Generation and application of second messenger molecules by SMODS and SAVED

项目摘要/摘要 基于核苷酸的第二信使在生物体中发挥着重要的生物学功能。其中 各种第二信使分子，其中几个是由属于以下的NTase家族产生的 波尔布超级家族。在动物中，胞质NTase OAS和cGAs产生2‘-5’-连接的寡腺苷和2‘- 5‘-连接的c-GAMP，两者都能触发针对病毒和细菌的先天免疫反应 感染。在细菌中，NTase DncV产生3‘-5’连接的cGAMP，激活CapV抑制细胞 成长。利用比较基因组学、序列保守和结构分析的组合， Aravind和他的同事在细菌中发现了一个巨大的核苷酸中心系统网络。一家人 名为SMODS(次级信使寡核苷酸和二核苷酸合成酶)的NTase被预测为 产生第二个信使。几个保守的结构域被预测为该受体 SMODS生成的第二个信使。在预测的受体中，名为SAVE的结构域 (SMODS相关并融合到各种效应域)是最丰富的。除了 最近对一种SMODS产生的产物的表征，然而，预测的生化 SMODS和SAVE的生物学功能还没有得到实验测试。用人 生物信息学、生物化学和结构生物学的方法，我们的目标是追求以下两条路线 SMODS和SAVE的研究：1)我们将在体外重建SMODS和SAVE的酶活性 探测SMODS生成的Saved和Second Messenger之间的交互；以及2)我们将携带 对SMODS及其激活剂和底物的络合物SMODS和SAVE，SMODS的结构进行了研究 与SMODS生成的第二信使一起保存在复合体中。

Project Summary/Abstract Nucleotide-based second messengers play important biological functions in living organisms. Among various second messenger molecules, several of them are generated by families of NTases that belong to Polb superfamily. In animals, cytosolic NTases OAS and cGAS generate 2’-5’-linked oligoadenylates and 2’- 5’-linked c-GAMP, respectively, both of which trigger innate immune response against viral and bacterial infection. In bacteria, NTase DncV generates 3’-5’-linked cGAMP, which activates CapV to inhibit cell growth. Using a combination of comparative genomics, sequence conservation, and structure analysis, Aravind and coworkers uncovered a vast network of nucleotide-centric systems in bacteria. A family of NTases named SMODS (secondary messenger oligo and dinucleotides synthase) was predicted to generate second messengers. And several conserved domains were predicted to be receptors of the second messengers generated by SMODS. Among the predicted receptors, the domain named SAVED (SMODS-associated and fused to various effector domains) is the most abundant. With the exception of a recent characterization of the product generated by one of SMODS, however, the predicted biochemical and biological functions of SMODS and SAVED have not been experimentally tested. Employing approaches of bioinformatics, biochemistry, and structural biology, we aim to pursue the following two lines of investigation of SMODS and SAVED: 1) We will in vitro reconstitute the enzymatic activity of SMODS and probe interaction between SAVED and the second messengers generated by SMODS; and 2) We will carry out structural studies of SMODS and SAVED, SMODS in complex with its activator and substrates, and SAVED in complex with the second messengers generated by SMODS.