Chemical genetic analysis of intracellular Transport

描述(申请人提供)：细胞内转运是一个基本的生物学过程，对于细胞器和大分子复合体在每个真核细胞的细胞质中的适当分布和定位是必需的。我们项目的目标是利用化学遗传学来研究细胞内转运的分子机制。这种方法模仿经典遗传学作为一种发现工具，涉及到选择可以扰乱细胞功能的化学探针，识别这些探针的目标，然后使用这些探针来澄清已知或新的蛋白质对细胞功能的贡献。我们认为，化学遗传学方法在识别和研究脊椎动物细胞内运输的分子机制方面将特别有效，在这种情况下，应用遗传筛选是困难的，而且在时间尺度上干扰蛋白质功能的方法比运输过程慢得多。我们已经确定了一个强大而高效的系统，它将允许选择作用于马达蛋白的化合物，或最直接参与调节马达蛋白在细胞内运输功能的蛋白。在这项工作的过程中，我们将：(1)使用非洲爪哇黑素载体系统来识别细胞内转运的小分子探针。(2)使用二级筛选来选择直接抑制运动蛋白驱动的细胞器运动的小分子，或针对与这些运动最密切相关的调节机制。(3)确定细胞靶点，检查化学遗传探针研究细胞内转运的特异性和普遍适用性。在我们的研究中发现和验证的小分子探针将成为检查细胞内运输机制的强大工具。这些小分子很可能会导致发现治疗药物的新靶点，并改进人类疾病的治疗方法。

DESCRIPTION (provided by applicant): Intracellular transport is a fundamental biological process that is required for the proper distribution and positioning of organelles and macromolecular complexes in the cytoplasm of every eukaryotic cell. The goal of our project is to examine molecular mechanisms of intracellular transport using chemical genetics. This approach emulates classical genetics as a discovery tool, and involves the selection of chemical probes that can perturb a cellular function, identification of targets of these probes, followed by the use of the probes to clarify the contribution of known or novel proteins to the cellular function. We believe that the chemical genetic approach will be particularly effective in identifying and examining molecular mechanisms of vertebrate intracellular transport, where the application of genetic screens is difficult and approaches Perturbing protein function on time-scales much slower than the transport processes can be of limited use. We have identified a robust and efficient system that will allow the selection of compounds that either act on motor proteins, or proteins most directly involved in regulating motor protein function during intracellular transport. In the course of this work we will: (1) Use the Xenopus melanophore system to identify small molecule probes for intracellular transport. (2) Use secondary screens to select small molecules that either directly inhibit motor protein-driven organelle movement or target the regulatory mechanisms most closely associated with these movements. (3) Identify cellular targets, examine specificity and general applicability of chemical genetic probes for studying intracellular transport. The small molecules probes discovered and validated in our studies will be powerful tools to examine mechanisms of intracellular transport. It is likely that these small molecules will lead to the identification of new targets for therapeutic agents and improved treatments of human diseases.