Lipid modification of secreted signaling proteins

描述（由研究者提供）： 发育过程中的模式形成和形态发生是由在细胞之间传递信号的分泌蛋白质组织的。脂肪酸修饰已显示影响细胞内蛋白质的运输和膜靶向。最近发现三种分泌的信号蛋白也携带脂肪酸修饰是出乎意料的，因为这些蛋白质必须穿过水性细胞外环境。由酰基转移酶Rasp和Porcupine催化的棕榈酰化促进Hedgehog和Wnt家族蛋白的分泌、运输和信号传导活性。我们实验室最近的一项研究表明，Rasp也棕榈酰化Spitz，一种果蝇表皮生长因子受体的配体。棕榈酰化限制Spitz扩散，但增强其生物活性。这些结果表明，其他分泌信号蛋白也可能携带脂质修饰的可能性。本提案的目的是使用两种互补的方法来确定在发育过程中作为细胞间信号的分泌蛋白的脂质修饰的流行率。第一个目的是突变三个未知的果蝇基因编码酰基转移酶同源Rasp和豪猪，并测试突变体的信号蛋白功能的缺陷。第二个目标是调查样本的分泌信号蛋白从所有已知的家庭的疏水性时，在培养细胞中表达，疏水蛋白将被测试其能力，将放射性棕榈酸。如果获得了新配体的脂肪酸修饰的证据，则将使用生物化学和遗传学方法来鉴定修饰和负责的酰基转移酶。这种修饰在体内的重要性将通过酰化位点的突变进行测试。这些实验将提高我们对这种新的分泌发育信号修饰的功能作用的理解。 公共卫生相关性：最近的研究揭示了一个令人惊讶的发现，即细胞在发育过程中相互传递信号的几种分泌蛋白质都附着了脂肪酸。虽然这样的疏水性修饰似乎与通过细胞外环境的扩散不一致，但它们对于这些信号蛋白的功能是必不可少的。我们建议调查是否额外的分泌蛋白作为发育信号也进行类似的修改。生物化学和遗传技术的结合将被用来调查潜在的修饰蛋白质和可能添加修饰的酶。由于果蝇具有快速、经济和强大的遗传工具的优势，我们将果蝇作为模型系统。进化保护表明，这些结果也适用于人类，并可能阐明出生缺陷的基础。

DESCRIPTION (provided by investigator): Pattern formation and morphogenesis during development are organized by secreted proteins that transmit signals between cells. Fatty acid modifications have been shown to affect the trafficking and membrane targeting of intracellular proteins. The recent discovery that three classes of secreted signaling proteins also carry fatty acid modifications was unexpected, since these proteins have to travel through the aqueous extracellular environment. Palmitoylation catalyzed by the acyltransferases Rasp and Porcupine promotes the secretion, transport and signaling activity of Hedgehog and Wnt family proteins. A recent study from our lab demonstrated that Rasp also palmitoylates Spitz, a ligand for the Drosophila Epidermal growth factor receptor. Palmitoylation restricts Spitz diffusion, but enhances its biological activity. These results suggest the possibility that other secreted signaling proteins might also carry lipid modifications. The objective of this proposal is to use two complementary approaches to determine the prevalence of lipid modifications of secreted proteins that act as intercellular signals during development. The first aim is to mutate three uncharacterized Drosophila genes encoding acyltransferases homologous to Rasp and Porcupine, and to test the mutants for defects in signaling protein function. The second aim is to survey a sample of secreted signaling proteins from all the known families for hydrophobicity when expressed in cultured cells; hydrophobic proteins will then be tested for their ability to incorporate radioactive palmitate. If evidence for fatty acid modification of a novel ligand is obtained, both biochemical and genetic methods will be used to identify the modification and the acyltransferase responsible. The importance of this modification in vivo will be tested through mutation of the acylation site. These experiments will improve our understanding of the functional roles of this novel modification of secreted developmental signals. Public Health Relevance: Recent research has revealed the surprising finding that several secreted proteins used by cells to signal to each other during development have attached fatty acids. Although such hydrophobic modifications seem inconsistent with diffusion through the extracellular environment, they are essential for the functions of these signaling proteins. We propose to investigate whether additional secreted proteins that act as developmental signals also carry similar modifications. A combination of biochemical and genetic techniques will be used to survey both potentially modified proteins and the enzymes likely to add the modifications. We will use Drosophila as a model system, due to its advantages of speed, economy and powerful genetic tools. Evolutionary conservation suggests that the results will also be applicable to humans and may illuminate the basis of birth defects.