TRANSPORT FUNCTION OF THE MELANOGENIC P PROTEIN

眼皮肤白化病（OCA）的研究已经确定了两种类型的 OCA，酪氨酸酶突变（I型OCA）， 负责黑色素产生的第一步， OCA的常见形式，II型或OCA 2。 OCA 2是由于 染色体15 q11-q13基因，P，编码一个假定的黑素细胞特异性 蛋白 虽然，P不直接参与酶促反应， 黑色素的产生，从结构分析来看，P 蛋白质序列可能有12个跨膜结构域， 代表了一类新的转运蛋白。 我们认为P可以 负责传递黑色素的底物或辅因子 生物合成，如酪氨酸或L-DOPA（3，4-二羟基苯丙氨酸）。 在 在这个试点方案中，我们将研究P蛋白的功能， 细胞内定位，并确定是否差异表达的 P蛋白与不同程度的色素减退有关 在一些病人身上看到。 由于黑素细胞的亚细胞分级是 这是困难的，并且经常导致黑素体的交叉污染。 与其他细胞器（例如溶酶体）的组分，我们将使用 黑素细胞[正常（Melan-A），P-缺乏（Melan-P），和酪氨酸酶- 在全细胞免疫细胞化学分析中， 多克隆P抗体。 用黑素体和 亚细胞器特异性抗体将用于直接 确定P蛋白的细胞内定位。 为了 直接测定P蛋白的功能， 黑色素的氨基酸前体和辅因子的分析 将对Melan-A、Melan-P和Melan-C进行生物合成 用人P cDNA稳定转染的黑素细胞。 虽然P OCA 2中的突变是隐性的，Prader中P的单等位基因缺失， Willi和Angelman综合征与轻度至中度 在某些但不是所有情况下色素减退。 我们建议一个发起人 多态性导致P的差异表达，因此， 在这些患者中观察到不同的色素减退。 这将受到考验 通过克隆完整的P启动子和鉴定 受影响和正常个体的多态性。 成效为何 启动子多态性将在表达构建体中建模 利用荧光素酶作为报告基因。 我们的长期目标是 理解P函数是小说的理性设计 用于治疗患有OCA 2和P-gp的患者的药理学药剂 Prader-Willi和Angelman缺失型色素减退患者 综合征

Studies of oculocutaneous albinism (OCA) have identified two types of OCA, those with mutations in tyrosinase (Type I OCA), the enzyme responsible for the first step in melanin production, and the most common form of OCA, type II or OCA2. OCA2 results from defects in a chromosome 15q11-q13 gene, P, encoding a putative melanocyte-specific protein. Although, P is not directly involved in the enzymatic production of melanin, it appears from structural analysis that the P protein sequence likely has 12 membrane-spanning domains and may represent a novel class of transporter proteins. We suggest that P may be responsible for delivery of substrate or cofactors for melanin biosynthesis, such tyrosine or L-DOPA (3,4-dihydroxyphenylalanine). In this pilot proposal, we will examine the function of the P protein, its intracellular localization, and determine if differential expression of the P protein is associated with the variable degree of hypopigmentation seen in some patients. As subcellular fractionation of melanocytes is difficult and often results in cross-contamination of melanosomal fractions with other organelles (e.g. lysosomes), we will use melanocytes [normal (Melan-A), P-deficient (Melan-P), and tyrosinase- deficient (Melan-C)] in whole cell immunocytochemical analyses with a polyclonal P antibody. Double-labeling with melanosomal- and subcellular organelle-specific antibodies will be utilized to directly determine the intracellular localization of the P protein. In order to directly assay the function ascribed to the P protein, transport analyses with amino acid precursors and cofactors of melanin biosynthesis will be conducted on Melan-A, Melan-P, and Melan-C melanocytes stable-transfected with the human P cDNA. Although P mutations in OCA2 are recessive, single allele deletions of P in Prader- Willi and Angelman syndrome are associated with mild to moderate hypopigmentation in some but not all cases. We suggest that a promoter polymorphism results in differential expression of P and hence, the variable hypopigmentation seen in these patients. This will be tested by cloning of the complete P promoter and identification of polymorphisms in affected and normal individuals. The effects of these promoter polymorphisms will be modeled in expression constructs utilizing luciferase as a reporter gene. Our long term goal in the understanding of P function is the rational design of novel pharmacological agents for the treatment of patients with OCA2 and P- deletion patients with hypopigmentation in Prader-Willi and Angelman syndromes.