Signal Transduction by Oxysterols

描述(申请人提供)：氧固醇是一类源自胆固醇的内源性细胞脂，与动脉粥样硬化、先天代谢障碍、炎症和癌症的病理生理学有关。在许多情况下，这些神秘分子发挥其强大生物效应的蛋白质和分子途径尚不清楚。我们建立了一位合成有机化学家和一位细胞生物学家之间的合作，以了解氧固醇如何激活Hedgehog(HH)信号系统，这是一条在发育、再生和癌症中发挥重要作用的途径。我们发现，一种特定的氧固醇，20(S)-OHC，是7-通道跨膜蛋白Smoothned(SMO)的变构激活剂，Smo是一种人类癌蛋白，也是肿瘤学的关键药物靶点。这一发现极大地扩大了氧化甾醇作为信号分子的调节范围，证明了它们作为人类辅蛋白和信号受体的直接激动剂的能力。在前期工作的基础上，我们假设内源性20(S)-OHC在HH信号转导中起第二信使的作用。利用突变、光亲和标记和质谱学的组合，我们将绘制与20(S)-OHC相互作用的Smo区域，以提供这类新的受体-配体相互作用的生化画像(目标1)。vbl.使用 通过定量质谱学和基于点击化学的成像分析，我们将询问Hedgehog信号是否可以改变20(S)-OHC(目标2)的细胞水平或分布。最后，我们会 开发和表征受氧固醇支架启发的新型HH途径抑制剂(目标3)。我们预计这个项目的成功完成将产生三个主要结果：(1)Smo是如何在细胞中调节的问题的答案，这可能是HH途径中存在时间最长的谜团，(2)对氧固醇如何参与和调节7-PASS信号受体的生化理解，以及(3)开发一个可以用于剖析任何其他氧固醇调节的细胞过程的综合工具包。为了实现这些目标，我们招募了一支在细胞生物学、蛋白质生物化学、合成化学和质谱学方面具有互补专业知识的研究团队。

DESCRIPTION (provided by applicant): Oxysterols are a class of endogenous cellular lipids derived from cholesterol that have been implicated in the pathophysiology of atherosclerosis, inborn errors of metabolism, inflammation and cancer. In many cases, the proteins and molecular pathways through which these enigmatic molecules exert their powerful biological effects remain unknown. We established collaboration between a synthetic organic chemist and a cell biologist to understand how oxysterols activate the Hedgehog (Hh) signaling system, a pathway that plays important roles in development, regeneration and cancer. We discovered that a specific oxysterol, 20(S)-OHC, is an allosteric activator of the 7-pass transmembrane protein Smoothened (Smo), a human oncoprotein and key drug target in oncology. This finding significantly expands the regulatory scope of oxysterols as signaling molecules, demonstrating their capacity to function as direct agonists for both a human on co-protein and a signaling receptor. Based on preliminary work, we hypothesize that endogenous 20(S)-OHC functions as a second-messenger in Hh signaling. Using a combination of mutagenesis, photo affinity labeling, and mass spectrometry, we will map the region of Smo that interacts with 20(S)-OHC to provide a biochemical portrait of this novel class of receptor-ligand interaction (Aim 1). Using quantitative mass spectrometry and a click chemistry-based imaging assay, we will ask if Hedgehog signaling can alter cellular levels or distribution of 20(S)-OHC (Aim 2). Finally, we will develop and characterize novel Hh pathway inhibitors that are inspired by oxysterol scaffolds (Aim 3). We expect three major outcomes to emerge from the successful completion of this project: (1) An answer to the question of how Smo is regulated in cells, perhaps the longest-standing mystery in the Hh pathway, (2) a biochemical understanding of how oxysterols engage and regulate 7-pass signaling receptors, and (3) the development of an integrative toolkit that can be deployed to dissect any other oxysterol- regulated cellular process. To accomplish these goals, we have recruited a team of investigators with complementary expertise in cell biology, protein biochemistry, synthetic chemistry, and mass spectrometry.