Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis

BACKGROUND Nicotinamide adenine dinucleotide (NAD) plays a central role in energy metabolism and integrates cellular metabolism with signalling and gene expression. NAD biosynthesis depends on the enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18), in which converge the de novo and salvage pathways. OBJECTIVE The purpose of this study was to analyse the protein-protein interactions (PPI) of NMNAT of Leishmania braziliensis (LbNMNAT) in promastigotes. METHODS Transgenic lines of L. braziliensis promastigotes were established by transfection with the pSP72αneoαLbNMNAT-GFP vector. Soluble protein extracts were prepared, co-immunoprecipitation assays were performed, and the co-immunoprecipitates were analysed by mass spectrometry. Furthermore, bioinformatics tools such as network analysis were applied to generate a PPI network. FINDINGS Proteins involved in protein folding, redox homeostasis, and translation were found to interact with the LbNMNAT protein. The PPI network indicated enzymes of the nicotinate and nicotinamide metabolic routes, as well as RNA-binding proteins, the latter being the point of convergence between our experimental and computational results. MAIN CONCLUSION We constructed a model of PPI of LbNMNAT and showed its association with proteins involved in various functions such as protein folding, redox homeostasis, translation, and NAD synthesis.

背景：烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD）在能量代谢中处于核心地位，可将细胞代谢与信号传导及基因表达相整合。烟酰胺腺嘌呤二核苷酸的生物合成依赖于烟酰胺/烟酸单核苷酸腺苷酰转移酶（nicotinamide/nicotinate mononucleotide adenylyltransferase, NMNAT; EC: 2.7.7.1/18），该酶是从头合成途径与补救合成途径的交汇节点。
研究目的：本研究旨在分析巴西利什曼原虫（Leishmania braziliensis）前鞭毛体中巴西利什曼原虫NMNAT（LbNMNAT）的蛋白质-蛋白质相互作用（protein-protein interactions, PPI）。
方法：通过转染pSP72αneoαLbNMNAT-GFP载体，构建巴西利什曼原虫前鞭毛体转基因株系。制备可溶性蛋白提取物，开展免疫共沉淀实验，并利用质谱技术对免疫共沉淀产物进行分析。此外，借助网络分析等生物信息学工具构建PPI网络。
结果：研究发现，参与蛋白质折叠、氧化还原稳态及翻译过程的蛋白质可与LbNMNAT蛋白发生相互作用。PPI网络显示，烟酸与烟酰胺代谢通路中的酶类以及RNA结合蛋白均可与LbNMNAT结合，其中RNA结合蛋白为本研究实验结果与计算分析结果的交汇点。
主要结论：本研究构建了巴西利什曼原虫LbNMNAT的蛋白质相互作用模型，并证实其可与参与多种生物学功能的蛋白质产生关联，包括蛋白质折叠、氧化还原稳态、翻译过程以及NAD合成。