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的基本过程。酿酒酵母是鉴定和表征蛋白质转运机制中许多成分的宝贵工具，也是研究蛋白质分泌机制的一个成熟模型。然而，酵母和哺乳动物细胞中运输途径的分支使得对晚期（后高尔基体）分泌途径的研究变得复杂，而胞外货物分拣和从高尔基体和内体中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。在此步骤中确定中介传输的组件对于实现此目标至关重要。由于货物可以从阻塞的路线中分离出来，并通过另一条路线分泌，因此仅在一条路线上有缺陷的突变体不会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，成功地识别了结构相关的化合物，这些化合物导致分泌货和高尔基体膜在低（低至500 nM）浓度下快速（在15分钟内）积累，而不会导致内质网到高尔基体或高尔基体到溶酶体的缺陷，表明它们可能针对高尔基体的运输机制。我们建议使用我们目前的策略鉴定的化合物作为一种新的高通量筛选工具，旨在鉴定高度通路特异性的化合物。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，旨在描述多种运输途径如何相互作用，无论是否存在突变。许多化合物可能有特定的靶点，识别这些靶点将识别和确定分泌机制的新成分的作用。此外，通路特异性抑制剂有潜力作为治疗药物的先导，因为它们对分泌没有一般影响，毒性低，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对晚期分泌转运机制和涉及这些机制扰动的疾病的更好理解。

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.