ON THE ROLE OF THE HPS GENE PRODUCT IN MELANOCYTES

Hermansky-Pudlak综合征（HPS）是一种先天性的、潜在致命性的多系统疾病，表现为眼皮肤白化病、轻度至重度出血性素质和神经样沉积病。这些特征是由一组特定细胞器（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/残体）的畸变引起的。HPS是一种常染色体隐性遗传病，在美国比较流行，在波多黎各比较常见（1:1800患病）。据推测，HPS中受影响的单个基因调节了三种特定细胞器的生物合成和/或维持的共同特征。通常，这些细胞器或产生或接受来自高尔基体的特定糖蛋白。因此，假设HPS基因产物调节从反高尔基网络到不同靶细胞器的选择性运输。HPS的基因最近被克隆出来了。它编码的氨基酸序列与任何已知的蛋白质没有显著的同源性。因此，这种分子似乎是一种独特的蛋白质。HPS基因产物确实包含提示信号序列的基序、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/核内体运输基序。因此，假设它是在内质网合成的糖蛋白，并被运输到所讨论的特定细胞器的限制膜上。我们建议利用黑素细胞系统来确定HPS基因产物的最终定位位点及其合成方式。此外，我们将研究其在黑色素化过程中的作用，并确定其在选择性运输功能中必须用于融合/对接过程的候选结合伙伴。具体来说，培养的人类黑素细胞将被评估用于免疫细胞化学定位和HPS基因产物的生物合成分析，使用一组针对HPS基因产物产生的抗体。此外，将转染反义HPS cDNA的黑色素瘤细胞和HPS患者培养的黑素细胞分别作为人工和自然敲除模型，分析各种黑素细胞特异性基因产物的运输同时发生的变化。此外，将HPS黑素细胞转染sense HPS cDNA，试图纠正形态/易位缺陷。最后，免疫沉淀/免疫印迹实验以及酵母双杂交系统将被用来鉴定与HPS基因产物结合的蛋白质，作为在靶向过程中合作的候选分子。这些研究将有助于我们对细胞运输的一般知识和HPS的具体病理生理学。

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.