Novel mechanisms for oxysterols in cell-cell signaling

描述(申请人提供)：氧固醇是细胞从胆固醇合成的分子，它影响许多生物过程，如新陈代谢，细胞-细胞信号和细胞迁移。令人惊讶的是，尽管它们具有强大的活性，但我们对氧化类固醇所扮演的角色以及它们如何作用于细胞实现各种影响知之甚少。氧化甾醇与Hedgehog信号通路有关，Hedgehog信号通路是胚胎发育、成人干细胞维持所必需的细胞-细胞信号通路，并与许多人类癌症密切相关。众所周知，氧固醇结合并激活一种膜蛋白Smoothens，它是触发Hedgehog信号转导的关键。然而，角色的许多关键方面 氧化甾醇在Hedgehog信号中的作用仍然不清楚：我们不知道正常的Hedgehog信号是否需要氧固醇，它们是如何调节的，以及阻断氧固醇是否可能是抑制癌症Hedgehog信号的好方法。此外，需要新的和强大的化学方法来可视化和分析体内的氧化甾醇；这种方法将极大地帮助我们了解它们在Hedgehog信号转导和其他方面的作用。我们开发了一种新的化合物，称为氮杂甾醇，它可以阻断氧固醇与Smoothed的结合；结果，Hedgehog信号被抑制，这表明拮抗氧固醇是抑制Hedgehog途径的有效策略。使用氮杂甾醇和氧固醇化学探针，我们精确地确定了氧固醇与Smoothens结合的位置，这使得我们能够构建不再对Ox-sterol产生反应的Smoothed突变体。有趣的是，我们发现这些光滑的突变体不能正确地发出信号；这表明氧固醇是正常刺猬信号所必需的。最后，我们已经合成并表征了新的化学探针，使我们能够以比以前更好的灵敏度和特异度来可视化和分析细胞中的氧化甾醇。我们计划使用这些探针来更好地了解氧固醇在Hedgehog信号中的作用。我们建议结合化学生物学、生物化学和细胞生物学来完成以下目标：A)阐明Hedgehog信号如何在我们的新型氧固醇探针的帮助下调节氧固醇；B)准确地确定哪些氧固醇参与Hedgehog信号；C)发现参与Hedgehog信号的氧甾醇是如何在细胞中合成的。这些研究很重要，原因如下：1)通过阐明氧固醇的关键作用，它们将增进我们对Hedgehog信号的理解；2)它们将基于氧固醇抑制，识别在癌症中阻断Hedgehog信号的新靶点；3)我们的新型化学探针将广泛应用于研究氧化甾醇在健康和疾病中的作用机制，将为甾醇紊乱提供诊断工具，并将有助于识别甾醇的小分子抑制剂。 功能。

DESCRIPTION (provided by applicant): Oxysterols are molecules that cells synthesize from cholesterol, which affect many biological processes such as metabolism, cell-cell signaling and cellular migration. Surprisingly, in spite of their potent activities, we understand only poorly th roles that oxysterols play, and how they act to accomplish various effects on cells. Oxysterols have been implicated in Hedgehog signaling, a cell-cell signaling pathway essential for embryonic development, for adult stem cell maintenance, and deeply involved in many human cancers. It is known that oxysterols bind and activate a membrane protein, Smoothened, which is critical for triggering Hedgehog signal transduction. However, many critical aspects of the role of oxysterols in Hedgehog signaling have remained obscure: we do not know if oxysterols are required for normal Hedgehog signaling, how they are regulated, and whether blocking oxysterols might be a good way to inhibit Hedgehog signaling in cancer. Additionally, there is a need for new and powerful chemical approaches to visualize and assay oxysterols in vivo; such methods would greatly aid our understanding of their role in Hedgehog signaling and beyond. We developed novel chemicals, called azasterols, which block binding of oxysterols to Smoothened; as a result, Hedgehog signaling is inhibited, which shows that antagonizing oxysterols is an effective strategy to inhibit the Hedgehog pathway. Using azasterol and oxysterol chemical probes, we pinpointed where oxysterols bind to Smoothened, which allowed us to build Smoothened mutants that no longer respond to oxysterols. Interestingly, we discovered that these Smoothened mutants cannot signal properly; this showed that oxysterols are required for normal Hedgehog signaling. Finally, we have synthesized and characterized novel chemical probes that allow us to visualize and assay oxysterols in cells, with better sensitivity and specificity than before. We plan to use these probes to better understand the role of oxysterols in Hedgehog signaling. We propose to use a combination of chemical biology, biochemistry and cell biology, to accomplish the following aims: A) To elucidate how Hedgehog signaling regulates oxysterols, with the aid of our novel oxysterol probes B) To determine precisely which oxysterols are involved in Hedgehog signaling C) To discover how oxysterols involved in Hedgehog signaling are synthesized in cells These studies are important for the following reasons: 1) They will advance our understanding of Hedgehog signaling, by elucidating the critical role of oxysterols; 2) They will identify novel targets for blocking Hedgehog signaling in cancer, based on oxysterol inhibition; and 3) Our novel chemical probes will be broadly applicable to study oxysterol mechanisms in health and in disease, will provide diagnostic tools for sterol disturbances and will help identify small molecule inhibitors of sterol function.