AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability
AP2M1 mediates autophagy-induced CLDN2 (claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability
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AP2M1通过内吞作用和与LC 3的相互作用介导自噬诱导的CLDN2（claudin 2）降解，并降低肠上皮紧密连接通透性
DOI：
10.1080/15548627.2021.2016233
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发表时间：
2021-12-26
期刊：
影响因子：
13.3
通讯作者：
Nighot, Prashant
中科院分区：
文献类型：
--
作者：
Ganapathy, Ashwinkumar Subramenium;Saha, Kushal;Nighot, Prashant
肠上皮紧密连接（TJ）提供了防止腔内抗原细胞旁渗透的屏障。 TJ 屏障缺陷，例如成孔 TJ 蛋白 CLDN2 (claudin-2) 水平升高，与炎症性肠病相关。我们之前报道过饥饿诱导的巨自噬/自噬通过降解成孔的 CLDN2 来增强 TJ 屏障。在这项研究中，我们研究了自噬诱导 CLDN2 降解的分子机制。 CLDN2 降解在多种自噬诱导模式中持续存在。免疫定位、膜分级和药理学抑制研究表明，饥饿时网格蛋白介导的 CLDN2 内吞作用增加。抑制网格蛋白介导的内吞作用可消除自噬诱导的 CLDN2 降解和 TJ 屏障的增强。免疫共沉淀研究显示，饥饿时 CLDN2 与网格蛋白和衔接蛋白 AP2（AP2A1 和 AP2M1 亚基）以及 LC3 和溶酶体的关联增加，表明网格蛋白介导的内吞作用在自噬诱导的 CLDN2 降解中的作用。饥饿时 AP2M1 的表达和磷酸化增加。 AP2M1 激活的体外、体内（小鼠结肠）和离体（人结肠）抑制可防止 CLDN2 降解。 AP2M1 敲除通过减少 CLDN2-LC3 相互作用来阻止自噬诱导的 CLDN2 降解。定点诱变显示 AP2M1 与 CLDN2 酪氨酸基序（YXXCYRILLIC 大写字母 EF）结合（67-70 和 148-151）。 ATG7 敲除细胞中 CLDN2 和 TJ 通透性基线表达增加以及 CLDN2-AP2M1-LC3 相互作用减少，验证了自噬在调节 CLDN2 水平中的作用。小鼠中 Atg7 的急性缺失会增加 CLDN2 水平以及对实验性结肠炎的易感性。连接 CLDN2、AP2M1 和 LC3 的自噬调节分子机制可能提供对抗肠道炎症的治疗工具。
The intestinal epithelial tight junctions (TJs) provide barrier against paracellular permeation of lumenal antigens. Defects in TJ barrier such as increased levels of pore-forming TJ protein CLDN2 (claudin-2) is associated with inflammatory bowel disease. We have previously reported that starvation-induced macroautophagy/autophagy enhances the TJ barrier by degrading pore-forming CLDN2. In this study, we examined the molecular mechanism underlying autophagy-induced CLDN2 degradation. CLDN2 degradation was persistent in multiple modes of autophagy induction. Immunolocalization, membrane fractionation, and pharmacological inhibition studies showed increased clathrin-mediated CLDN2 endocytosis upon starvation. Inhibition of clathrin-mediated endocytosis negated autophagy-induced CLDN2 degradation and enhancement of the TJ barrier. The co-immunoprecipitation studies showed increased association of CLDN2 with clathrin and adaptor protein AP2 (AP2A1 and AP2M1 subunits) as well as LC3 and lysosomes upon starvation, signifying the role of clathrin-mediated endocytosis in autophagy-induced CLDN2 degradation. The expression and phosphorylation of AP2M1 was increased upon starvation. In-vitro, in-vivo (mouse colon), and ex-vivo (human colon) inhibition of AP2M1 activation prevented CLDN2 degradation. AP2M1 knockout prevented autophagy-induced CLDN2 degradation via reduced CLDN2-LC3 interaction. Site-directed mutagenesis revealed that AP2M1 binds to CLDN2 tyrosine motifs (YXXCYRILLIC CAPITAL LETTER EF) (67-70 and 148-151). Increased baseline expression of CLDN2 and TJ permeability along with reduced CLDN2-AP2M1-LC3 interactions in ATG7 knockout cells validated the role of autophagy in modulation of CLDN2 levels. Acute deletion of Atg7 in mice increased CLDN2 levels and the susceptibility to experimental colitis. The autophagy-regulated molecular mechanisms linking CLDN2, AP2M1, and LC3 may provide therapeutic tools against intestinal inflammation.