细胞自噬对昆虫脂肪体中脂肪和储存蛋白的代谢调控

Via the degradation of stored lipids and proteins to maintain cell survival, autophagy plays critical roles in normal development and diseases such as tumor progression and cancer in animals. The applicant has successfully completed the previous Youth Fund Project 31101670 in advance, with five SCI papers including one publication in Autophagy. Meanwhile, we have discoverd that decreasing autophagy in the Bombyx fat body results in abnormal larval-pupal transition, pupal development and reproductive ability in adults. Later studies revealed that decreasing autophagy disrupted normal metabolism of the stored lipids and proteins in the Bombyx fat body, solidifying the hypothesis that autophagy mobilizes the stored energy resources in the larval fat body to maintain adult organ formation and reproductive maturation. On the basis of these findings, we aim to continuously unravel the detailed molecular mechanisms using Bombyx and Drosophila as two model insects. First, we will provide direct cellular evidences how acid lipases, lipid droplet, and lysosome participate in autophagy-mediated lipid degradation. Second, we will illustrate how autophagy affects endocytosis of the storage proteins to disrupt their normal metabolism, bringing up a unique in vivo system to dissect out the sophisticated relationship between autophagy and endocytosis. The accomplishment of this project will help illustrate the detailed molecular mechanisms how autophagy mobilizes the stored lipids and proteins in the larval fat body for supporting adult organ formation and reproductive maturation. This project will shed lights on general studies on autophagy-regulated metabloism and development in mammals.

细胞自噬通过降解脂肪和蛋白等能量物质而维持细胞存活，与动物的正常生长发育及肿瘤和癌症等疾病密切相关。申请者提早圆满完成了青年基金项目31101670预定任务，并发现下调家蚕脂肪体内的细胞自噬可导致幼虫变态发育异常、蛹重下降减少、成虫生殖能力下降。后续研究发现细胞自噬中断后，脂肪体中脂肪和储存蛋白的代谢受阻，基本证实了细胞自噬可动用脂肪体储存的能量物质而维持成虫器官形成和生殖发育这一假说。我们将利用家蚕和果蝇两种模式昆虫：（1）剖析细胞自噬介导脂肪降解的分子机制，为酸性脂肪酶参与细胞自噬和溶酶体介导的脂肪降解提供直接的细胞生物学证据；（2）阐释细胞自噬通过调控细胞内吞而影响储存蛋白代谢的作用机理，并提供一个解答细胞自噬和细胞内吞相互关系的研究体系。本项目有助于揭示细胞自噬通过影响脂肪体脂肪和储存蛋白代谢而调控昆虫变态和生殖发育的生命本质，并为高等动物相关研究提供重要借鉴，具有普遍生物学意义。

本项目研究发现，RNA干扰（RNAi）或超表达失活形式关键自噬（Atg）基因阻碍其功能后，家蚕和果蝇脂肪体中细胞自噬小体形成和脂代谢受阻，而果蝇中超表达Atg1基因则造成脂代谢的加剧；此外，氯喹处理阻碍自噬小体与溶酶体的融合后，同样可以造成上蔟时期家蚕脂肪体内脂肪的累积，进一步研究发现参与自噬的泛素化蛋白能与骨架蛋白结合参与脂滴的降解，解析了自噬小体的形成及其与溶酶体的融合参与自噬介导的脂代谢的分子机制。证实了中性脂类和酸性的功能差异：中性脂酶brummer不影响溶酶体的酸性，而家蚕假定的酸性脂酶acid lipase1基因RNAi后则影响家蚕脂肪体溶酶体的酸化，进一步证实acid lipase1只有在脂质和饥饿信号存在时，才会与酸性的溶酶体共定位，证实其是真正的酸性脂酶发挥功能。首次证实蜕皮激素（20E）及其信号在转录和翻译水平诱导家蚕v-ATPases的上调，从而促进溶酶体的酸化及v-ATPases参与的细胞自噬进程，发现了20E除诱导Atg基因表达和抑制mTOR活性外调控细胞自噬发生的新方式。.家蚕预蛹时期，脂肪体从血淋巴中吸收的蛋白质主要分布在55-100kDa之间，蛋白质谱分析在家蚕雌虫的血淋巴和脂肪体中一共鉴定到6个家蚕血蓝蛋白家族成员，其中4个为首次鉴定到转录本。细胞自噬基因RNAi后家蚕脂肪体对55-100kDa蛋白的吸收减少，进一步研究发现储存蛋白的吸收主要依赖于网格蛋白介导的内吞，而网格蛋白介导的内吞受阻后，内吞途径上的关键rab蛋白水平降低，细胞自噬显著上调。本项目已发表受资助英文SCI文章6篇、中文文章1篇；并且有2篇SCI英文、1篇中文文章正在审稿和投稿之中；与此同时，通过本项目的开展，项目组还在溶酶体、内吞与自噬的相互作用机制方面进行了深入的挖掘，获得了非常重要的原始数据和积累，为今后的相关延续工作的开展奠定了基础。