Mitogen-Activated Protein Kinase Kinase 5 Regulates Proliferation and Biosynthetic Processes in Procyclic Forms of Trypanosoma brucei

The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated protein kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light on the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acids biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acids biosynthesis in procyclic T. brucei.

致病性原生动物布氏锥虫（Trypanosoma brucei, T. brucei）可在哺乳动物与昆虫宿主中切换为不同的发育阶段。丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路能够将胞外刺激转导为一系列细胞应答，最终介导生物体对外界环境的适应。本研究将功能丧失（loss of function）实验策略与基于细胞培养氨基酸稳定同位素标记（stable isotope labeling with amino acids in cell culture, SILAC）的质谱（mass spectrometry, MS）技术联用，探究丝裂原活化蛋白激酶激酶5（mitogen-activated protein kinase kinase 5, MKK5）在布氏锥虫中的功能。实验结果显示，MKK5基因沉默（silencing）会显著降低布氏锥虫循环期形态的增殖能力。为阐明该表型相关的分子改变，我们对MKK5沉默细胞的总蛋白质组（proteome）与磷酸化蛋白质组（phosphoproteome）进行了检测。在总蛋白质组中，我们观察到与核糖体及翻译过程相关的蛋白质整体表达下调，同时脂肪酸生物合成通路的多个组分也呈现表达降低。此外，我们还发现关键代谢酶的蛋白水平与磷酸化修饰均发生改变，这提示氧化代谢（oxidative metabolism）受到抑制。综上，本研究结果表明，MKK5基因沉默会改变布氏锥虫循环期形态的细胞增殖、能量代谢、蛋白质及脂肪酸生物合成过程。