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.

项目摘要/摘要 刺猬（HH）蛋白是调节正常细胞分化以及 恶性细胞生长。脂肪酸棕榈酸酯在HH的N末端的共价附着 对于HH功能至关重要。与几乎所有其他含有硫酯链接的棕榈酰化蛋白不同的不同 棕榈酸酯，棕榈酸酯通过酰胺（N-）键与HH相关。这项研究的总体目标是 阐明发育中N-膜酰化的酶学和生化机制 重要的信号蛋白，并了解N-膜酰基的表达和功能如何 调节转移酶。我们将使用HH蛋白作为模型系统来解决以下问题： 1。使用纯化的刺猬和RASP蛋白重建n-甲酰化 蝇和小鼠的遗传实验表明，多质膜蛋白RASP是 HH棕榈酰化所必需的。迄今 同源性mart-2，作为棕榈酰转移酶独立和催化地发挥作用。我们现在有 成功地以主动形式净化了mart -2。生化参数和 将确定SHH和HH N-膜酰化的酶促机制。 2。RASP/MART-2和HH/SHH的结构/功能分析：Mboat蛋白如何工作和 他们如何识别其底物？使用缺失分析和嵌合蛋白 在RASP与另一名M船家庭成员豪猪之间形成，我们将确定 RASP和MART-2的跨膜和/或细胞质环区域包括活性位点， 对于催化很重要。我们将确定最小n-膜酰化序列基序 HH/SHH并使用此信息来识别RASP和MART-2的其他基板。 3。确定如何在细胞内调节rasp/mart-2的n-膜酰化 将进行亚细胞定位和贩运实验，以确定何时和 HH的位置是棕榈树。将设计抑制MART-2介导的棕榈酰化的方法。高 吞吐量筛选将被利用以鉴定新型的HH棕榈酰化的小分子抑制剂。 这些试剂在HH驱动的恶性肿瘤中可能会在临床上作为临床有用。 将测定MART-2抑制对SHH依赖性胰腺癌细胞生长的影响。项目叙述/与公共卫生相关： HH信号传导已被证明可以驱动许多人类癌的生长，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工作，并将旨在开发可能是可能是的HH抑制剂 在HH驱动的恶性肿瘤中作为抗肿瘤药物在临床上有用。