Mechanism of activation of a new cGAS-like enzyme in humans

项目摘要 小的rna第二信使分子很快被认为是有效的免疫调节。 具有治疗潜力的因素。在人类中，内源性第二信使2‘3’cGAMP是由 CGAS(环状GMP-AMP合成酶)是对细胞质中双链DNA感应的反应。 激活cGAS的DNA的来源可以是细胞内病原体的复制，也可以是细胞应激的结果， 在细胞分裂过程中不适当地分离基因组DNA，或通过摄取死亡来传递肿瘤细胞DNA 癌症中的细胞碎片。STING(干扰素基因刺激物)是人类必需的适配分子 能够触发I型干扰素基因下游转录的先天免疫系统 促炎细胞因子是病原菌感染后最早的细胞反应之一 病毒。STING下游信号反应直接依赖于其检测细菌循环的能力 二核苷酸和人源性cGAMP-有效地整合了细菌、病毒和自身免疫途径。 CGAS是最近发现的人类核苷酸转移酶亚家族的一个新成员 被称为Mab-21的酶。令人惊讶的是cGAS第二信使的保守和重要作用 在动物中的信号表明，其他孤儿Mab-21家族酶肯定也具有类似的功能 目的并可能产生新的RNA产物。MB21D2是一个可能的RNA第二信使合成酶 与cGAS具有较低的序列保守性，但具有较高的预测结构同源性。内部差异 CGAS和MB21D2之间的DNA结合位点暗示了不同的激活模式。我假设 MB21D2不仅是人类cGAs的结构同源物，而且是一个潜在的功能同源物，可以 合成一种新的第二信使，以响应细菌攻击引发下游免疫 回应。我的目标是用放射性标记的核苷酸测试纯化的重组MB21D2的功能 根据先前的结构和结合研究，筛选一组潜在的激活配体 关于细菌的相关同系物。我有初步证据证明MB21D2是一种活性酶，但还没有 以标识它生成的一个或多个产品。正在进行的实验旨在使用一种酵母蛋白 利用高分辨质谱仪检测裂解产物中新核苷酸产物的表达系统 分析。我还计划用X射线结晶学方法确定人MB21D2的结构 将有助于我们对其激活机制的理解。我的另一个主要目标是观察 MB21D2过表达、敲除和小分子激活的信号转导后果 活细胞的背景。我计划监测不同人类和小鼠细胞系的转录图谱，以 寻找mRNA表达的变化，寻找先天免疫信号的标志性信号 回应。我还计划测试MB21D2依赖于刺痛途径的免疫反应的激活。

Project Summary Small RNA second messenger molecules are quickly becoming recognized as potent immune regulatory factors with therapeutic potential. In humans, the endogenous second messenger 2′3′ cGAMP is generated by the enzyme cGAS (cyclic GMP–AMP synthase) in response to double stranded DNA sensing in the cytoplasm. The source of cGAS-activating DNA can be replicating intracellular pathogens or as a result of cellular stress, inappropriate separation of genomic DNA during cell division, or tumor cell DNA delivered by uptake of dying cell debris in cancer. STING (Stimulator of Interferon Genes) is an essential adaptor molecule in the human innate immune system capable of triggering downstream transcription of type-I interferon genes and proinflammatory cytokines as one of the earliest cellular responses to infection by pathogenic bacteria and viruses. STING downstream signaling responses are directly dependent on its ability to detect bacterial cyclic dinucleotides and human derived cGAMP– effectively integrating bacterial, viral, and autoimmunity pathways. cGAS is a recently identified and characterized member of a sub-family of human nucleotidyltransferase enzymes known as Mab-21. The surprisingly well conserved and important role of cGAS second messenger signaling in animals suggests that other orphan Mab-21 family enzymes must be fulfilling similar functional purposes and likely generate novel RNA products. MB21D2 is a putative RNA second messenger synthase that has low sequence conservation with cGAS yet has high predicted structural homology. Differences within the DNA binding site between cGAS and MB21D2 imply a different mode of activation. I hypothesize that MB21D2 is not only a structural but also a potential functional homolog of human cGAS which can synthesize a novel second messenger in response to bacterial insult triggering a downstream immune response. I aim to test the functionality of purified recombinant MB21D2 using radiolabeled nucleotide substrates and screen a panel of potential activating ligands based on previous structural and binding studies on related homologs from bacteria. I have preliminary evidence that MB21D2 is an active enzyme but have yet to identify the product or products that it generates. Ongoing experiments are designed to use a yeast protein expression system to detect novel nucleotide products in lysates using high resolution mass spectrometry analysis. I also plan to determine the structure of human MB21D2 using X-ray crystallographic methods which will inform our understanding behind the mechanism of its activation. My other major aim is to observe the signaling consequences of MB21D2 overexpression, knockout, and potential activation by small molecules in the context of living cells. I plan to monitor transcriptional profiles of different human and mouse cell lines to look for changes in mRNA expression and search for hallmark signatures of innate immune signaling responses. I also plan to test for MB21D2 activation of immune response dependent on the STING pathway.