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）的形式，影响超过6.8 全世界有100万患者。由于目前尚无治愈方法，治疗仅限于减轻IBD症状 如严重腹泻、体重减轻、疲劳和疼痛。尽管调节细胞凋亡的确切机制 疾病的发病机制尚不清楚，肠道中过度的蛋白水解与失调的信号传导相结合 蛋白酶激活受体（PAR）已被确定为IBD的重要驱动因素， 胃肠道（GI）疾病。PAR是一类独特的真核G蛋白偶联受体 GPCR是由细胞外N-乙酰基转移酶中的肽序列的蛋白水解切割直接调节的。 末端结构域（NTD）。裂解揭示了一个拴系激活配体或改变受体构象， 诱导活化。PAR信号可以通过破坏肠上皮细胞的完整性来促进炎症 在生理条件下允许营养物质渗透但限制细菌进入的屏障 病原体和毒素。许多胃肠道疾病伴随着屏障功能的丧失和肠道生态失调 微生物组来源于肠道细菌的蛋白酶可能是肠道的重要调节因子 稳态和发病机制，从而使它们成为潜在的治疗靶点。然而，目前还不清楚 胞外蛋白酶是由肠道中的益生菌菌株产生的，以及这些酶如何影响健康 和共定位PAR的蛋白水解引起的疾病。我们假设有益的肠道细菌分泌 通过PAR的基础激活或蛋白水解脱敏来控制过度炎症的蛋白酶。 相反，致病菌物种分泌蛋白酶，通过增加PAR促进炎症 activation.因此，由肠道微生物群以及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。来测试我们 假设，我们将开发一种稳健的体外测定来广泛筛选具有PAR加工活性的蛋白酶 在寄生虫和致病性细菌物种中。然后，我们将确定特定的PAR加工蛋白酶 并使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 系统和肠道类器官。最后，我们将通过以下方式确定所鉴定的蛋白酶的治疗相关性： 证实了它们在分离自活动性IBD患者的临床样品中的存在和升高的活性。 最终，这项工作将确定特定的PAR加工蛋白酶产生的细菌菌株，并定义 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.