Sterol Probes of Hedgehog Protein Processing

项目总结/摘要 Hedgehog蛋白是唯一的被胆固醇共价修饰的蛋白。这对于其 胚胎发育中的信号作用，也在调节成体干细胞。故障 刺猬信号导致严重的出生缺陷，并参与许多癌症。 胆固醇化对于Hedgehog功能是必需的，并且由胆固醇化的C-末端结构域催化。 刺猬前体蛋白胆固醇突变的许多有害影响 生物合成被认为涉及Hedgehog信号传导。甾醇结合区的催化 域是了解甚少，因为它本质上是太无序的服从 晶体学或蛋白质NMR。本提案的总体目标是提供关于 通过使用修饰的甾醇类来修饰甾醇结合域。一个含有23种甾醇的文库， 在甾醇核的每个位置上的基团将通过有机合成组装。甲基 基团将通过相应酮的亚甲基化，然后还原而就位。 两种立体异构体将在每个二级位置获得。角甲基将 采用文献法介绍。如果不存在优先级（位置8和9）， 将尝试应用用于C5-甲基化的方法。此库将由 合作者在一个基于FRET的刺猬加工试验。结果将确定哪些站点 与蛋白质的结合位点接触。合成策略允许添加空间体积 （甲基至乙基）以更好地限定结合区域。同一合作者的诱变研究 与被阻断的固醇重新起作用的蛋白质将决定哪些氨基酸与哪些区域接触 甾醇分子的结构另一个由23种甾醇组成的库，这次每个甾醇都带有氟原子， 位置，将使用与用于制造甲基的基本相同的前体合成。 甾醇文库。该文库将用于基于19 F-NMR的研究，以绘制 蛋白基于此库的加工反应的共价抑制剂也将被合成 和研究核磁共振研究将由一位合作的结构生物学家进行。刺猬 被甾醇类似物修饰的蛋白质可用于研究下游信号相互作用。 这两个甾醇库可能是有用的其他蛋白质-甾醇相互作用的研究。

PROJECT SUMMARY/ ABSTRACT Hedgehog protein is unique by being covalently modified by cholesterol. This is critical to its signaling role in embryonic development, and also in regulating adult stem cells. Malfunction of hedgehog signaling leads to severe birth defects and is involved in many cancers. Cholesterylation is essential to Hedgehog function and is catalyzed by the C-terminal domain of the Hedgehog precursor protein. Many of the deleterious effects of mutations in cholesterol biosynthesis are thought to involve Hedgehog signaling. The sterol binding region of the catalytic domain is poorly understood because it is intrinsically too disordered to be amenable to crystallography or protein NMR. The overall goal of this proposal to provide information about the sterol-binding domain through the use of modified sterols. A library of 23 sterols bearing a methyl group at each position of the sterol nucleus will be assembled by organic synthesis. The methyl groups will be put in place by methylenation of the corresponding ketones, followed by reduction. Both stereoisomers will be obtained at each secondary position. The angular methyl groups will be introduced by literature methods. Where precedence does not exist (positions 8 and 9), attempts will be made to apply methods for C5-methylation. This library will be tested by a collaborator in a FRET-based Hedgehog processing assay. The results will establish which sites of the sterol contact the binding site of the protein. The synthetic strategy allows adding steric bulk (methyl to ethyl) to better define the binding region. Mutagenesis studies by the same collaborator which regain function with a blocked sterol will determine which amino acids contact which regions of the sterol molecule. Another library of 23 sterols, this time bearing fluorine atoms at each position, will be synthesized using largely the same precursors that were used to make the methyl sterol library. This library will be used for 19F-NMR based studies to map the binding site of the protein. Covalent inhibitors of the processing reaction based on this library will also be synthesized and studied. The NMR studies will be carried by a collaborating structural biologist. Hedgehog protein modified by the sterol analogs may be useful to study downstream signaling interactions. The two sterol libraries may be useful in the study of other protein-sterol interactions.