Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.
Quadram Institute, Norwich Research Park, Norwich NR4 7UA, UK.
Dis Model Mech. 2019 Mar 18;12(3):dmm037069. doi: 10.1242/dmm.037069.
Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.
潘氏细胞是提供抗菌屏障并维持小肠干细胞龛完整性的关键上皮细胞。潘氏细胞异常不幸对肠道健康有害，并且常与消化病理学相关，如克罗恩病或感染。在自噬受损的个体中也观察到类似的改变，自噬是一种回收细胞成分的过程。自噬损伤对潘氏细胞的直接影响尚未被分析。为了研究这一点，我们生成了一种特异性在肠上皮细胞中缺失的小鼠模型，使这些细胞的自噬受损。我们使用富含潘氏细胞的三维肠类器官，比较了野生型和自噬受损类器官的蛋白质组图谱。我们使用了一种综合计算方法，结合蛋白质-蛋白质相互作用网络、自噬靶向蛋白和功能信息，来识别自噬损伤和中断途径之间的机制联系。在 284 个改变的蛋白质中，有 198 个（70%）在自噬受损的类器官中更丰富，表明蛋白质降解减少。有趣的是，这些差异丰富的蛋白质包括 116 个（41%）预测为自噬蛋白 p62、LC3 和 ATG16L1 的选择性自噬靶标。我们的综合分析揭示了自噬介导的机制，这些机制降解了潘氏细胞功能中的关键蛋白质，如胞吐作用、细胞凋亡和 DNA 损伤修复。对其他类器官的转录组谱分析证实，自噬改变后观察到的 90%变化在蛋白质水平上而不是在基因表达水平上有影响。我们进行了进一步的验证实验，显示溶菌酶分泌的差异，证实了我们通过计算推断的胞吐作用下调。我们的观察结果可以解释自噬损伤如何影响潘氏细胞的内稳态功能，导致蛋白质水平的改变。本文有一篇与该论文的共同第一作者的第一人称采访。
