Highly Conserved Cysteines Are Involved in the Oligomerization of Occludin-Redox Dependency of the Second Extracellular Loop
Highly Conserved Cysteines Are Involved in the Oligomerization of Occludin-Redox Dependency of the Second Extracellular Loop
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DOI：
10.1089/ars.2013.5288
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发表时间：
2014-02-20
影响因子：
6.6
通讯作者：
Blasig, Ingolf E.
中科院分区：
文献类型：
--
作者：
Bellmann, Christian;Schreivogel, Sophie;Blasig, Ingolf E.
紧密连接(TJ)标志物occludin是一种4-跨膜区(TMD)蛋白，其生理和病理功能尚不清楚，可与其他TJ蛋白相互作用。它是寡聚的，对氧化还原敏感。然而，齐聚的位置和机理尚不清楚。目的：为了确定低氧敏感的结合部位，我们研究了在正常和低氧孵育条件下，人类封闭蛋白中高度保守的半胱氨酸的氨基酸替换的后果。结果：(1)细胞外环2(ECL2)在对向细胞之间和细胞膜上分别显示出同亲的反式和顺式结合，这是由于一个环通过屏蔽的C216和C237之间的环内二硫键正确折叠而引起的。低氧和还原剂阻止了这种联系。(Ii)TMD1中的C82直接顺式缔合，不形成二硫键。(Iii)TMD1中的C76和TMD2中的C148限制了反式相互作用；C76还限制了封闭蛋白相关的细胞旁紧密度，并改变了claudin-1的链形态。(Iv)替代C82、C216或C237后，结合强度降低伴随着细胞膜中封闭蛋白流动性的增加。创新：这些数据使第一个实验证明的occludin及其亲性相互作用位点的结构模型成为可能，其中ECL2通过环内二硫键的形成，在occludin的低氧敏感寡聚和调节TJ的结构中发挥核心作用。结论：我们的发现支持了occludin作为低氧感受器的新概念，并有助于依赖地调节TJ装配氧化还原。这与组织屏障损伤的病原学相关。ECL2二硫键可能是TJ中四个TMD蛋白的模型，一个ECL中有两个保守的半胱氨酸。
The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. Aims: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. Results: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. Innovation: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. Conclusion: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL.