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

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 利用化学生物学和定量代谢蛋白质组学方法。在过去的几年里，我们有 开发了一种基于化学探针的技术组合，液相色谱串联质谱仪 光谱(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 微生物蛋白质组中的特异酶家族。我们最近表明，结肠炎小鼠有戏剧性的 与健康小鼠相比，使用这些组合后远端肠道中微生物组分泌的蛋白水解酶的升高 方法：研究方法。相应地，我们观察到宿主抗蛋白水解蛋白(APP)显著增加。 在结肠炎小鼠模型中，引入人类APP a-1-抗胰蛋白酶或 伊拉芬。虽然我们假设a-1-抗胰蛋白酶和弹力素(和其他APP)通过抑制 异常的蛋白分解活性，这两个APP也具有抗菌活性。因此，抗炎机制 通过这种作用，抗胰蛋白酶和弹力蛋白的作用可能主要或部分归因于改变细菌组成。 我们的主要问题是，应用程序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) 六个APP的蛋白质靶标因远端肠道炎症而过度产生。这六款应用程序加在一起 丝氨酸、半胱氨酸和金属亚家族的靶蛋白。我们已经净化了所有感兴趣的应用程序 目的利用这些蛋白质作为“诱饵”来确定这些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 代谢蛋白质组学方法。我们建议将我们的应用程序丰富战略和元蛋白质组学集中在一口井上- 建立过继T细胞转移慢性结肠炎小鼠模型并与饲养的对照小鼠直接比较 在相同的条件下。我们的初步数据证实，a-1-抗胰蛋白酶不可逆转地结合一个独特的集合 与对照相比，大肠菌微生物群样本中分泌蛋白的数量。这一点意义重大，就像表达 在结肠炎组和对照组之间，宿主弹性蛋白酶和胰蛋白酶(a-1-抗胰蛋白酶的靶标)水平相似。 在接下来的两年里，我们预计将确定几个宿主应用程序目标细菌家族 蛋白水解酶在结肠炎小鼠中上调，按序列和/或结构同源性分类。这是一组蛋白水解酶 将帮助指导和集中未来的生化、细胞和动物模型研究特定的蛋白水解酶家族 作为潜在的炎症剂(和治疗靶点)，以及提供优化的APP-浓缩 翻译成人类IBD患者和其他微生物组相关疾病的方法。

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.