TARGETING OF LYSOSOMAL ENZYMES IN DICTYOSTELIUM

我实验室的长期目标是确定 其调节细胞内溶酶体的生物合成和功能 含有水解酶的细胞器。 这些细胞器 通常具有降解细胞外和细胞内物质的功能， 与人类疾病有关 超过30种人类溶酶体遗传性疾病 已经描述过，许多是由于缺乏一个或多个 溶酶体酶 其他疾病涉及一般溶酶体功能障碍 (e.g. Chediak-Higashi综合征）。 溶酶体水解酶分泌到 外部环境也可能是炎症的部分原因， 疾病（包括关节炎）以及肿瘤转移。 前几 我们已经广泛地研究了生物合成，运输， 3种溶酶体酶的修饰和蛋白水解加工 简单的真核生物盘基网柄藻，一种适合于这些 研究，因为它可以很容易地在基因上操纵， 生物化学 这种生物体也缺乏靶向受体（man-6- 磷酸盐受体）用于在许多哺乳动物细胞中发现的溶酶体酶， 因此，它已成为一个有用的系统，以研究替代排序 机制等 我们现在建议使用重组DNA技术来确定 蛋白质结构域的位置是必要的，足以 将水解酶靶向至溶酶体。 这也将涉及网站 定向诱变以改变蛋白酶切割位点，以确定 需要溶酶体酶前体的蛋白水解加工， 正确的排序。 我们还将使用单克隆抗体来表征 溶酶体膜蛋白的生物合成和转运。 最后我们 建议从生物化学和遗传学的角度分析诱导的过程， 溶酶体酶分泌，这将包括解剖信号 负责分泌的转导途径，以及试图 定义我们拥有的突变体集合中的生化缺陷 分离出的酶分泌不足。 这些综合办法将 增加我们对溶酶体生物合成机制的了解 并在所有细胞中发挥作用。

The long term goal of my laboratory is to define the molecular mechanisms which regulate the biosynthesis and function of lysosomes, intracellular organelles which contain hydrolytic enzymes. These organelles which normally function to degrade extracellular and intracellular material have been implicated in human disease. Over 30 human lysosomal genetic diseases have been described, many resulting from the deficiency of one or more lysosomal enzyme. Other diseases involve general lysosomal dysfunction (e.g. Chediak-Higashi Syndrome). Secretion of lysosomal hydrolases into the external milieu also may be partially responsible for inflammatory diseases (including arthritis) as well as tumor metastasis. In previous studies we have extensively characterized the biosynthesis, transport, modification and proteolytic processing of 3 lysosomal enzymes in the simple eukaryote Dictyostelium discoideum, an organism amenable to these studies because of the ease in which it can be manipulated genetically and biochemically. This organism also lacks the targeting receptors (man-6- phosphate receptors) for lysosomal enzymes found in many mammalian cells, thus it has been a useful system to investigate alternative sorting mechanisms. We now propose to use recombinant DNA techniques to determine the location of the protein domains necessary and sufficient for the targeting of the hydrolases to lysosomes. This will also involve site directed mutagenesis to alter proteinase cleavage sites to determine if the proteolytic processing of lysosomal enzyme precursors is required for proper sorting. We will also use monoclonal antibodies to characterize the biosynthesis and transport of lysosomal membrane proteins. Finally we propose to biochemically and genetically analyze the process of induced lysosomal enzyme secretion which will include dissecting the signal transduction pathway responsible for secretion as well as attempting to define the biochemical defect(s) in a collection of mutants we have isolated which undersecrete enzymes. These combined approaches will increase our knowledge concerning the mechanisms of lysosome biosynthesis and function in all cells.