Lipid Modification of Extracellular Signaling Ligands

描述（由申请人提供）：细胞间通讯对于多细胞生物体的发育、生长和功能至关重要，这种通讯主要通过生长因子和其他细胞外配体进行。虽然许多工作都集中在了解细胞如何从细胞外信号中接收和传递信息，但对于配体如何在细胞外空间中导航仍有许多未知之处。最近已经发现几种突出的细胞外配体携带脂肪酸附着，包括主要的果蝇表皮生长因子受体配体Spitz。由于棕榈酰化已被证明发生在几个不相关的信号分子从不同的途径，它似乎可能是其他这样的分子也可能被棕榈酰化，这种修饰的共同机制功能可能会出现从不同的途径的研究。为了进一步了解Spitz棕榈酰化的功能，将在体内测试几种突变的Spitz蛋白。具体而言，将测定通过不可切割的跨膜结构域人工膜系连的未棕榈酰化Spitz构建体，以确定Spitz酰化的唯一目的是否是膜缔合。还将测定未棕榈酰化的糖基磷脂酰肌醇连接的Spitz，以确定Spitz与脂筏的结合是否对功能重要。为了研究其他细胞外配体是否被酰化，将研究果蝇基因组中未表征的假定酰基转移酶的功能。已知被酰化的三种细胞外信号分子，即Spitz、Hedgehog和Wingless，是膜结合O-酰基转移酶家族的相关酰基转移酶的底物。这个家族的七种酶存在于果蝇中，其中三种尚未被鉴定。编码这些蛋白质的基因将被突变并测试表型，表明在其他细胞外信号配体的棕榈酰化中的作用。还将使用细胞培养和生物化学实验直接测试各种类别的果蝇信号配体的脂化，以表征这些分子的疏水性。对于脂化的配体，将在体内测定干扰其脂化或突变其候选酰基转移酶的影响。细胞间通讯的缺陷与许多癌症有关。了解介导这种通信的分子的生物化学性质将有助于我们对这些疾病的理解。这些研究还可能揭示治疗药物的新途径，这些药物可以阻断细胞间信号传导，从而减弱癌症的扩散。

DESCRIPTION (provided by applicant): Intercellular communication is essential for the development, growth, and function of multicellular organisms, and this communication occurs predominantly via growth factors and other extracellular ligands. Although much work has focused on understanding how cells receive and transduce information from extracellular signals, much remains unknown about how ligands navigate the extracellular space. Several prominent extracellular ligands recently have been found to carry fatty acid attachments, including the primary Drosophila Epidermal Growth Factor Receptor ligand, Spitz. Because palmitoylation has been shown to occur on several unrelated signaling molecules from disparate pathways, it seems likely that other such molecules may also be palmitoylated and that a common mechanistic function for this modification may emerge from the study of different pathways. In order to further understand the function of Spitz palmitoylation, several mutant Spitz proteins will be tested in vivo. Specifically, an unpalmitoylated Spitz construct that is artificially membrane-tethered via an uncleavable transmembrane domain will be assayed in order to determine whether the sole purpose of Spitz acylation is membrane association. An unpalmitoylated glycosylphosphatidylinositol-linked Spitz also will be assayed to determine whether Spitz association with lipid rafts is important for function. To investigate whether other extracellular ligands are acylated, the function of uncharacterized putative acyltransferases in the Drosophila genome will be examined. The three extracellular signaling molecules that are known to be acylated, namely Spitz, Hedgehog, and Wingless, are substrates for related acyltransferase enzymes of the membrane bound O- acyl transferase family. Seven putative enzymes of this family exist in Drosophila, of which three have not yet been characterized. The genes encoding these proteins will be mutated and tested for phenotypes suggesting a role in palmitoylation of other extracellular signaling ligands. Drosophila signaling ligands of various classes also will be tested directly for lipidation using cell culture and biochemical experiments to characterize the hydrophobicity of these molecules. For ligands that are lipidated, the effects of perturbing their lipidation or of mutating their candidate acyltransferases will be assayed in vivo. Defects in intercellular communication have been implicated in many cancers. Understanding the biochemical nature of the molecules that mediate this communication will contribute to our understanding of these diseases. These studies also potentially will reveal new avenues for therapeutic agents that can block intercellular signaling and therefore attenuate the spread of cancer.