Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation.
Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation.
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与钙调蛋白的相互作用对于刺激后硫美特寡肽酶的分泌很重要。
DOI：
10.1111/j.1742-4658.2009.07144.x
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Ferro,EmerS
中科院分区：
文献类型：
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作者：
Russo,LilianC;Goñi,CamilaN;Castro,LeandroM;Asega,AmandaF;Camargo,AntonioCM;Trujillo,CleberA;Ulrich,Henning;Glucksman,MarcJ;Scavone,Cristoforo;Ferro,EmerS
蒂美特寡肽酶（EC 3.4.24.15；EP24.15）最初被描述为一种神经肽代谢酶，在大脑、肾脏和神经内分泌组织中高度表达。 EP24.15缺乏进入分泌途径的典型信号肽序列，并通过非常规且未知的机制由细胞分泌。在这项研究中，我们发现了 EP24.15 和钙调蛋白之间的一种新型钙依赖性相互作用，这对于 EP24.15 的刺激分泌（但不是组成型分泌）很重要。我们证明，在体外，EP24.15 和钙调蛋白仅在存在 Ca2+ 的情况下才会发生物理相互作用，估计 Kd 值为 0.52μm。共聚焦显微镜证实 EP24.15 与钙调蛋白共定位于静息 HEK293 细胞的细胞质中。当细胞用钙离子载体 A23187 或蛋白激酶 A 激活剂毛喉素处理时，这种共定位显着增加。 HEK293 细胞中钙调蛋白的过度表达足以大大增加 A23187 刺激的 EP24.15 的分泌，而这种分泌可以被钙调蛋白抑制剂卡咪唑鎓抑制。 KT5720 对蛋白激酶 A 的特异性抑制可减少 A23187 刺激的 EP24.15 分泌，并抑制毛喉素与 A23187 的协同作用。用卡米达唑和 KT5720 治疗几乎消除了 A23187 对 EP24.15 分泌的刺激作用。总之，这些数据表明 EP24.15 和钙调蛋白之间的相互作用在细胞内受到调节，对于刺激 HEK293 细胞分泌 EP24.15 很重要。结构化数字摘要·MINT-7148420:EP24.15(uniprotkb:P52888) 和钙调蛋白 (uniprotkb:P62161) 结合 (MI:0407) 通过表面等离子体共振(MI:0107)•MINT-7148437:EP24.15(uniprotkb:P52888)和钙调蛋白(uniprotkb:P62158)通过表面等离子体共定位(MI:0403) 共振(MI:0107)•MINT-7148406：钙调蛋白(uniprotkb:P62161)通过下拉(MI:0096)与EP24.15(uniprotkb:P52888)结合(MI:0407)
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide‐metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium‐dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that,in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimatedKdvalue of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187‐stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187‐stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.Structured digital abstract•MINT‐7148420:EP24.15(uniprotkb:P52888) andCalmodulin(uniprotkb:P62161)bind(MI:0407) bysurface plasmon resonance(MI:0107)•MINT‐7148437:EP24.15(uniprotkb:P52888) andCalmodulin(uniprotkb:P62158)colocalize(MI:0403) bysurface plasmon resonance(MI:0107)•MINT‐7148406:Calmodulin(uniprotkb:P62161)binds(MI:0407) toEP24.15(uniprotkb:P52888) bypull down(MI:0096)