Novel mechanisms for oxysterols in cell-cell signaling

描述（由申请人提供）：氧固醇是细胞从胆固醇合成的分子，其影响许多生物过程，如代谢、细胞-细胞信号传导和细胞迁移。令人惊讶的是，尽管它们具有强大的活性，但我们对氧化固醇所起的作用以及它们如何对细胞产生各种影响的了解甚少。氧固醇与Hedgehog信号传导有关，Hedgehog信号传导是胚胎发育、成体干细胞维持所必需的细胞-细胞信号传导途径，并且与许多人类癌症密切相关。已知氧固醇结合并激活膜蛋白Smoothened，其对于触发Hedgehog信号转导至关重要。然而，该角色的许多关键方面 关于氧化固醇在Hedgehog信号传导中的作用仍然不清楚：我们不知道氧化固醇是否是正常Hedgehog信号传导所必需的，它们是如何调节的，以及阻断氧化固醇是否可能是抑制癌症中Hedgehog信号传导的好方法。此外，需要新的和强大的化学方法来可视化和测定体内氧固醇;这些方法将极大地帮助我们理解它们在Hedgehog信号传导及其他方面的作用。 我们开发了一种称为氮杂甾醇的新型化学物质，它可以阻断氧化甾醇与Smoothened的结合;因此，Hedgehog信号被抑制，这表明拮抗氧化甾醇是抑制Hedgehog通路的有效策略。使用azasterol和oxysterol化学探针，我们确定了oxysterol与Smoothened结合的位置，这使我们能够构建不再对oxysterol产生反应的Smoothened突变体。有趣的是，我们发现这些Smoothened突变体不能正确地发出信号;这表明氧化固醇是正常Hedgehog信号传导所必需的。最后，我们合成并表征了新型化学探针，使我们能够可视化和测定细胞中的氧化固醇，具有比以前更好的灵敏度和特异性。我们计划使用这些探针来更好地了解氧固醇在Hedgehog信号传导中的作用。 我们建议使用化学生物学、生物化学和细胞生物学的组合来实现以下目标：A）在我们的新型氧固醇探针的帮助下，阐明Hedgehog信号传导如何调节氧固醇B）精确地确定哪些氧固醇参与Hedgehog信号传导C）发现参与Hedgehog信号传导的氧固醇如何在细胞中合成这些研究是重要的，原因如下：1）他们将通过阐明氧化固醇的关键作用来促进我们对Hedgehog信号传导的理解; 2）他们将基于氧化固醇抑制来确定阻断癌症中Hedgehog信号传导的新靶点;和3）我们的新型化学探针将广泛适用于研究健康和疾病中的氧固醇机制，将为甾醇紊乱提供诊断工具，并将有助于确定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.