应用脂质组学/代谢组学研究果蝇dAcsl影响个体发育缺陷的机制

Long-chain acyl-coenzyme A synthetases (ACSL) play a pivotal role in the biosynthesis of membrane lipids (e.g. phospholipids) and neutral lipids, and participate in numerous pathways controlling essential aspects of physiology such as lipid metabolism, signal transduction and cold-induced thermogenesis. ACSL4 represents the very first gene found to be implicated in the manifestation of non-specific X-1inked mental retardation（MRX）that is associated with fatty acid metabolism. Mutants of dAcsl, the Drosophila ortholog of human ACSL3/4, exhibit severe developmental defects spanning the entire body. The precise mechanisms pertaining to how mutation in dAcsl perturbs lipid homeostasis that underlie the whole-body developmental defects observed in dAcsl mutants, however, have remained unclear. Global lipidomic and metabolomic analyses can facilitate a better understanding of the mechanistic function of dAcsl via unravelling quantitative perturbations in the levels of its enzymatic substrates, products, as well as metabolic intermediates in dAcsl mutants relative to controls. Thus, an omics-oriented approach is expected to confer a non-biased and wholesome view of the metabolic landscape altered by dAcsl mutation, providing novel insights to phenotypic defects instigated by dAcsl mutation. We propose to apply systematic lipidomic and metabolomic analyses to dissect the global lipidomic/metabolomic profiles of Drosophila dAcsl mutants across development. Based on the omics profiles, molecular candidates implicated in dAcsl-driven metabolic dysfunction will be deciphered. Next, in-depth functional assays will be conducted by fine-tuning the expression of essential metabolites. By combining approaches in systems biology, genetics as well as molecular and cellular biology, we strive to derive the precise pathogenic mechanisms that essentially underlie developmental defects resulting from dAcsl mutation.

长链脂酰辅酶A（ACSL）是调控膜脂（磷脂等）及中性脂等合成的一个枢纽，参与脂类代谢、信号转导、寒冷刺激下产热等多个分子途径。ACSL4基因是第一个被发现与机体脂肪酸代谢相关的非综合征型X染色体连锁智力障碍致病基因。果蝇ACSL3/4的同源基因dAcsl突变体存在严重的全身发育缺陷，但dAcsl如何通过影响脂代谢及其他代谢过程进而影响全身发育缺陷的分子细胞机制目前还不清楚。探索dAcsl对细胞代谢与及机体发育障碍的影响需要分析脂质组以及主要代谢物的影响。根据其产物、底物以及其他代谢产物的含量变化，从全面代谢的角度阐明dacsl缺失的致病机理。本项目拟计划应用系统脂质组学、代谢组学方法分析果蝇突变体发育过程中的全脂谱图、代谢变化规律，选取相关的关键因子，对它们进行深入的功能分析和表达调控的研究。集合生物信息学、遗传、分子和细胞生物学、生物化学手段，明确dAcsl缺失的致病机理。

能量稳态和脂质稳态的维持对于生命体来说十分重要，脂质稳态的失衡会导致体内的代谢紊乱，并进一步诱发生理疾病，例如血脂的紊乱与肥胖、糖尿病和心血管疾病直接相关。本课题以果蝇为模型探索肠道、血淋巴与脂肪体之间的脂质稳态。我们发现果蝇长链脂酰辅酶A合酶(dAcsl)在脂肪体中敲降后会影响肠道和血淋巴中的中性脂质水平，导致血淋巴中中性脂含量减少，而肠道中积累大量的中性脂。同时还伴随着其他器官，如脑、成虫盘中中性脂含量的减少。通过脂组学手段我们发现，除肠道中中性脂的变化外，在脂肪体中出现了鞘糖脂代谢异常，大量鞘糖脂含量减少，而底物神经酰胺大量积累。. 为探索相关机制，我们对脂肪体进行蛋白组学检测，通过通路分析发现脂肪体中内质网、内质网蛋白进程和蛋白糖基化修饰等通路在dAcsl敲降组中降低，同时内质网中出现明显的内质网应激现象。并且我们发现血淋巴和脂肪体中的脂蛋白含量明显降低，由于果蝇的脂蛋白仅在脂肪体中形成，所以我们推测，由于脂肪体中的脂蛋白含量降低导致血淋巴中脂蛋白含量相应减少，最终不足以从肠道中吸收脂质，导致肠道从食物中吸收的营养滞留在肠道中，并大量积累。当我们在dAcsl RNAi背景下过表达载脂蛋白apoLpp时可以部分恢复肠道中性脂质积累的表型，表明脂蛋白介导了dAcsl敲降导致肠道脂质堆积的过程。由于脂蛋白需要糖基化修饰才能形成成熟的蛋白，如果其不能正常糖基化则会被降解。我们通过小范围的遗传筛选发现当在脂肪体中敲降糖基化相关酶CG9035后也会出现肠道中性脂脂质积累的表型，表明了CG9035可能参与到了脂蛋白的成熟过程中。同时dAcsl的敲降会导致内质网膜系统紊乱，并导致内质网膜形态异常。. 综上所述，我们认为dAcsl在脂肪体敲降后导致内质网结构异常，脂蛋白糖基化过程受到影响，不能正确折叠，进一步导致脂蛋白降解，最终分泌到血淋巴中的脂蛋白不足以从肠道中转运其从食物中吸收的脂质，最终导致肠道出现中性脂质异位积累的现象。