Signal Transduction by Oxysterols

描述（由申请人提供）：氧化甾醇是一类来源于胆固醇的内源性细胞脂类，与动脉粥样硬化、先天性代谢错误、炎症和癌症的病理生理有关。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仍然未知。我们在合成有机化学家和细胞生物学家之间建立了合作关系，以了解氧甾醇如何激活Hedgehog （Hh）信号系统，这是在发育，再生和癌症中发挥重要作用的途径。我们发现一种特殊的氧甾醇，20(S)-OHC，是7-pass跨膜蛋白Smoothened （Smo）的变构激活剂，Smo是一种人类肿瘤蛋白和肿瘤中的关键药物靶点。这一发现显著扩展了氧甾醇作为信号分子的调控范围，证明了它们作为人类共蛋白和信号受体的直接激动剂的能力。基于前期工作，我们假设内源性20(S)-OHC在Hh信号传导中起第二信使的作用。利用诱变、光亲和标记和质谱的组合，我们将绘制Smo与20(S)-OHC相互作用的区域，以提供这种新型受体-配体相互作用的生化肖像（目的1）。使用

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.