Distal gut microbiome targets of host anti-proteolytic proteins during colitis

结肠炎期间宿主抗蛋白水解蛋白的远端肠道微生物组目标

• 批准号: 10320030
• 负责人: Howard C Hang
• 金额: $ 22.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-18 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320030
• 关键词: Abate; Address; Affinity; Algorithms; Animal Model; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antiinflammatory Effect; Binding; Binding Proteins; Bioinformatics; Biological; Biology; C57BL/6 Mouse; Cells; Chemicals; Chronic; Colitis; Collaborations; Collection; Colorectal Cancer; Communities; Control Groups; Cysteine; Data; Development; Disease; Distal; Drug Targeting; Elastases; Enzymes; Experimental Designs; Family; Female; Future; Genetic; Genomics; Goals; Hand; Harvest; Homeostasis; Human; Ileitis; Immune; Immunocompromised Host; In Vitro; Infiltration; Inflammation; Inflammatory; Injections; Intestinal Content; Intestinal Mucosa; Intestines; Knowledge; Laboratories; Letters; Libraries; Liquid Chromatography; Maps; Mass Spectrum Analysis; Measures; Metabolism; Methodology; Methods; Modeling; Mus; Obesity; PI3 gene; Parents; Patients; Peptide Hydrolases; Property; Proteins; Proteome; Proteomics; Publishing; Rag1 Mouse; Research; Role; Sampling; Serine; Serine Protease; T cell therapy; T-Lymphocyte; Technology; Time; Translations; Trypsin; antimicrobial; base; biochemical model; chemically induced colitis; commensal bacteria; drug discovery; gut inflammation; gut microbiome; in vivo; interest; metaproteomics; microbial; microbial host; microbiome; mouse model; murine colitis; pathogenic bacteria; response; small molecule; stem; tandem mass spectrometry; therapeutic target

Title: Distal gut microbiome targets of host anti-proteolytic proteins during colitis ABSTRACT Our long-term goal is to advance our understanding of the distal gut microbiome from its basis in genomics with chemical biology and quantitative metaproteomics approaches. Over the last several years, we have developed a combination of chemical probe-based technologies, liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics, and bioinformatic analyses as approaches to interrogate and quantitate specific enzyme families in microbiome proteomes. We recently showed that colitic mice have a dramatic elevation in microbiome-secreted proteases within the distal gut relative to healthy mice using these combined methods. Correspondingly, we observe a significant increase in host anti-proteolytic proteins (APPs). Gut inflammation can be abated in colitis mouse models upon introduction of human APPs a-1-antitrypsin or elafin. While we posit that a-1-antitrypsin and elafin (and other APPs) reduce inflammation via inhibition of aberrant proteolytic activities, both APPs also have antimicrobial activity. Thus, the anti-inflammatory mechanism by which antitrypsin and elafin act could be primarily, or in part, attributable to altering bacterial composition. Our primary question is if APPs target microbial proteases. Here, we will elucidate the microbiome (and host) protein targets of six APPs overproduced in response to distal gut inflammation. These six APPs collectively target proteases from the serine, cysteine, and metallo subfamilies. We have purified all APPs of interest and aim to employ the proteins as “bait” to determine if these APPs primarily target the inhibition of bacterial proteases. We will subsequently identify and quantitate the APP-captured proteins with our LC-MS/MS metaproteomics methods. We propose to focus our APP-enrichment strategy and metaproteomics on a well- established adoptive T cell transfer murine model of chronic colitis and directly compare to control mice raised under identical conditions. Our preliminary data confirms that a-1-antitrypsin irreversibly binds a unique collection of secreted proteins within colitic microbiome samples compared to controls. This is significant, as expression levels of host elastase and trypsin (targets of a-1-antitrypsin) are similar between the colitis and control groups. Over the next two years, we anticipate that we will identify several families of host APP-targeted bacterial proteases upregulated in colitic mice, as classified by sequence and/or structural homology. This list of proteases will help guide and focus future biochemical, cell-based, and animal model research on specific protease families as potential inflammatory agents (and therapeutic targets), as well as provide an optimized APP-enrichment methodology for translation into human IBD patients and other microbiome-related diseases.

标题：结肠炎期间宿主抗蛋白水解蛋白的远端肠道微生物组靶标 摘要 我们的长期目标是从基因组学的基础上推进我们对远端肠道微生物组的理解 化学生物学和定量元蛋白质组学方法。在过去的几年里，我们 开发了基于化学探针的技术，液相色谱串联质谱 质谱（LC-MS/MS）蛋白质组学和生物信息学分析作为询问和定量的方法 微生物组蛋白质组中的特定酶家族。我们最近发现，结肠炎小鼠有一个戏剧性的， 相对于使用这些组合的健康小鼠，远端肠道内微生物组分泌的蛋白酶的升高 方法.相应地，我们观察到宿主抗蛋白水解蛋白（APP）的显著增加。 在结肠炎小鼠模型中，在引入人APP α-1-抗胰蛋白酶或 弹性蛋白酶。虽然我们认为α-1-抗胰蛋白酶和弹力蛋白酶（和其他APP）通过抑制炎症来减少炎症， 除了异常的蛋白水解活性外，两种APP还具有抗微生物活性。因此，抗炎机制 抗胰蛋白酶和弹性蛋白酶活性主要或部分归因于细菌组成的改变。 我们的主要问题是APP是否针对微生物蛋白酶。在这里，我们将阐明微生物组（和宿主） 6种APP的蛋白靶点在对远端肠道炎症的反应中过度产生。这六个APP共同 靶向丝氨酸、半胱氨酸和金属亚家族的蛋白酶。我们已经纯化了所有感兴趣的APP， 目的是利用蛋白质作为“诱饵”，以确定这些APP是否主要针对细菌的抑制， 蛋白酶随后，我们将用LC-MS/MS鉴定和定量APP捕获的蛋白质 元蛋白质组学方法。我们建议将我们的APP富集策略和元蛋白质组学集中在一个良好的- 建立了过继性T细胞转移慢性结肠炎小鼠模型并与对照小鼠直接比较 在相同的条件下。我们的初步数据证实，a-1-抗胰蛋白酶不可逆地结合一个独特的集合 大肠菌群样本中分泌蛋白的含量。这一点很重要，因为表达 宿主弹性蛋白酶和胰蛋白酶（α-1-抗胰蛋白酶的靶标）的水平在结肠炎组和对照组之间相似。 在接下来的两年里，我们预计我们将确定几个宿主APP靶向细菌家族， 如通过序列和/或结构同源性分类的，在结肠炎小鼠中上调的蛋白酶。这份蛋白酶清单 将有助于指导和集中未来的生物化学，细胞为基础的，和动物模型研究特定的蛋白酶家族 作为潜在的炎症因子（和治疗靶点），以及提供优化的APP富集 用于翻译成人类IBD患者和其他微生物组相关疾病的方法。