Pathway-Specific Inhibitors of Post-Golgi Transport

描述（由申请人提供）：细胞表面和分泌蛋白的细胞内转运是所有真核细胞中的基本过程。酵母酿酒酵母（Saccharomyces cerevisiae）是鉴定和表征蛋白质转运机制中许多组分的宝贵工具，也是研究分泌机制的成熟模型。然而，在酵母和哺乳动物细胞中的运输途径的分支使晚期（后高尔基体）分泌途径的研究变得复杂，并且外泌货物分选和从高尔基体和内体退出所需的分子机制在很大程度上是未知的。我们的长期目标是确定货物从高尔基体运输到细胞表面的过程。鉴定在这一步骤中介导转运的组分对于实现这一目标至关重要。由于货物可以从阻断的途径中分选出来并通过替代途径分泌，因此仅在一种途径中有缺陷的突变体不表现出容易筛选的分泌表型，因此难以分离。作为突变体筛选的有效替代方案，我们设计了一种化学遗传筛选策略，该策略已成功地鉴定出结构相关的化合物，这些化合物在低浓度（低至500 nM）下引起分泌性货物和高尔基体膜的快速（15分钟内）积累，而不会引起ER-高尔基体或高尔基体-溶酶体缺陷，表明它们可能靶向高尔基体的转运机制。我们建议使用我们目前的策略确定的化合物作为一种工具，在一个新的高通量筛选，旨在确定化合物是高度路径特异性。这些化合物，特别是当以各种组合使用时，将在未来的研究中有用，这些研究旨在描绘许多运输途径如何相互作用，无论是否存在突变。 许多化合物可能具有特定的靶点，识别这些靶点将识别和确定分泌机制的新组分的作用。此外，途径特异性抑制剂具有作为治疗药物的先导的潜力，因为它们对分泌没有一般影响并且具有低毒性，这是我们筛选的要求。从我们的工作中获得的知识和工具将导致后期分泌运输机制和疾病，涉及这些机制的扰动的理解得到改善。

DESCRIPTION (provided by applicant): Intracellular transport of cell surface and secreted proteins is a fundamental process in all eukaryotic cells. The yeast Saccharomyces cerevisiae has been an invaluable tool in identifying and characterizing many of the components in the protein transport machinery and is a well-established model in the study of secretory mechanisms. However, the branching of transport routes in both yeast and mammalian cells has complicated studies of the late (post-Golgi) secretory pathway, and the molecular machinery required for exocytic cargo sorting and exit from the Golgi and endosomes is largely unknown. Our long-term goal is to define the processes by which cargo is transported from the Golgi to the cell surface. The identification of the components that mediate transport at this step is critical for reaching this goal. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit an easily screened secretory phenotype and are therefore difficult to isolate. As an efficient alternative to a mutant screen, we devised a chemical genetic screen strategy that has succeeded in identifying structurally related compounds that cause a rapid (within 15 min) accumulation of secretory cargo and Golgi membranes at low (as little as 500 nM) concentration, without causing ER-to-Golgi or Golgi-to-lysosome defects, indicating that they likely target the transport machinery at the Golgi. We propose to use a compound identified by our current strategy as a tool in a new high-throughput screen aimed at identifying compounds that are highly pathway-specific. Such compounds, especially when used in various combinations, will be useful in future studies aimed at delineating how the numerous transport routes interact, with or without the presence of mutations. Many compounds will likely have specific targets, and identifying these targets will identify and determine the roles of novel components of the secretory machinery. Furthermore, pathway-specific inhibitors have potential as leads for therapeutic drugs, since they do not have a general effect on secretion and have low toxicity, a requirement in our screen. The knowledge and tools gained from our work will result in an improved understanding of late secretory transport mechanisms and of diseases that involve perturbations of these mechanisms.