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类以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潜在的色素缺陷的黑素小体功能障碍。黑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的伤害。在黑素小体中，黑色素沉积在纤维状淀粉样基质上，高度保守的色素细胞特异性蛋白PMel17是其主要成分。为了形成纤维，PMel17--就像几种病理性淀粉样前体一样--必须首先在内体中间体到腔内小泡(ILV)内进行分选，但负责这种分选的机制尚不清楚。在模式生物中，异常长的Pmel17 TMD中自然发生的突变会导致色素减退；虽然人类Pmel17中的类似突变尚未在色素疾病中被发现，但它们可能存在，因为脊椎动物Pmel17同源基因之间高度同源性，以及与Pmel17突变相关的相似表型在广泛的物种中存在。与这些突变相关的色素减退表型表明，TMD对Pmel17的功能至关重要。这一建议提出了一种假设，即Pmel17 TMD介导了脂筏的分配，并且这种分配是将Pmel17分选到ILV以便随后形成纤维所必需的，因为长TMD倾向于在脂筏中分配，而ILV富含脂筏成分。这一假设将以下列具体目标加以阐述： 1)使用经典的生化分级法确定PMel17是否与脂筏相关，并测试胆固醇耗竭是否影响PMel17脂筏的结合、分类和/或纤维的形成。 2)确定Pmel17 TMD的突变是否影响ILV的分选和/或纤维形成。 拟议的工作是相关的，因为它涉及到了解控制PMel17靶向黑素小体和淀粉样基质形成的机制，这对PrneM 7的功能和黑素小体的完整性是必不可少的。这一结果也可能与理解神经退行性疾病中淀粉样蛋白的形成有关。

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.