ON THE ROLE OF THE HPS GENE PRODUCT IN MELANOCYTES

Hermansky-Pudlak综合征（HPS）是一种先天性、潜在致命的多系统疾病，表现为眼皮肤白化病、轻度至重度出血素质和蜡样物质沉积病。 这些特征是由一组特定的细胞器中的畸变引起的，（即，黑素细胞的色素颗粒、血小板的致密体和网状细胞的溶酶体/残余体）。 HPS是一种常染色体隐性遗传疾病，在美国较为普遍，在波多黎各相对常见（1：1800患病）。 在HPS pupulum中受影响的单个基因调节所涉及的三种类型的特定细胞器的生物合成和/或维持中的共同特征。 通常，这些细胞器起源于高尔基体和/或从高尔基体接收特定的糖蛋白。 因此，假设HPS基因产物调节从transGolgi网络到不同靶细胞器的选择性运输。 HPS的基因最近已被克隆。 其编码的氨基酸序列与已知的蛋白质没有明显的同源性。 因此，这种分子似乎是一种独特的蛋白质。HPS基因产物确实含有提示信号序列、跨膜结构域、糖基化位点、富含半胱氨酸的区域和几个溶酶体/内体运输基序的基序。 因此，它被假设为在RER合成的糖蛋白，并被运输到所讨论的特定细胞器的限制膜。我们建议使用黑素细胞系统确认HPS基因产物的最终定位位点及其合成模式。 此外，我们将调查它在黑化中的作用，并确定一个候选的结合伙伴，它必须使用的融合/对接过程中，其选择性贩运功能。 具体地，将使用针对HPS基因产物产生的一组抗体，评估培养的人黑素细胞的HPS基因产物的免疫细胞化学定位和生物合成分析。 此外，用反义HPS cDNA转染的黑色素瘤细胞和从HPS患者培养的黑色素细胞将分别用作人工和天然敲除模型，以分析各种黑色素细胞特异性基因产物运输的同时改变。 此外，HPS黑素细胞将用有义HPS cDNA转染，以试图纠正形态学/易位缺陷。 最后，免疫沉淀/免疫印迹实验沿着酵母双杂交系统将用于鉴定结合HPS基因产物的蛋白质作为在靶向过程中协同作用的候选分子。 这些研究将有助于我们的知识，细胞贩运的一般和病理生理学的HPS的具体。

The Hermansky-Pudlak Syndrome (HPS) is a congenital, potentially fatal, multi-system disorder presenting with oculocutaneous albinism, a mild to severe bleeding diathesis, and ceroid storage disease. These characteristics result from aberrations in a specific group of cellular organelles, (i.e., the pigment granule of the melanocyte, the dense bodies of the platelet, and the lysosome/residual body of the reticular cell respectively). HPS is an autosomal recessive disease that is somewhat prevalent in the United States and relatively frequent in Puerto Rico (1:1800 affected). The single gene affected in HPS putatively regulates a common feature in the biosynthesis and/or maintenance of the three types of specific cellular organelles involved. In common, these organelles either originate and/or receive specific glycoproteins from the Golgi apparatus. Thus it is hypothesized that the HPS gene product regulates selective trafficking from the trans Golgi network to distinct target organelles. The gene for HPS has recently been cloned. The deduced amino acid sequence it encodes has no significant homologies with any known protein. Therefore, this molecule appears to be a unique protein. The HPS gene product does contain motifs suggestive of a signal sequence, transmembrane domains, glycosylation sites, cysteine rich areas, and several lysosome/endosome trafficking motifs. Thus it is hypothesized to be a glycoprotein synthesized at the RER and trafficked to the limiting membranes of the specific organelles discussed. We propose to confirm the ultimate site of localization of the HPS gene product and its mode of synthesis using the melanocyte system. In addition, we will investigate its role in melanization and identify a candidate binding partner it must use for the fusion/docking process in its selective trafficking function. Specifically, cultured human melanocytes will be assessed for the immunocytochemical localization and biosynthesis analysis of the HPS gene product using a panel of antibodies generated against the HPS gene product. In addition, melanoma cells transfected with the anti-sense HPS cDNA and melanocytes cultured from patients with HPS will be used as artificial and natural knock-out models respectively to analyze the concurrent alteration in trafficking of various melanocyte specific gene products. In addition, HPS melanocytes will be transfected with the sense HPS cDNA in an attempt to correct the morphological/translocation defect. Finally, immunoprecipitation/immunoblotting experiments along with the Yeast Two-Hybrid system will be utilized to identify proteins that bind to the HPS gene product as candidate molecules that co-operate in the targeting process. These studies will contribute to our knowledge of cellular trafficking in general and the pathophysiology of HPS in specific.