with the inhibitory effect of the Ala mutation where hydrogen bonding donor (-OH) and acceptor (C=O) are removed. However, the difference in residue size, i.e. sidechain packing, cannot be ruled out as a contributing factor in kinetics modulation. By comparison, hydrogen bonding and/or size are not as critical at E404 where Ala/Gln both stimulate aggregation. Mutations at both E425 and E430 have a similar negative effect on aggregation, prolonging fibril growth. Upon protonation, these residues influence the self-assembly process perhaps through the formation of local noncovalent interactions and thus, retarding aggregation. Only E422 mutants had a substantial impact where fibrils now form at pH 6.5. Consistently, dissolution experiments conducted on E422Q fibrils verified that it is more stable than WT fibrils and are resistant to disassembly up to pH 7. We note that E404A/Q occasionally aggregated at pH 6 suggesting that it may play an ancillary role. Taken together, our data suggest that residue 422 is the critical sidechain in controlling the pH sensitivity of RPT amyloid formation. From a structural perspective, we propose that E404 and E422 reside within the amyloid-forming region of RPT. Here, Glu sidechains are oriented with E404 positioned outside and E422 inside the filament. Having E422 sidechains within the filament core, suggests that upon protonation, both intra- and inter-sheet contacts are facilitated and essential in stabilizing filament structure. In the absence of a net charge, filaments can form at higher pH. The reduced aggregation rates associated with E422A indicate that either hydrogen bonding or size is involved in inter-sheet packing and stability. Protonation of the outwardly facing E404 would prevent intra-sheet electrostatic repulsion. The increased aggregation propensity associated with E404A also may suggest a role for sidechain interdigitation as the small methyl groups would allow tighter packing between filaments.
| 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 |
