胆固醇是生物体内含量最丰富的甾醇分子，它主要调控膜的性质。1996年首次发现胆固醇可以共价修饰Hh蛋白，这一修饰在胚胎发育、细胞增殖和肿瘤发生中起重要作用。长期以来学术界一直认为Hh是唯一的胆固醇修饰蛋白。我们合作发展了一模拟胆固醇的小分子探针CP，结合点击化学反应，能够分析并鉴定胆固醇连接蛋白。利用该方法，已经发现一新的胆固醇共价修饰蛋白Smo，它是Hh通路中的关键成员，其胆固醇修饰对Hh信号转导和胚胎发育至关重要。基于该研究基础，本项目将进一步优化胆固醇探针CP及分析方法以提高效率和灵敏度；鉴定新的胆固醇共价修饰蛋白；发现催化Smo胆固醇修饰的酶及调控因子，阐明Smo进行胆固醇修饰的分子机制；揭示Smo的胆固醇修饰在Hh信号转导中的功能调控；发展干预Smo胆固醇修饰的小分子，分析其抑制Hh信号转导与抗肿瘤的功能。本项目将开拓蛋白质胆固醇动态修饰的研究领域，并为抗肿瘤药物研发提供新思路。

Cholesterol is the most abundant sterol in mammals. It mainly regulates membrane properties. Hh was found to be covalently modified by cholesterol in 1996. Hh plays essential roles in embryonic development, cell proliferation and cancer. Since then, Hh has been considered as the only cholesterol-modified protein in proteome. During long-term collaboration, we have developed a cholesterol-mimicking chemical probe, called CP. Using CP coupled with click chemistry, we were able to analyze and identify cholesterol covalently linked proteins. We found that Smo was modified by cholesterol on the D95 residue. Smo is a key member in Hh pathway. Homozygous modification site mutated mouse was embryonic lethal with severe developmental defects, indicating cholesterylation is critical for Smo’s function. Based on the previous work, we will optimize the chemical probes of cholesterol and the method to identify new cholesterol-modified proteins. We will characterize the enzymes and regulatory factors for cholesterol modification of Smo. The functional regulation of Smo cholesterylation in Hh signaling will be investigated. We will develop small molecules that interfere with Smo cholesterylation and inhibit Hh signaling. The anti-tumor activity of the chemicals will be measured. In sum, this project will deepen our understanding on the cholesterol modification of proteins and pave a new way to treat cancers.