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

人体中新型 cGAS 样酶的激活机制

• 批准号: 9759505
• 负责人: Benjamin Robert Morehouse
• 金额: $ 6.12万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9759505
• 关键词: Agonist; Animals; Atlases; Autoimmune Diseases; Autoimmunity; Bacteria; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; C-terminal; Cell Line; Cell division; Cells; Cellular Stress; Chemicals; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Combined Modality Therapy; Cyclic GMP; Cytoplasm; DNA; DNA Binding; Dinucleoside Phosphates; Disease; Enzyme Activation; Enzymes; Expression Profiling; Family; Folic Acid; Future; Gene Expression; Genes; Genetic Transcription; Genomic DNA; Global Change; Goals; Growth and Development function; Hodgkin Disease; Homologous Gene; Housing; Human; Human Biology; Human Genome; IRF3 gene; Immune; Immune response; Immune signaling; Immunologics; Incubated; Infection; Inflammatory; Innate Immune System; Interferon Type I; Kidney; Knock-out; Laboratories; Lead; Left; Length; Ligand Binding; Ligands; Luciferases; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Messenger RNA; Metabolism; Methods; Molecular; Monitor; Mouse Cell Line; Natural Immunity; Nucleic Acid Binding; Nucleotides; Organism; Orphan; PC3 cell line; Pathway interactions; Periodicity; Phenotype; Play; Positioning Attribute; Protein Overexpression; Proteins; RNA; Radiolabeled; Reaction; Recombinants; Regulation; Reporter; Research; Resolution; Resources; Roentgen Rays; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Small RNA; Source; Stimulator of Interferon Genes; Sting Injury; Structure; Suggestion; System; Testing; Therapeutic; Therapeutic Intervention; Thin Layer Chromatography; Tissues; Transcriptional Activation; Vertebrates; Viral; Work; X-Ray Crystallography; Yeasts; base; cancer immunotherapy; cytokine; design; ds-DNA; experimental study; immune activation; immune checkpoint blockade; in vitro testing; mRNA Expression; member; microbial; microbiome; mouse model; new therapeutic target; novel; nucleotidyltransferase; overexpression; pathogen; pathogenic bacteria; pathogenic virus; programs; protein expression; response; sensor; small molecule; three dimensional structure; transcriptome sequencing; tripolyphosphate; tumor DNA; uptake; yeast protein

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.

项目摘要 小RNA第二信使分子很快被认为是有效的免疫调节分子。 具有治疗潜力的因素。在人类中，内源性第二信使2′3′ cGAMP是通过以下途径产生的： 酶cGAS（环状GMP-AMP合酶）响应于细胞质中的双链DNA传感。 cGAS激活DNA的来源可以是复制的细胞内病原体或作为细胞应激的结果， 细胞分裂过程中基因组DNA的不适当分离，或通过摄取染料递送的肿瘤细胞DNA 癌细胞碎片STING（干扰素基因刺激因子）是人类免疫系统中必需的衔接分子。 能够触发I型干扰素基因下游转录的先天免疫系统， 促炎细胞因子作为病原菌感染的最早细胞反应之一， 病毒STING下游信号传导应答直接依赖于其检测细菌循环的能力。 二核苷酸和人源性cGAMP-有效整合细菌、病毒和自身免疫途径。 cGAS是最近鉴定和表征的人核苷酸转移酶亚家族的成员 称为Mab-21的酶。cGAS第二信使的保守性和重要性令人惊讶 动物中的信号传导表明，其他孤儿Mab-21家族酶必须实现类似的功能， 目的并可能产生新的RNA产物。MB 21 D2是一种RNA第二信使合酶 其与cGAS具有低序列保守性，但具有高预测的结构同源性。内部分歧 cGAS和MB 21 D2之间DNA结合位点意味着不同的激活模式。我假设 MB 21 D2不仅是人cGAS的结构同源物，而且是人cGAS的潜在功能同源物，其可以 合成新第二信使以响应细菌损伤触发下游免疫 反应我的目的是使用放射性标记的核苷酸来测试纯化的重组MB 21 D2的功能性。 底物，并根据先前的结构和结合研究筛选一组潜在的活化配体 与细菌同源的基因。我有初步证据表明MB 21 D2是一种活性酶，但还没有 来识别它所产生的一个或多个产品。正在进行的实验旨在使用酵母蛋白 使用高分辨率质谱法检测裂解物中新核苷酸产物的表达系统 分析.我还计划使用X射线晶体学方法确定人类MB 21 D2的结构， 将有助于我们理解其激活机制。我的另一个主要目的是观察 MB 21 D2过表达、敲除和小分子潜在激活的信号传导后果， 活细胞的背景。我计划监测不同人类和小鼠细胞系的转录谱， 寻找mRNA表达的变化，并寻找先天免疫信号的标志性特征 应答我还计划测试MB 21 D2对依赖于STING途径的免疫应答的激活。