Palmitoylation of Hedgehog Proteins

描述（由申请人提供）：Hedgehog（Hh）蛋白是调节正常细胞分化以及恶性细胞生长的分泌型形态发生素。脂肪酸棕榈酸酯与Hh的N-末端的共价连接对于Hh功能至关重要。与几乎所有其他含有硫酯连接的棕榈酸酯的棕榈酰化蛋白质不同，棕榈酸酯通过酰胺（N-）键与Hh连接。本研究的总体目标是阐明发育重要信号蛋白N-棕榈酰化的酶学和生化机制，并了解N-棕榈酰转移酶的表达和功能是如何调节的。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，一个多通道膜蛋白，是所需的Hh棕榈酰化。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和催化地发挥作用。我们现在已经成功地将Mart-2纯化为活性形式的同质性。确定Shh和Hh N-棕榈酰化的生化参数和酶促机理。 2.骨锉/Mart-2和Hh/Shh的结构/功能分析 MBOAT蛋白如何工作以及它们如何识别其底物？使用缺失分析和Rasp和另一个MBOAT家族成员豪猪之间形成的嵌合蛋白，我们将确定Rasp和Mart-2的跨膜和/或细胞质环区域，这些区域包含活性位点，并且对催化很重要。我们将确定Hh/Shh内的最小N-棕榈酰化序列基序，并使用此信息来确定Rasp和Mart-2的其他底物。 3.确定Rasp/Mart-2如何在细胞内调节N-棕榈酰化 将进行亚细胞定位和运输实验，以确定Hh棕榈酰化的时间和位置。将设计抑制Mart-2介导的棕榈酰化的方法。将利用高通量筛选来鉴定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。将测定Mart-2抑制对Shh依赖性胰腺癌细胞生长的影响。 公共卫生相关性：Hh信号已被证明驱动许多人类癌症的生长，包括成神经管细胞瘤、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，并旨在开发Hh抑制剂，这些抑制剂可能在临床上用作Hh驱动的恶性肿瘤的抗肿瘤药物。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.