Palmitoylation of Hedgehog Proteins

Hedgehog 蛋白的棕榈酰化

• 批准号: 7888608
• 负责人: MARILYN D RESH
• 金额: $ 21.45万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-10 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888608
• 关键词: Active Sites; Address; Amides; Biochemical; Biological Assay; Biological Models; Catalysis; Cell Differentiation process; Cells; Chimeric Proteins; Enzymatic Biochemistry; Erinaceidae; Family member; Fatty Acids; Genetic; Goals; Growth; Homologous Gene; Human; Link; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Membrane Proteins; Methods; Molecular; Mus; Normal Cell; Palmitates; Porcupines; Proteins; Public Health; Reagent; Research; Signal Transduction; Signaling Protein; Structure; Transferase; Work; abstracting; antitumor agent; cancer cell; cell growth; deletion analysis; fly; high throughput screening; inhibitor/antagonist; medulloblastoma; melanoma; morphogens; novel; palmitoylation; pancreatic neoplasm; reconstitution; research study; thioester; trafficking

PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) proteins are secreted morphogens that regulate normal cell differentiation as well as malignant cell growth. Covalent attachment of the fatty acid palmitate to the N-terminus of Hh is critical for Hh function. Unlike nearly all other palmitoylated proteins, that contain thioester linked palmitate, palmitate is linked to Hh via amide (N-) bond. The overall goals of this research are to elucidate the enzymology and biochemical mechanism of N-palmitoylation of developmentally important signaling proteins, and to understand how expression and function of N-palmitoyl transferases is regulated. We will use Hh proteins as model systems to address the following issues: 1. To reconstitute N-palmitoylation using purified Hedgehog and Rasp proteins Genetic experiments in flies and mice indicate that Rasp, a multipass membrane protein, is required for Hh palmitoylation. To date, there is no biochemical evidence that Rasp, or its mammalian homolog Mart-2, functions independently and catalytically as a palmitoyl transferase. We have now succeeded in purifying Mart -2 to homogeneity in active form. The biochemical parameters and enzymatic mechanism of Shh and Hh N-palmitoylation will be determined. 2. Structure/Function analysis of Rasp/Mart-2 and Hh/Shh: How do MBOAT proteins work and how do they recognize their substrates? Using deletion analysis and chimeric proteins formed between Rasp and another MBOAT family member Porcupine, we will identify the transmembrane and/or cytoplasmic loop regions of Rasp and Mart-2 that comprise the active site and are important for catalysis. We will identify the minimum N-palmitoylation sequence motif within Hh/Shh and use this information to identify other substrates for Rasp and Mart-2. 3. To determine how N-Palmitoylation by Rasp/Mart-2 is regulated within the cell Subcellular localization and trafficking experiments will be performed to determine when and where Hh is palmitoylated. Methods to inhibit Mart-2 mediated palmitoylation will be devised. A high throughput screen will be exploited to identify novel small molecular inhibitors of Hh palmitoylation. These reagents could potentially be clinically useful as anti-tumor agents in Hh driven malignancies. The effects of Mart-2 inhibition on growth of Shh-dependent pancreatic cancer cells will be assayed. Project Narrative/Relevance to Public Health: Hh signaling has been shown to drive the growth of many human cancers, including medulloblastoma, melanoma, and pancreatic tumors. The proposed studies will help us understand how Hh proteins work in normal and malignant cells and will aim to develop Hh inhibitors that could potentially be clinically useful as anti-tumor agents in Hh driven malignancies.

项目概要/摘要 Hedgehog (Hh) 蛋白是分泌的形态发生素，可调节正常细胞分化以及 恶性细胞生长。脂肪酸棕榈酸酯与 Hh N 末端的共价连接是 对于 Hh 功能至关重要。与几乎所有其他棕榈酰化蛋白质不同，这些蛋白质含有硫酯连接的 棕榈酸酯，棕榈酸酯通过酰胺 (N-) 键与 Hh 连接。这项研究的总体目标是 阐明发育中N-棕榈酰化的酶学和生化机制 重要的信号蛋白，并了解 N-棕榈酰的表达和功能 转移酶受到监管。我们将使用 Hh 蛋白作为模型系统来解决以下问题： 1. 使用纯化的 Hedgehog 和 Rasp 蛋白重建 N-棕榈酰化 果蝇和小鼠的遗传实验表明，Rasp（一种多通道膜蛋白） Hh 棕榈酰化所需。迄今为止，没有生化证据表明 Rasp 或其哺乳动物 同源物 Mart-2，作为棕榈酰转移酶独立发挥催化作用。我们现在有 成功地将Mart -2纯化至活性形式的同质化。生化参数和 将确定Shh和Hh N-棕榈酰化的酶促机制。 2. Rasp/Mart-2 和 Hh/Shh 的结构/功能分析：MBOAT 蛋白如何工作和 它们如何识别它们的底物？使用缺失分析和嵌合蛋白 由 Rasp 和另一位 MBOAT 家族成员 Porcupine 组成，我们将确定 Rasp 和 Mart-2 的跨膜和/或细胞质环区域，包含活性位点和 对催化很重要。我们将确定最小的 N-棕榈酰化序列基序 Hh/Shh 并使用此信息来识别 Rasp 和 Mart-2 的其他底物。 3. 确定 Rasp/Mart-2 的 N-棕榈酰化在细胞内的调节方式 将进行亚细胞定位和运输实验以确定何时和 其中 Hh 是棕榈酰化的。将设计抑制 Mart-2 介导的棕榈酰化的方法。一个高 将利用通量筛选来鉴定 Hh 棕榈酰化的新型小分子抑制剂。 这些试剂可能在临床上用作 Hh 驱动的恶性肿瘤的抗肿瘤剂。 将检测 Mart-2 抑制对 Shh 依赖性胰腺癌细胞生长的影响。项目叙述/与公共卫生的相关性： Hh 信号传导已被证明可促进许多人类癌症的生长，包括髓母细胞瘤、 黑色素瘤和胰腺肿瘤。拟议的研究将帮助我们了解 Hh 蛋白如何 在正常和恶性细胞中发挥作用，目标是开发 Hh 抑制剂，该抑制剂有可能成为 临床上可用作 Hh 驱动的恶性肿瘤的抗肿瘤剂。