Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL

Susan M Mitchell,Morven Graham,Ralf M Leonhardt,Xinran Liu

doi:10.1038/s41598-021-87259-y

Susan M Mitchell, Morven Graham + Show 2 more

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https://doi.org/10.1038/s41598-021-87259-y

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Abstract

The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

Highlights

PMEL is a pigment cell protein expressed in melanocytes and retinal pigment epithelium[1]
Prior work has demonstrated that the 21 amino acid long regulatory N-terminal fragment (NTF) region within PMEL is structurally sensitive to mutations[44]
In order to reduce broad structural effects on PMEL folding as much as possible and to identify individual amino acids that are functionally critical for fibril formation, we constructed a set of point mutants, in which individual residues were substituted with alanine or glycine

Summary

Introduction

PMEL is a pigment cell protein expressed in melanocytes and retinal pigment epithelium[1]. P1 is rapidly exported from the ER and undergoes extensive O-glycosylation, in the repeat (RPT) domain, as well as maturation of various N-glycans in the Golgi[19,20] This gives rise to a ~ 120 kDa form of PMEL named P2, which is subsequently cleaved by proprotein convertases[21] during passage through secretory c ompartments[22,23]. Amyloid formed in vitro by artificial unglycosylated RPT domain fragments dissolves at neutral p H37. This is non-physiological, because pH neutralization during melanosome development[38,39,40] is required for tyrosinase to be enzymatically active and produce melanin[41,42]. Acidic pH-addicted RPT fibrils would be predicted to fall apart in vivo in the melanosome right in the moment when they are most needed, at the time when the pigment is beginning to be synthesized

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