ELD607 specifically traffics Orai1 to the lysosome leading to inhibition of store operated calcium entry

IF 4.3 2区 生物学 Q2 CELL BIOLOGY Cell calcium Pub Date : 2024-08-14 DOI:10.1016/j.ceca.2024.102945
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Abstract

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1′s (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1’s α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607’s mechanism of action. We overexpressed either Orai1-HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.

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ELD607 能特异性地将 Orai1 运送到溶酶体,从而抑制储存操作的钙离子输入
Orai1 是一种质膜 Ca2+ 通道,参与贮存操作的钙离子进入(SOCE)。SOCE 可调控细胞生长、外吞、基因表达和炎症。我们之前发现,短腭肺和鼻上皮细胞克隆 1′s(SPLUNC1)的第六个α-螺旋(α6)与 Orai1 结合,从而抑制了 SOCE。SPLUNC1 蛋白水解不稳定,因此我们开发了基于 SPLUNC1 α6 区域的 11 个氨基酸肽 ELD607,它比 SPLUNC1/α6 更稳定、更有效。在此,我们研究了ELD607的作用机制。我们在 HEK293T 细胞中过表达 Orai1-HA 或 Orai1-YFP,通过共聚焦显微镜探究 ELD607 与 Orai1 的相互作用。我们还在多板阅读器中测量了作为细胞质 Ca2+ 水平标记的 Fluo-4 荧光的变化。ELD607 内化 Orai1 与 STIM1 无关。暴露于ELD607 15分钟和3小时都同样耗尽了质膜中的Orai1。然而,暴露于 ELD607 3 小时对 SOCE 的抑制作用更大。ELD607在内化后继续与Orai1共定位,这一过程依赖于泛素连接酶NEDD4.2的存在。同样,ELD607 增加了 Orai1 与泛素的共定位。ELD607还增加了Orai1与Rab5和7的共定位,但没有增加Rab11的共定位,这表明Orai1通过早期和晚期内体而不是循环内体进行运输。最后,ELD607导致Orai1,而不是Orai2、Orai3或STIM1迁移到溶酶体。我们的结论是,ELD607 能迅速与 Orai1 结合,并以与全长 SPLUNC1 相同的方式内化 Orai1 并将其送往溶酶体,从而导致 SOCE 的减少。
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来源期刊
Cell calcium
Cell calcium 生物-细胞生物学
CiteScore
8.70
自引率
5.00%
发文量
115
审稿时长
35 days
期刊介绍: Cell Calcium covers the field of calcium metabolism and signalling in living systems, from aspects including inorganic chemistry, physiology, molecular biology and pathology. Topic themes include: Roles of calcium in regulating cellular events such as apoptosis, necrosis and organelle remodelling Influence of calcium regulation in affecting health and disease outcomes
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