Biochemical and Functional Analysis of Wnt Acyltransferase, Porcupine

描述（由申请人提供）：Wnt蛋白包含一个家族分泌的信号分子，其在胚胎发生期间的组织发育和细胞命运决定以及成人的组织维持和肿瘤发生中起主要作用。为了正确地发出信号，Wnt蛋白需要被加工、修饰和分泌。Wnt加工涉及两个脂肪酰基部分，棕榈酸酯和棕榈油酸酯的连接。不能掺入这些脂肪酸使得Wnt不能启动细胞内信号级联或被有效地分泌。豪猪蛋白（Porcupine，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被认为是开发抑制剂的一个有吸引力的靶点，这些抑制剂可以调节Wnt相关疾病如癌症和骨骼异常中的Wnt信号传导活性。不幸的是，关于豪猪的生物化学和功能特性的有限知识阻碍了这一目标。该项目的长期目标是表征豪猪识别其底物（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。目的1中的实验将确定棕榈酸酯掺入Porcn中是否代表酰基-酶中间体的形成，或者是另一种棕榈酰酰基转移酶修饰的结果。将鉴定和突变Porc上的棕榈酰化位点，并监测对Porcn稳定性、定位和酰基转移酶活性的影响。具体目标2涉及使用诱变方法对Porcn进行全面的结构-功能分析，以鉴定脂肪酸结合、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豪猪的生化性质和活性以及对Wnt分泌和活性的影响来确定。此外，膜拓扑结构和寡聚状态的Porcn将被确定。这种方法将使我们能够确定催化残基位于周围的脂质双层的平面内，以及映射预测的跨膜结构域。这些研究将有助于我们了解一个相对未开发的多通道膜蛋白，调节一个关键的信号分子家族。

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.