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Pmel 17的跨膜结构域（TMD）如何调节其对黑素体的分选及其形成纤维状黑素体基质的能力，以及该结构域中的突变如何导致色素沉着缺陷背后的黑素体功能障碍。黑素体是色素细胞特有的细胞器，其中合成和储存保护皮肤和眼睛免受紫外线辐射的黑色素。在黑素体内，黑色素沉积在纤维状淀粉样基质上，其中高度保守的色素细胞特异性蛋白Pmel 17是主要成分。为了形成原纤维，Pmel 17-像几种病理性淀粉样蛋白前体-必须首先在内体中间体内分选为腔内小泡（ILV），但负责这种分选的机制尚不清楚。在异常长的Pmel 17 TMD中自然发生的突变导致模式生物中的色素减退;尽管在色素性疾病中尚未鉴定出人Pmel 17中的类似突变，但考虑到脊椎动物Pmel 17直系同源物之间的高度同源性以及与广泛物种中的Pmel 17突变相关的相似表型，它们可能存在。与这些突变相关的色素减退表型表明TMD对Pmel 17功能至关重要。该提议提出了这样的假设，即Pmel 17 TMD介导分配成脂筏，并且这种分配是Pmel 17分选成ILV以用于随后的原纤维形成所需的，因为长TMD倾向于分配在脂筏中，并且ILV富含脂筏组分。将以下列具体目标探讨这一假设： 1)使用经典的生物化学分级法确定Pmel 17是否与脂筏相关，并测试胆固醇消耗是否影响Pmel 17脂筏缔合、分选和/或原纤维形成。 2)确定Pmel 17 TMD中的突变是否影响分选至ILV和/或原纤维形成。 拟议的工作是相关的，因为它涉及到理解控制Pmel 17靶向黑素体和淀粉样基质形成的机制，这对PrneM 7功能和黑素体完整性至关重要。该结果也可能与了解神经退行性疾病中淀粉样蛋白的形成方式有关。

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.