基质金属蛋白酶解离果蝇幼虫脂肪体的分子基础

During metamorphosis in holometabolic insects, such as the fruitfly, Drosophila melanogaster, and the silkworm, Bombyx mori, the larval organs have distinct developmental fates. Some larval organs undergo massive programmed cell death (PCD, including apoptosis and autophagy) and eventually disappear, some larval organs remodel to adult ones, while the larval fat body tissues undergo PCD and gradually remodel to individual cells, and some dissociated fat body cells survive until the adult stages. The applicants have previously found that fat body PCD and cell dissociation in Drosophila and Bombyx larvae are mainly regulated by 20-hydroxyecdysone (20E), juvenile hormone (JH) and nutritional signal, and that two matrix metalloproteinases (Mmp1 and Mmp2) cooperatively induce Drosophila larval fat body cell dissociation with distinct roles. With the synergism of structural biology and Drosophila genetics, we aim to solve the molecular basis of fat body cell dissociation by Mmps in Drosophila larvae in this project: (1) To define the crystal structure of Mmp1 and Mmp2 and to understand their substrate specificity and molecular cooperation. (2) To illustrate the molecular interactions between Mmp-induced cell dissociation and PCD after the identification of key coupling components. (3) To demonstrate how hormones and nutrition coordinately regulate fat body dissociation by controlling the mRNA and protein levels of Mmps. The accomplishment of this project will solve the molecular basis of an important physiological event during insect metamorphosis, and moreover, will shed lights on the structural, functional, and regulatory mechanism of Mmps in higher animals.

在果蝇和家蚕等完全变态昆虫的变态过程中，幼虫器官发育命运不同：一些幼虫器官因程序化细胞死亡（PCD；凋亡和自噬）大量发生而消失；一些幼虫器官重塑为成虫器官；而幼虫脂肪体则同时发生PCD和重塑，解离成单个细胞并部分存活到成虫期。申请者前期发现果蝇和家蚕的幼虫脂肪体PCD和重塑过程主要受激素和营养信号的双重调控，而两种基质金属蛋白酶（Mmp1和Mmp2）则协同解离果蝇的幼虫脂肪体。拟整合结构生物学和果蝇遗传学，深入阐释Mmp解离果蝇幼虫脂肪体的分子基础：（1）解析Mmp1和Mmp2的晶体结构，探索二者底物特异性的结构差别及其协作关系；（2）挖掘和探究偶联Mmp促进的脂肪体解离与PCD的关键中间分子；（3）阐明激素和营养信号如何精细调控Mmp的mRNA和蛋白水平而最终决定脂肪体解离。本项目既能阐释昆虫变态发育的一个重要生理事件，还将为高等动物中Mmps的结构、功能和调控研究提供借鉴和参考。

完全变态昆虫（如果蝇、家蚕、棉铃虫等）在幼虫-蛹变态期间，不完全变态昆虫在羽化期间（如美洲大蠊、德国小蠊等），许多器官会发生组织重塑，主要受营养、JH和20E调控。在本项目的资助下，我们就昆虫组织重塑的机理、调控及其应用发表SCI论文8篇，取得专利7项，主要取得如下重要结果：（1）阐析了JH和20E通过调节Mmps的表达和酶活精准控制脂肪体解离的分子网络，并发现Mmps在美洲大蠊的羽化过程中也起着关键作用；（2）揭示了JH信号和20E信号对20E合成的调控机理，JH信号诱导Kr-h1直接调控前胸腺中ecdysone的合成，20E信号的初级响应基因E75抑制前胸腺ecdysone合成，进一步筛选到具有明显效果的20E信号拮抗剂葫芦素B，并对动物界中20E前体胆固醇的生物合成途径做了进化分析；（3）在基因组和功能基因组水平上，从营养信号、JH信号和20E信号等方面，揭示了美洲大蠊超强适应能力和发育可塑性的机制；（4）发现Ras-Raf-MAPK信号通过磷酸化FoxA家族转录因子SGF1来促进细胞增殖和存活；（5）基于Dsx、Hedgehog、Wingless信号基因，取得美洲大蠊防治专利3项，基于Grh、P450基因，取得德国小蠊防治专利4项。这些研究结果对揭示昆虫发育的机理具有重要的科学意义，对益虫利用和害虫防治也具有潜在的应用价值。