TARGETING OF LYSOSOMAL ENZYMES IN DICTYOSTELIUM

我的实验室的长期目标是确定分子机制 它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 正常功能是降解细胞外和细胞内物质 与人类疾病有牵连。30多种人类溶酶体遗传病 已经描述了，许多是由于缺少一个或多个 溶酶体酶。其他疾病包括全身溶酶体功能障碍。 (例如切迪亚克-东氏综合征)。溶酶体水解酶的分泌 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。在以前的 我们已经广泛地描述了生物合成、运输、 乳酸菌中3种溶酶体酶的修饰和蛋白降解加工 简单的真核生物盘基网眼菌，一种适合于这些的生物体 研究是因为它可以很容易地被基因和 生化方面的。这种生物也缺乏靶向受体(MAN-6- 在许多哺乳动物细胞中发现的溶酶体酶的磷酸受体)， 因此，研究替代排序已成为一种有用的系统 机械装置。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和充分的 将水解酶定位于溶酶体。这也将涉及Site 通过定向突变来改变蛋白水解酶的裂解位点 需要对溶酶体酶前体进行蛋白水解性加工 适当的分类。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运输。最后我们 建议从生物化学和遗传学的角度分析诱生的过程。 溶酶体酶的分泌，包括对信号的剖析 负责分泌的信号转导途径以及尝试 在我们拥有的一组突变中定义生化缺陷(S) 分离出分泌不足的酶。这些结合起来的方法将 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.