The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis.
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ABSTRACT:
SUBMITTER: McGlinchey RP
PROVIDER: S-EPMC2728962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Pmel17 is a human amyloid involved in melanin synthesis. A fragment of Pmel17, the repeat domain (RPT) rich in glutamic acids, forms amyloid only at mildly acidic pH. Unlike pathological amyloids, these fibrils dissolve at neutral pH, supporting a reversible aggregation-disaggregation process. Here, we study RPT dissolution using atomic force microscopy and solution-state nuclear magnetic resonance spectroscopy. Our results reveal asymmetric fibril disassembly proceeding in the absence of intermediates. We suggest that fibril unfolding involves multiple deprotonation events resulting in electrostatic charge repulsion and filament dissolution.
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Project description:Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ?5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel ?-sheet architecture with two RPT molecules per layer, similar to amyloid of the A? peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.
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Project description:The pre-melanosomal protein (Pmel17) aggregates within melanosomes to form functional amyloid fibrils that facilitate melanin polymerization. The repeat domain (RPT) of Pmel17 fibrillates under strict acidic melanosomal pH. Alternative splicing results in a shortened repeat domain (sRPT), which also forms amyloid fibrils. Here, we explored the effects of pH and protein concentration on sRPT aggregation by monitoring the intrinsic fluorescence of the sole tryptophan at position 381 (381W). 381W emission properties revealed changes of local environment polarity for sRPT fibrils formed at different pH. At pH?4, fibrils formed rapidly with no lag phase. A high 381W intensity was observed with a slight blue shift (10?nm). These fibrils underwent further structural rearrangements at intermediate pH (5-6), mirroring that of melanosome maturation, which initiates at pH?4 and increases to near neutral pH. In contrast, typical sigmoidal kinetics were observed at pH?6 with slower rates and 381W exhibited quenched emission. Interestingly, biphasic kinetics were observed at pH?5 in a protein concentration-dependent manner. A large 381W blue shift (23?nm) was measured, indicating a more hydrophobic environment for fibrils made at pH?5. Consistent with 381W fluorescence, Raman spectroscopy revealed molecular level perturbations in sRPT fibrils that were not evident from circular dichroism, transmission electron microscopy, or limited proteolysis analysis. Finally, sRPT fibrils did not form at pH ?7 and preformed fibrils rapidly disaggregated under these solution conditions. Collectively, this work yields mechanistic insights into pH-dependent sRPT aggregation in the context of melanosome maturation.
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Project description:Pmel17 is a functional amyloidogenic protein whose fibrils act as scaffolds for pigment deposition in human skin and eyes. We have used the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system to study conformational changes from soluble unstructured monomers to ?-sheet-containing fibrils. Specifically, we report on the effects of solution pH (4 ? 7) mimicking pH conditions of melanosomes, acidic organelles where Pmel17 fibrils are formed. Local, secondary, and fibril structure were monitored via intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. We find that W423 is a highly sensitive probe of amyloid assembly with spectral features reflecting local conformational and fibril morphological changes. A critical pH regime (5 ± 0.5) was identified for fibril formation suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. Moreover, we demonstrate that RPT fibril morphology can be transformed directly by changing solution pH. Based on these results, we propose that intramelanosomal pH regulates Pmel17 amyloid formation and its subsequent dissolution in vivo.
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Project description:Amyloids are traditionally observed in the context of disease. However, there is growing momentum that these structures can serve a beneficial role where the amyloid carries out a specific function. These so called 'functional amyloids' have all the structural hallmarks of disease-associated amyloids, raising the question as to what differentiates a well-behaved benign amyloid from a lethally destructive one. Here, we review our work on the repeat domain (RPT) from Pmel17, an important functional amyloid involved in melanin biosynthesis. Particularly, we focused our attention on the unique reversible aggregation-disaggregation process of RPT that is controlled strictly by solution pH. This pH dependence of RPT amyloid formation functions as a switch to control fibril assembly and maintains the benign nature that is associated with functional amyloids.
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Project description:For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins ? 2-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.
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Project description:Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.
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Project description:Many tissues of the human body encounter hyperosmotic stress. The effect of extracellular osmotic changes on melanin production has not yet been elucidated. In this study, we determined that hyperosmotic stress induced by organic osmolytes results in reduced melanin production in human melanoma MNT-1 cells. Under hyperosmotic stress, few pigmented mature melanosomes were detected, but there was an increase in swollen vacuoles. These vacuoles were stained with an anti-M6PR antibody that recognizes late endosomal components and with anti-TA99 and anti-HMB45 antibodies, implying that melanosome formation was affected by hyperosmotic stress. Electron microscopic analysis revealed that the M6PR-positive swollen vacuoles were multi-layered and contained melanized granules, and they produced melanin when L-DOPA was applied, indicating that these vacuoles were still capable of producing melanin, but the inner conditions were not compatible with melanin production. The vacuolation phenomenon induced by hyperosmotic conditions disappeared with treatment with the PI3K activator 740 Y-P, indicating that the PI3K pathway is affected by hyperosmotic conditions and is responsible for the proper formation and maturation of melanosomes. The microarray analysis showed alterations of the vesicle organization and transport under hyperosmotic stress. Our findings suggest that melanogenesis could be regulated by physiological conditions, such as osmotic pressure.
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Project description:Amyloid fibrils have evolved from purely pathological materials implicated in neurodegenerative diseases to efficient templates for last-generation functional materials and nanotechnologies. Due to their high intrinsic stiffness and extreme aspect ratio, amyloid fibril hydrogels can serve as ideal building blocks for material design and synthesis. Yet, in these gels, stiffness is generally not paired by toughness, and their fragile nature hinders significantly their widespread application. Here we introduce an amyloid-assisted biosilicification process, which leads to the formation of silicified nanofibrils (fibril-silica core-shell nanofilaments) with stiffness up to and beyond ∼20 GPa, approaching the Young's moduli of many metal alloys and inorganic materials. The silica shell endows the silicified fibrils with large bending rigidity, reflected in hydrogels with elasticity three orders of magnitude beyond conventional amyloid fibril hydrogels. A constitutive theoretical model is proposed that, despite its simplicity, quantitatively interprets the nonmonotonic dependence of the gel elasticity upon the filaments bundling promoted by shear stresses. The application of these hybrid silica-amyloid hydrogels is demonstrated on the fabrication of mechanically stable aerogels generated via sequential solvent exchange, supercritical [Formula: see text] removal, and calcination of the amyloid core, leading to aerogels of specific surface area as high as 993 [Formula: see text]/g, among the highest values ever reported for aerogels. We finally show that the scope of amyloid hydrogels can be expanded considerably by generating double networks of amyloid and hydrophilic polymers, which combine excellent stiffness and toughness beyond those of each of the constitutive individual networks.
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