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 315-444) 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. Most recently, our mutational study showed that a single C-terminal glutamic acid, E422, is predominantly responsible for this pH behavior; neutralization of the negative charge at E422 accelerates fibril formation and increases fibril stability by a full pH unit. This highly reversible aggregation/disaggregation process under physiological pH is a unique property of RPT and contrasts those exhibited by disease-related amyloids, which only upon the harshest treatments will disassemble, e.g. chemical denaturants and non-physiological pH. We speculate that this is a potential way for keeping these amyloids benign if they were to escape melanosomes into the cytosol. Moreover, it may be plausible to recycle amyloid fibrils via this mechanism. While this is a compelling hypothesis, there is no current data supporting fibril dissolution in vivo and other domains may be involved. Studies have shown that Pmel17 trafficking and its subsequent proteolytic processing in melanosomes is important for melanin formation; however, the precise series of events and players involved in initiating and propagating fibril formation remain to be defined. Electron tomographic analyses of early stage melanosomes suggest that fibril formation is initiated on intralumenal membrane vesicles (ILVs). In another study, we sought to determine the effects of membrane lipids on RPT fibril formation, including vesicles and micelles formed from phospholipids and lysophospholipids (lysolipids), respectively. Lysolipids are particularly interesting due to their high content in melanosomal membranes (> 10%) as compared to plasma membranes (< 2%). With a combination of biophysical techniques, including circular dichroism and tryptophan fluorescence spectroscopy, dynamic light scattering as well as transmission electron microscopy, mechanistic insights were gained for the modulation of RPT amyloid formation by two specific lysolipids, negatively-charged lysophosphatidylglycerol (LPG) and zwitterionic lysophosphatidylcholine (LPC). Negatively charged LPG has dual and opposing effects on RPT aggregation: monomers accelerate whereas micelles retard kinetics. For the zwitterionic LPC, both monomers and micelles stimulate fibril formation, with micelles exerting a stronger effect. Lysolipids are structurally closer to surfactants than to phospholipids. Lysolipids are inverted cone-shaped due to their large headgroup and relatively small acyl chain, a structure that contributes to positive spontaneous curvature. As a result, it is not surprising to see a higher lysolipid content in melanosomes because these structural features of lysolipids would favor the formation of highly curved small ILVs and stabilize the ellipsoid-shaped melanosomes. Therefore, it supports the proposal that the fibril initiation occurs on the highly curved ILV membrane surface. Moreover, the preference of lysolipids for positive spontaneous curvature has been shown to promote membrane fusion by minimizing the bending energy. We conjecture that a high concentration of lysolipids is needed in ILVs to aid their merging with melanosomal membranes. Because the intermediate species (oligomers) en-route to fibrils are proposed to be the most potent cytotoxic agents, one way for melanosomes to cope with the adverse effect of amyloid formation is through the spatial and temporal regulation of aggregation. By using different lysolipids and their relative distribution and concentrations on ILVs, both the location and the speed of fibrillation can be controlled. With faster aggregation kinetics, over-population of oligomers may also be circumvented as well as the prompt sequestration of melanin and its associated toxic intermediates on the fibrils may be achieved. Taken together, our data suggest that lysolipids may play a key role in modulating Pmel17 fibril formation and possibly be involved in melanosome maturation.
| McGlinchey, Ryan P; Jiang, Zhiping; Lee, Jennifer C (2014) Molecular origin of pH-dependent fibril formation of a functional amyloid. Chembiochem 15:1569-72 |
| 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; 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 |
