Palmitoylation of Hedgehog Proteins

项目摘要/摘要 Hedgehog(HH)蛋白是一种分泌的形态原，调节正常的细胞分化以及 恶性细胞生长。脂肪酸棕榈酸酯与HH的N-末端的共价连接 对HH功能至关重要。与几乎所有其他棕榈酰化蛋白质不同的是，这些蛋白质含有硫代酯连接 棕榈酸酯，棕榈酸酯通过酰胺(N-)键与HH相连。这项研究的总体目标是 发育过程中N-棕榈酰化的酶学和生化机制 重要的信号蛋白，并了解N-棕榈酰基的表达和功能 转移酶是受调控的。我们将使用HH蛋白质作为模型系统来解决以下问题： 1.用纯化的Hedgehog和Rasp蛋白重建N-棕榈酰化 在苍蝇和小鼠身上的遗传学实验表明，rasp是一种多通道膜蛋白， HH棕榈酰化所必需的。到目前为止，还没有生化证据表明Rasp或它的哺乳动物 同系物MART-2，作为棕榈酰基转移酶独立和催化作用。我们现在有了 成功地将MART-2纯化为活性形式的均一。生化指标和 Shh和HH N-棕榈酰化的酶机理将被确定。 2.rasp/MART-2和HH/Shh的结构/功能分析：MBOAT蛋白是如何发挥作用的 它们如何识别它们的底物？利用缺失分析和嵌合蛋白 在Rasp和另一个MBOAT家族成员豪猪之间形成的，我们将确定 Rasp和MART-2的跨膜和/或细胞质环区，组成活性部位和 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，并使用该信息确定rasp和MART-2的其他底物。 3.确定rasp/MART-2在细胞内如何调节N-棕榈酰化 将进行亚细胞定位和贩运实验，以确定何时和 其中HH是棕榈酰化的。抑制MART-2介导的棕榈酰化的方法将被设计出来。一次高潮 吞吐量筛选将被用来鉴定新型的HH棕榈酰化小分子抑制剂。 这些试剂可能在临床上用作HH驱动的恶性肿瘤的抗肿瘤药物。 MART-2抑制对Shh依赖的胰腺癌细胞生长的影响将被检测。项目说明/与公共卫生的相关性： HH信号已被证明推动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蛋白是如何 在正常和恶性细胞中工作，目标是开发HH抑制剂，这种抑制剂可能是 作为抗肿瘤药物在HH驱动的恶性肿瘤中的临床应用。

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.