幼稚和发炎的鼠结肠的N末端分析揭示了豆科素的蛋白水解特征
N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain
影响因子:4.00000
分区:生物学3区 / 细胞生物学3区 生理学3区
发表日期:2025 Jan
作者:
Alexander R Ziegler, Bethany M Anderson, Rocco Latorre, Rachel M McQuade, Antoine Dufour, Brian L Schmidt, Nigel W Bunnett, Nichollas E Scott, Laura E Edgington-Mitchell
摘要
豆类蛋白酶是一种与炎症广泛相关的半胱氨酸蛋白酶。据报道，它裂解并激活蛋白酶激活的受体2，以促进与口腔癌相关的疼痛。在胃癌和结肠癌之外，几乎没有报道过豆蔻在胃肠道中的作用。使用基于豆科蛋白酶的活性探针LE28，我们报告说，豆科蛋白酶在鼠结肠的结肠细胞和巨噬细胞中被激活，并且在急性实验性结肠炎的模型中被上调。我们证明，通过药理抑制或基因缺失，结肠细胞中豆科蛋白酶活性的丧失对体外上皮渗透性没有影响。此外，豆类蛋白的抑制作用或缺失对与硫酸葡萄糖钠诱导的结肠炎相关的症状或组织学特征没有明显的影响，这表明其蛋白水解活性对于结肠炎的开始是可支配的。为了深入了解结肠内的豆科蛋白酶的潜在功能，我们对野生型和果蝇缺乏的小鼠对幼稚和发炎的结肠组织进行了野外波形离子迁移率迁移率细胞的定量蛋白质组学和N-末端分析。我们确定了16个改变的裂解位点，具有天冬酰胺内肽酶特征，可能是Legumain的直接底物，另外16个裂解位点可能是由Legumain间接介导的。我们还分析了肠道中与结肠炎广泛相关的蛋白质丰度和蛋白水解事件的变化，这与最近对炎性肠病患者的粘膜活检的分析进行了比较。总的来说，这些结果阐明了豆科蛋白酶的潜在功能，并突出了其从炎症到结直肠癌的过渡中的潜在作用。
Abstract
Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.