Repeat Domains of Melanosome Matrix Protein Pmel17 Orthologs Form Amyloid Fibrils at the Acidic Melanosomal pH

Abstract

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 ϳ5.0). 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.Amyloid is a filamentous ␤-sheet-rich protein polymer usually associated with Alzheimer disease, Parkinson disease, type II diabetes, and other pathological conditions, but several functional amyloids have been identified. The external curli fibers of Escherichia coli are involved in biofilm formation and adhesion to host cells (1); the hydrophobin amyloids protect fungal cell surfaces (2), and the [Het-s] prion of Podospora anserina appears to help the host by facilitating heterokaryon incompatibility (3).Pmel17 is a protein important for melanin synthesis and deposition that forms intralumenal filaments in melanosomes, the endosome/lysosome-like organelle in which melanin synthesis takes place (reviewed in Refs. 4, 5). Pmel17 is made as a transmembrane protein, processed by a series of proteolytic steps and glycosylations and assembled into a filamentous form in stage II melanosomes. There, Pmel17 filaments facilitate melanization, perhaps acting as a template on which melanin is deposited or by adsorbing toxic by-products of the melanin biosynthetic process (reviewed in Ref. 6).The gene for Pmel17 was first identified by a single gene mouse mutant called Silver, in which the normal black coat color was lost as animals grew and aged (7). The mouse Pmel17 cDNA, mapping at the Silver locus, defined the protein with its single transmembrane region and repeat domain (8). Comparisons of orthologs from a number of vertebrate species have revealed the domain structure shown in supplemental Fig. 1 (6, 9). Human Pmel17 consists of (N-to C-terminal) a signal peptide (residues 1-23 in the human protein), an N-terminal domain, the polycystic kidney disease-like domain (PKD), 2 the repeat domain (RPT), the Kringle-like domain (KLD or KRG), the transmembrane domain, and the cytoplasmic C-terminal domain. Most parts of Pmel17 are well aligned acros...

Amyloid-based nanosensors and nanodevices

“…Briefly, the melanin precursor (MP) is activated by a tyrosinase (T) resulting in the activated melanin precursor (AMP), which interacts with the amyloidogenic sequence PmeI17 (PmeI) leading to mature melanin. [91][92][93] assembled to microtubes, has been explored for effectively deliver drugs. Using rhodamine as a model drug, it was shown that the FF microtubes released the drug in a steady-state profile, following first-order kinetics.…”

“…Here, the Pmel17 amyloid fibers, found in melanosomes, serve as a template for the polymerization of the pigment melanin. 92 Hence, Pmel17 strikes a remarkable balance, being a non-pathological functional amyloid in humans. Also, the peptide hormones corticotropin-releasing hormone (CRH), human prolactin (hPRL), and somatostatin, the growth hormone inhibitor hormone (GHIH), are other examples that transform at high concentrations into amyloid fibrils.…”

“…The amyloid sequence PmeI drives a fast amyloid formation process: mature melanin is formed with PmeI serving as a template that organizes the melanin precursors and accelerates the covalent polymerization of these molecules into the mature melanin molecule. [91][92][93] Amyloidogenic protein sequences have been suggested to play other physiological roles, namely involving fibrin and tissue-type plasminogen activator. Fibrin results from thrombin-mediated proteolysis of fibrinogen.…”

The Role of Glycosylation in the Control of Processing and Cellular Transport of the Functional Amyloid PMEL17

“…However, there is no evidence of amyloid accumulation either in skin samples or in skin keratinocytes when co-cultured with normal human melanocytes (JVC, unpublished data). One possible reason for this is that PMEL amyloid formation is sensitive to pH [7]. Thus, the neutral pH in the extracellular compartment most likely undermines PMEL amyloid fiber ability or if any amyloid is formed it is dissolved; while the intra organelle acidic pH (5.0) will favor amyloid formation.…”

“…Early reports suggested that two domains inside PMEL are contributors to fibril formation [5]. Today, unequivocal evidence recognizes that the so called repeat (RPT) domain is the only region capable to form amyloid fibrils in vivo and in vitro [6][7][8]. Unlike disease-associated amyloids, functional amyloids are the product of coordinated and regular cellular processes that ensure that amyloidogenesis does not result in cell damage or death [2;9].…”