Fatty Acylation of Hedgehog and Wnt Proteins

 描述(申请人提供)：我们研究的目标是阐明分泌蛋白质脂肪酰化的生化机制。我们的重点是Sonic hedgehog(Shh)和Wnts，这两种信号蛋白在胚胎发育和肿瘤发生中发挥关键作用。为了发出信号，Shh必须通过连接棕榈酸酯来修饰，这是一个由HHAT(刺猬酰基转移酶)催化的反应。HHAT是膜结合O-酰基转移酶(MBOAT)家族的一员。我们开发了基于细胞的和体外的Shh棕榈酰化实验，纯化了HHAT，并进行了高通量筛选以确定HHAT的小分子抑制剂。HHAT抑制剂可以阻止人胰腺癌细胞和乳腺癌细胞的生长，目前正在作为潜在的化疗药物进行开发。平行的研究探索了Wnt蛋白的脂肪酰化作用。WNT被棕榈油酸的MBOAT酰基转移酶豪猪碱(PORCN)修饰，这是一种产生活跃WNT信号所需的不寻常的脂类修饰。尽管我们有HHAT和PORCN介导的脂质修饰的工作模型，但我们对不同的脂肪酰基转移酶如何修饰分泌的形态原的了解仍然存在关键差距。我们的目标是使用计算机和经验方法来生成HHAT和PORCN的2D和3D结构模型，并以此为基础揭示MBOAT酶如何识别它们的脂肪酸和蛋白质底物。目的1.HHAT的结构、功能及其调控。我们将与Chris Sander合作，使用EVfold膜法预测硅胶中HHAT的三维结构，并对该模型进行实证检验。结合预测的HHAT细胞质和腔内环的底物和/或蛋白质将通过GST下拉实验来鉴定。将探索在正常细胞和癌细胞中通过翻译后修饰、扩增和/或突变来调节HHAT。目标2.底物如何传递给HHAT并被HHAT识别？目前尚不清楚棕榈酰辅酶A是如何通过生物膜传递到内质网管腔内的HHAT的。我们将确定棕榈酰辅酶A是否通过肉碱依赖系统进入内质网管腔，以及HHAT是否调节这一过程。一种基于蛋白质组学的方法将通过结合“点击化学”和SILAC(细胞培养中氨基酸的稳定同位素标记)来识别额外的HHAT底物。将分析新发现的HHAT底物在调节乳腺癌细胞生长中的作用。目的3.破译PORCN介导的Wnt酰化的分子机制。我们将通过建立蛋白质和多肽底物的体外WNT酰化实验来检验PORCN在二硫键连接结构的背景下识别WNT中的丝氨酸酰化位点的假设。将对PORCN进行纯化，并对其酶学参数进行表征。PORCN的跨膜拓扑图将使用差异膜通透性和蛋白酶保护分析来生成。

 DESCRIPTION (provided by applicant): The goal of our studies is to elucidate the biochemical mechanism of fatty acylation of secreted proteins. Our focus is on Sonic hedgehog (Shh) and Wnts, signaling proteins that play key roles during embryonic development and tumorigenesis. In order to signal, Shh must be modified by attachment of palmitate, a reaction catalyzed by Hhat (hedgehog acyltransferase). Hhat is a member of the MBOAT (membrane bound O-acyltransferase) family of multipass membrane proteins. We developed cell-based and in vitro assays for Shh palmitoylation, purified Hhat and performed high throughput screening to identify small molecule inhibitors of Hhat. Hhat inhibitors block the growth of human pancreatic and breast cancer cells and are currently undergoing development as potential chemotherapeutics. Parallel studies have explored fatty acylation of Wnt proteins. Wnts are modified by the MBOAT acyltransferase Porcupine (Porcn) with palmitoleic acid, an unusual lipid modification required for production of an active Wnt signal. Although we have working models for Hhat and Porcn-mediated lipid modification, critical gaps remain in our knowledge of how secreted morphogens are modified by distinct fatty acyltransferases. We aim to use in silico and empirical methods to generate 2D and 3D structural models for Hhat and Porcn, and use this as a basis for revealing how MBOAT enzymes recognize their fatty acid and protein substrates. Aim 1. Structure, function and regulation of Hhat. In collaboration with Chris Sander, we will use the EVfold_membrane algorithm to predict the 3D structure of Hhat in silico and test the model empirically. Substrates and/or proteins that bind to predicted cytoplasmic and intralumenal loops of Hhat will be identified using GST pulldown experiments. Regulation of Hhat by posttranslational modification, amplification and/or mutation in normal and cancer cells will be explored. Aim 2. How are substrates delivered to and recognized by Hhat? It is not known how palmitoyl CoA, which is not permeable across biological membranes, is delivered to Hhat in the ER lumen. We will determine whether palmitoyl CoA enters the ER lumen through a carnitine-dependent system, and if Hhat regulates this process. A proteomics-based approach will be used to identify additional Hhat substrates by combining "click chemistry" with SILAC (stable isotope labeling with amino acids in cell culture). Newly identified Hhat substrates will be analyzed for their roles in regulating breast cancer cell growth. Aim 3. To decipher the molecular mechanism of Porcn-mediated Wnt acylation. We will test the hypothesis that Porcn recognizes the Serine acylation site within Wnt in the context of a disulfide- linked structure by establishing an in vitro Wnt acylation assay with protein and peptide substrates. Porcn will be purified and its enzymologic parameters will be characterized. A transmembrane topology map of Porcn will be generated using differential membrane permeabilization and protease protection assays.