Fatty Acylation of Hedgehog and Wnt Proteins

Hedgehog 和 Wnt 蛋白的脂肪酰化

• 批准号: 9197314
• 负责人: MARILYN D RESH
• 金额: $ 51.48万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197314
• 关键词: Acylation; Acyltransferase; Algorithms; Amino Acids; Binding Proteins; Biochemical; Biological; Biological Assay; Breast Cancer Cell; Carbon; Carnitine; Catalysis; Cell Culture Techniques; Cells; Chemistry; Collaborations; Computer Simulation; Development; Disease; Disulfides; Embryonic Development; Enzymes; Erinaceidae; Family; Fatty Acids; Goals; Growth; Human; In Vitro; Knowledge; Label; Link; Lipids; Malignant Neoplasms; Malignant neoplasm of pancreas; Mammalian Cell; Maps; Mediating; Membrane; Membrane Proteins; Metabolic; Methods; Modeling; Modification; Molecular; Mutation; Normal Cell; Palmitates; Palmitoyl Coenzyme A; Peptide Hydrolases; Peptides; Play; Porcupines; Post-Translational Protein Processing; Process; Production; Proteins; Proteomics; Reaction; Regulation; Role; SHH gene; Serine; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Stable Isotope Labeling; Structural Models; Structure; System; Testing; Transferase; Transmembrane Domain; WNT Signaling Pathway; Wnt proteins; base; cancer cell; cell growth; design; experimental study; fatty acylation; high throughput screening; in vitro Assay; inhibitor/antagonist; member; morphogens; palmitoleic acid; palmitoylation; preclinical development; public health relevance; reconstitution; small molecule inhibitor; smoothened signaling pathway; three dimensional structure; tool; tumorigenesis

 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.

 描述（由申请人提供）：我们研究的目标是阐明分泌蛋白脂肪酰化的生化机制，我们的重点是 Sonic Hedgehog (Shh) 和 Wnts，它们是在胚胎发育和肿瘤发生过程中发挥关键作用的信号蛋白。为了发出信号，Shh 必须通过棕榈酸酯的连接进行修饰，该反应由 Hhat（刺猬酰基转移酶）催化。我们开发了基于细胞的多通道膜蛋白 MBOAT（膜结合 O-酰基转移酶）家族，纯化了 Hhat，并进行了高通量筛选，以鉴定 Hhat 抑制剂的小分子抑制剂可阻断人胰腺的生长。和乳腺癌细胞，目前正在开发作为潜在的化疗药物，并探索了 MBOAT 修饰 Wnt 蛋白的脂肪酰化。酰基转移酶豪猪 (Porcn) 与棕榈油酸是产生活性 Wnt 信号所需的一种不寻常的脂质修饰。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，并以此作为揭示的基础。 MBOAT 酶如何识别其脂肪酸和蛋白质底物 目标 1. Hhat 的结构、功能和调节 我们将与 Chris Sander 合作，使用 EVfold_membrane 算法在计算机中预测 Hhat 的 3D 结构并根据经验测试模型。和/或结合预测的Hhat细胞质和腔内环的蛋白质将通过翻译后修饰、扩增对Hhat的调节进行鉴定。将探讨正常细胞和癌细胞中的突变和/或突变。 目标 2. 底物如何传递至 Hhat 并被 Hhat 识别？ 目前尚不清楚不可渗透生物膜的棕榈酰 CoA 是如何传递至 ER 腔中的 Hhat 的。我们将确定棕榈酰 CoA 是否通过肉碱依赖性系统进入 ER 腔，以及 Hhat 是否调节该过程。将使用基于蛋白质组学的方法来识别其他 Hhat 底物。通过将“点击化学”与 SILAC（细胞培养物中氨基酸的稳定同位素标记）相结合，将分析新鉴定的 Hhat 底物在调节乳腺癌细胞生长中的作用。 目标 3. 破译 Porcn 介导的 Wnt 的分子机制。我们将通过建立体外 Wnt 酰化测定来测试 Porcn 在二硫键连接结构中识别 Wnt 内丝氨酸酰化位点的假设。将纯化 Porcn 的肽底物，并使用差异膜透化和蛋白酶保护测定来表征 Porcn 的跨膜拓扑图。