Role of PMEL 17 in melanosome biogenesis

描述（由申请人提供）：本提案的总体目标是了解Pmel17的跨膜结构域（TMD）如何调节其对黑素小体的分选及其形成纤维状黑素小体基质的能力，以及该结构域的突变如何导致色素沉着缺陷背后的黑素小体功能障碍。黑色素小体是色素细胞特有的细胞器，黑色素在其中合成和储存，保护皮肤和眼睛免受紫外线辐射。在黑素体中，黑色素沉积在纤维样淀粉样基质上，其中高度保守的色素细胞特异性蛋白Pmel17是主要成分。为了形成原纤维，Pmel17 -像几种病理性淀粉样蛋白前体一样-必须首先在内体中间体中分选到腔内囊泡（ILVs），但负责这种分选的机制尚不清楚。在异常长的Pmel17 TMD中自然发生的突变导致模式生物色素沉着降低；尽管人类Pmel17的类似突变尚未在色素疾病中发现，但鉴于脊椎动物Pmel17同源物之间的高度同源性以及与广泛物种中Pmel17突变相关的相似表型，它们可能存在。与这些突变相关的低色素沉着表型表明，TMD对Pmel17的功能至关重要。这一提议提出了Pmel17 TMD介导脂质筏的分裂，并且这种分裂是Pmel17向ILVs分选并随后形成原纤维所必需的，因为长TMD倾向于在脂质筏中分裂，ILVs富含脂质筏成分。这一假设将以以下具体目标来解决：

DESCRIPTION (provided by applicant): The overall goals of this proposal are to understand how the transmembrane domain (TMD) of Pmel17 regulates its sorting to melanosomes and its ability to form the fibrillar melanosome matrix, and how mutations in this domain result in the melanosome dysfunction underlying pigmentation defects. Melanosomes are pigment-cell specific organelles in which melanins, which protect the skin and eyes from UV radiation, are synthesized and stored. Within melanosomes, melanins are deposited on a fibrillar amyloid-like matrix of which the highly conserved pigment cell-specific protein Pmel17 is the main component. To form fibrils, Pmel17 - like several pathological amyloid precursors - must first be sorted within endosomal intermediates to intralumenal vesicles (ILVs), but the mechanisms responsible for this sorting are unclear. Natural occurring mutations in the unusually long Pmel17 TMD cause hypopigmentation in model organisms; although similar mutations in human Pmel17 have not been identified in pigmentary diseases, they likely exist given the high degree of homology among vertebrate Pmel17 orthologues and the similar phenotypes associated with Pmel17 mutations in a broad range of species. The hypopigmentation phenotypes associated with these mutations suggest that the TMD is critical to Pmel17 function. This proposal poses the hypothesis that the Pmel17 TMD mediates partitioning into lipid rafts and that this partitioning is required for sorting of Pmel17 to ILVs for subsequent fibril formation because long TMDs tend to partition in lipid rafts and ILVs are enriched in lipid raft components. This hypothesis will be addressed with the following specific aims: 1) Determine if Pmel17 associates to lipid rafts using classical biochemical fractionation and test whether cholesterol depletion affects Pmel17 lipid raft association, sorting, and/or fibril formation. 2) Determine if mutations in the Pmel17 TMD affects sorting to ILVs and/or fibrillogenesis. The proposed work is relevant as it relates to understanding the mechanisms that control the targeting of Pmel17 to melanosomes and the formation of the amyloid-like matrix, essential to PrneM 7 function and melanosome integrity. The results may also be relevant to understanding how amyloid forms in neurodegenerative diseases.