Targeting bacterial proteases involved in PAR signaling to treat inflammatory bowel diseases

项目摘要 克罗恩病和溃疡性结肠炎是炎症性肠病(IBD)的一种形式，影响超过6.8 全球有一百万名患者。由于到目前为止还没有治愈的方法，治疗仅限于减轻IBD症状。 如严重腹泻、体重减轻、疲劳和疼痛。尽管调停的确切机制 该病的发病机制尚不清楚，肠道蛋白过度分解并伴有信号转导失调。 的蛋白水解酶激活受体(PARs)已被确定为IBD的重要驱动因素 胃肠道(GI)疾病。PARs是一类独特的四种真核G蛋白偶联受体 (GPCRs)是由胞外N-肽序列的蛋白水解性切割直接调节的 终端域(NTD)。切割揭示了一个被拴住的激活配体或改变了受体的构象 诱导激活。PAR信号可以通过破坏肠道上皮细胞的完整性来促进炎症 屏障，在生理条件下允许营养物质渗透，但限制细菌进入 病原体和毒素。许多胃肠道疾病都伴随着屏障功能丧失和肠道生物失调。 微生物组。共生菌来源的蛋白水解酶可能是肠道的重要调节因子 动态平衡和发病机制，从而使其成为潜在的治疗靶点。然而，目前还不清楚是哪一种 肠道中的共生菌株产生胞外蛋白水解酶以及这些酶如何影响健康 通过共定位的PARs的蛋白分解引起的疾病。我们假设有益的共生细菌分泌 通过基础激活或蛋白水解性脱敏来抑制过度炎症的蛋白水解酶。 相反，病原菌通过增加PAR来分泌促进炎症的蛋白酶。 激活。因此，肠道微生物群和病原菌产生的蛋白酶具有 有可能成为治疗各种形式IBD的有价值的新治疗靶点。测试我们的 假设，我们将开发一种强大的体外试验来广泛筛选具有PAR加工活性的蛋白酶 在共生和致病细菌物种中都是如此。然后我们将鉴定特定的PAR加工酶。 并利用细胞培养评估它们在调节上皮屏障完整性和炎症中的特定作用 系统和肠道器官。最后，我们将通过以下方式确定已识别的蛋白酶的治疗相关性 证实从活动期IBD患者分离的临床样本中它们的存在和活性升高。 最终，这项工作将鉴定由细菌菌株产生的特定的PAR加工酶，并确定 它们影响IBD发病机制的机制。

Project Summary Crohn’s disease and ulcerative colitis are forms of inflammatory bowel disease (IBD) that affect more than 6.8 million patients worldwide. Because no cure is available to date, treatment is limited to reducing IBD symptoms such as severe diarrhea, weight loss, fatigue and pain. Although the exact mechanisms that mediate the pathogenesis of the disease are unclear, excessive proteolysis in the gut combined with dysregulated signaling of protease-activated receptors (PARs) have been identified as important drivers of IBD and additional gastrointestinal (GI) diseases. PARs are a unique class of four eukaryotic G-protein coupled receptors (GPCRs) that are directly regulated by proteolytic cleavage of a peptide sequence in the extracellular N- terminal domain (NTD). Cleavage reveals a tethered activating ligand or alters the receptor conformation to induce activation. PAR-signaling can promote inflammation by disrupting the integrity of the intestinal epithelial barrier, which under physiological conditions allows permeability of nutrients but restricts the entry of bacterial pathogens and toxins. Many GI diseases are accompanied by loss of barrier function and dysbiosis of the gut microbiome. Proteases derived from commensal bacteria are likely to be important regulators of gut homeostasis and pathogenesis, thus making them potential therapeutic targets. However, it is unclear which extracellular proteases are produced by commensal strains in the gut and how these enzymes affect health and disease by proteolysis of co-localized PARs. We hypothesize that beneficial commensal bacteria secrete proteases that keep excessive inflammation in check by basal activation or proteolytic desensitization of PARs. Conversely, pathobiont bacteria species secrete proteases that promote inflammation via increased PAR activation. Therefore, proteases produced by the gut microbiota as well as pathogenic bacteria have the potential to be valuable new therapeutic targets for the treatment of various forms of IBD. To test our hypothesis, we will develop a robust in vitro assay to broadly screen for proteases with PAR-processing activity in both commensals and pathobiont bacterial species. We will then identify specific PAR processing proteases and assess their specific roles in regulating epithelial barrier integrity and inflammation using cell culture systems and gut organoids. Finally, we will establish the therapeutic relevance of the identified proteases by confirming their presence and elevated activity in clinical samples isolated from patients with active IBD. Ultimately, this work will identify specific PAR processing proteases produced by bacterial strains and define the mechanism by which they impact the pathogenesis of IBD.