The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

McGlinchey, Ryan P.; Shewmaker, Frank; McPhie, Peter; Monterroso, Begoña; Thurber, Kent R.; Wickner, Reed B.

doi:10.1073/pnas.0906509106

Cited by 133 publications

(167 citation statements)

References 55 publications

(66 reference statements)

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“…Specifically, aggregation kinetics are accelerated from pH 5.5 (20-30 h) to 4.0 (<1 h). We find that fibrils are formed only below pH 6.0, consistent with previous observation that RPT fibrils will dissolve in near neutral and basic solutions (24).…”

Section: Resultssupporting

confidence: 79%

“…RPT was expressed and purified as previously described with the following modification (24). Lysis buffer contained 8 M guanidinium hydrochloride, 100 mM NaCl, 100 mM K 2 HPO 4 , pH 7.5, and 10 mM imidazole.…”

Section: Methodsmentioning

confidence: 99%

“…Though there is no current consensus as to which polypeptide domain solely or partly constitutes the Pmel17 amyloid core, recent results showed that both isolated luminal regions, RPT, residues 315-444 ( Fig. 1) (24) and the polycystic kidney disease-1 like domain, residues 201-314 (25) are sufficient for amyloid formation in vitro. While both domains are important for fibril formation in vivo (26), we choose RPTas a model system to study aggregation kinetics because prior observation revealed that RPT fibril stability is particularly pH sensitive (24).…”

mentioning

confidence: 99%

“…1) (24) and the polycystic kidney disease-1 like domain, residues 201-314 (25) are sufficient for amyloid formation in vitro. While both domains are important for fibril formation in vivo (26), we choose RPTas a model system to study aggregation kinetics because prior observation revealed that RPT fibril stability is particularly pH sensitive (24). Further, the inability of a closely related, melanosomal glycoprotein, nonmetastatic melanoma protein b, which lacks a RPT domain, to form fibers also points to the role of this sequence in amyloid formation (27).…”

mentioning

confidence: 99%

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Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Pfefferkorn

McGlinchey²,

Lee³

2010

Proc. Natl. Acad. Sci. U.S.A.

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114

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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 β-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 AE 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.fibril | fluorescence | melanosome | tryptophan | melanin

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Section: Resultssupporting

confidence: 79%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Pfefferkorn

McGlinchey²,

Lee³

2010

Proc. Natl. Acad. Sci. U.S.A.

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114

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“…Using a variety of biophysical and spectroscopic approaches, we show that, contrary to a recent report (36), the unglycosylated RPT domain in isolation is very soluble, protease-sensitive, and unable to form amyloid fibrils in aqueous solution within the biologically relevant experimental time frames employed for the other Pmel17 domains. This finding suggests that the RPT domain likely plays a regulatory rather than a direct structural role in Pmel17 fibril formation.…”

mentioning

confidence: 51%

N-terminal Domains Elicit Formation of Functional Pmel17 Amyloid Fibrils

Watt

Niel

Fowler

et al. 2009

Journal of Biological Chemistry

103

154

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Pmel17 is a transmembrane protein that mediates the early steps in the formation of melanosomes, the subcellular organelles of melanocytes in which melanin pigments are synthesized and stored. In melanosome precursor organelles, proteolytic fragments of Pmel17 form insoluble, amyloid-like fibrils upon which melanins are deposited during melanosome maturation. The mechanism(s) by which Pmel17 becomes competent to form amyloid are not fully understood. To better understand how amyloid formation is regulated, we have defined the domains within Pmel17 that promote fibril formation in vitro. Using purified recombinant fragments of Pmel17, we show that two regions, an N-terminal domain of unknown structure and a downstream domain with homology to a polycystic kidney disease-1 repeat, efficiently form amyloid in vitro. Analyses of fibrils formed in melanocytes confirm that the polycystic kidney disease-1 domain forms at least part of the physiological amyloid core. Interestingly, this same domain is also required for the intracellular trafficking of Pmel17 to multivesicular compartments within which fibrils begin to form. Although a domain of imperfect repeats (RPT) is required for fibril formation in vivo and is a component of fibrils in melanosomes, RPT is not necessary for fibril formation in vitro and in isolation is unable to adopt an amyloid fold in a physiologically relevant time frame. These data define the structural core of Pmel17 amyloid, imply that the RPT domain plays a regulatory role in timing amyloid conversion, and suggest that fibril formation might be physically linked with multivesicular body sorting.

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Cutaneous Amyloidoses

Schreml

2016

Rook's Textbook of Dermatology, Ninth Edition

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Amyloids are common protein aggregates in nature. Some amyloids fulfil important biological tasks while others are known to cause diseases. Despite the fact that the ultrastructure of amyloid is highly conserved, the mechanism of amyloidogenesis remains a challenging research topic. In humans, amyloidoses can develop primarily in the skin, or systemic amyloidoses can lead to secondary cutaneous involvement. An accurate diagnostic procedure is crucial for planning therapy in this heterogeneous group of diseases. This chapter gives an overview of the different kinds of cutaneous amyloidoses as well as on diagnostic and therapeutic approaches. Furthermore, the contrast between functional and disease‐causing amyloid is discussed.

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The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

Cited by 133 publications

References 55 publications

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17

N-terminal Domains Elicit Formation of Functional Pmel17 Amyloid Fibrils

Cutaneous Amyloidoses

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