Biochemical and Functional Analysis of Wnt Acyltransferase, Porcupine

描述(申请人提供)：WNT蛋白包括一个分泌的信号分子家族，在胚胎发育期间的组织发育和细胞命运决定中发挥重要作用，以及在成人的组织维持和肿瘤发生中发挥重要作用。为了正确地传递信号，需要对Wnt蛋白进行加工、修饰和分泌。WNT加工涉及两个脂肪酰基部分的附着，即棕榈酸酯和棕榈油酸酯。不能结合这些脂肪酸使得Wnt不能启动细胞内的信号级联或有效地分泌。豪猪(PORCN)是一种多通道跨膜蛋白，是负责这些脂肪酸加合物附着的酰基转移酶。PORCN一直被认为是开发能够调节WNT相关疾病(如癌症和骨骼异常)中WNT信号活性的抑制剂的一个有吸引力的靶点。不幸的是，对豪猪的生化和功能特性的有限了解阻碍了这一目标的实现。该项目的长期目标是确定豪猪识别其底物(棕榈酸酯和WNT)并催化脂肪酸转移到WNT上的机制。在进行初步研究时，我发现PORCN本身是棕榈酰化的。我将通过确定PORCN棕榈酰化的功能意义来跟进这一新发现。目标1中的实验将确定棕榈酸酯掺入PORCN是否代表酰基酶中间体的形成，或者是另一种棕榈酰基转移酶修饰的结果。PORC上的棕榈酰化位点(S)将被识别和突变，并将对PORCN的稳定性、定位和酰基转移酶活性的影响进行监测。具体目标2涉及使用突变方法对PORCN进行全面的结构-功能分析，以确定脂肪酸结合、Wnt结合和棕榈酰酰基转移酶活性所需的关键残基。这些残基的功能相关性将通过分析点突变对猪的生化特性和活性以及对Wnt分泌和活性的影响来确定。此外，还将确定PORCN的膜拓扑结构和齐聚状态。这种方法将使我们能够确定催化残基在周围脂质双层平面内的位置，以及绘制预测的跨膜结构域。这些研究将有助于我们了解一种相对未被探索的多通道膜蛋白，该蛋白调节一个关键的信号分子家族。 公共卫生相关性：Wnt蛋白水平升高与多种人类癌症(乳腺癌、结肠癌、肺癌)以及人类骨骼异常有关。为了使WNT正常发挥作用，豪猪酶需要在WNT上添加两种脂肪酸(棕榈酸酯和棕榈油酸酯)。该项目的目标是破译豪猪酰化Wnt的机制，并研究豪猪作为癌基因的可能作用和开发选择性抑制剂的潜在靶点。

DESCRIPTION (provided by applicant): Wnt proteins comprise a family secreted signaling molecules that play major roles in tissue development and cell fate determination during embryogenesis, as well as tissue maintenance and oncogenesis in adults. In order to signal correctly, Wnt proteins need to be processed, modified and secreted. Wnt processing involves the attachment of two fatty-acyl moieties, palmitate and palmitoleate. Inability to incorporate these fatty acids renders Wnt unable to initiate the intracellular signaling cascade or to be efficiently secreted. Porcupine (Porcn), a multipass transmembrane protein, is the acyltransferase responsible for attachment of these fatty- acid adducts. Porcn has been postulated as an appealing target for the development of inhibitors that could modulate Wnt signaling activity in Wnt-related diseases such as cancer and skeletal abnormalities. Unfortunately, the limited knowledge about Porcupine's biochemical and functional properties has hampered this goal. The long-term objective of this project is to characterize the mechanism by which Porcupine recognizes its substrates (palmitate and Wnt) and catalyzes the transfer of fatty acids onto Wnt. While performing preliminary studies, I discovered that Porcn itself is palmitoylated. I will follow up on this novel finding by determining the functional significance of Porcn palmitoylation. Experiments in Aim 1 will ascertain whether palmitate incorporation into Porcn represents formation of an acyl-enzyme intermediate or is the result of modification by another palmitoyl acyltransferase. The site(s) of palmitoylation on Porc will be identified and mutated and effects on Porcn stability, localization and acyltransferase activity will be monitored. Specific Aim 2 involves a comprehensive structure-function analysis of Porcn, using a mutagenesis approach, to identify key residues required for fatty acid binding, Wnt binding and palmitoyl acyltransferase activity. The functional relevance of these residues will be determined by analyzing the effect of point mutations on Porcupines' biochemical properties and activity, and on Wnt secretion and activity. In addition, the membrane topology and oligomerization state of Porcn will be determined. This approach will allow us to determine where the catalytic residues lie within the plane of the surrounding lipid bilayer, as well as to map the predicted transmembrane domains. These studies will contribute to our knowledge of a relatively unexplored multipass membrane protein that regulates a critical family of signaling molecules. PUBLIC HEALTH RELEVANCE: Elevated levels of the protein Wnt have been linked to a wide variety of human cancers (breast, colon, lung), as well as in human skeletal abnormalities. In order for Wnt to function correctly, the enzyme Porcupine needs to add two fatty acids (palmitate and palmitoleate) onto Wnt. The goal of this project is to decipher the mechanism by which Porcupine acylates Wnt, as well as to investigate Porcupine's possible role as an oncogene and potential target for the development of selective inhibitors.