Mutations in the Drosophila melanogaster gene pyruvate kinase interfere with larval development

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster, however, are complicated by the fact the fly genome encodes six Pyk paralogs and the function of these individual genes remain poorly defined. To address this issue, we used a phylogenetic approach to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs while the other five Drosophila Pyk have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of two different Pyk mutant backgrounds revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectantly reveals that steady state pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomics findings, a complementary RNAseq analysis revealed that genes involved in lipid metabolism and peptidase activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight towards understanding how Drosophila larval metabolism adapts to disruption of glycolytic metabolism and immediate clinical relevance considering that Pyk deficiency is the most common congenital enzymatic defect in humans. RNA-seq of Pyk 23/+ heterozygous controls versus Pyk 23/31 CRISPR mutants, three whole-fly biological replicates

丙酮酸激酶（Pyruvate kinase, Pyk）是一类限速酶，可催化糖酵解的最后一步代谢反应。然而该酶的重要性远不止ATP生成，研究证实Pyk还可调控组织生长、细胞增殖与个体发育。不过在黑腹果蝇（Drosophila melanogaster）中开展该酶的相关研究却存在阻碍：果蝇基因组共编码6种Pyk旁系同源基因，且这些独立基因的功能仍未得到明确阐释。为解决这一问题，本研究采用系统发育学方法，证实唯一的Pyk基因所编码的酶与哺乳动物Pyk直系同源物相似度最高，而其余5种果蝇Pyk则与经典酶已发生显著分化。与该观察结果一致的是，针对两种不同Pyk突变背景的代谢组学研究显示，缺失Pyk的果蝇幼虫糖酵解过程出现严重阻滞，丙酮酸上游的糖酵解中间产物大量积累。然而本研究的分析还意外发现，Pyk突变体的稳态丙酮酸水平并未发生改变，这表明尽管代谢功能受限，果蝇幼虫仍可维持丙酮酸池的大小。与代谢组学结果相符，补充的RNA测序（RNA-seq）分析显示，Pyk突变体中参与脂质代谢与肽酶活性的基因表达水平上调，再次表明该糖酵解酶的缺失会诱导代谢其他方面发生代偿性变化。综上，本研究不仅为理解黑腹果蝇幼虫代谢如何适应糖酵解代谢紊乱提供了新见解，同时鉴于Pyk缺乏症是人类最常见的先天性酶缺陷疾病，本研究还具备直接的临床相关性。本实验的RNA-seq测序以Pyk 23/+杂合子作为对照，与Pyk 23/31 CRISPR突变体进行比对，共设置3份整虫生物学重复。