AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells
AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells
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DOI：
10.1016/j.biochi.2015.12.010
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发表时间：
2016-02-01
期刊：
影响因子：
3.9
通讯作者：
Arcari, Paolo
中科院分区：
文献类型：
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作者：
Di Stadio, Chiara Stella;Altieri, Filomena;Arcari, Paolo
AMP 18是胃特异性分泌蛋白，在正常胃粘膜中表达，但在胃癌中不表达。AMP 18在维持胃粘膜完整性中起主要作用，其特征在于存在由约100个氨基酸组成的BRICHOS结构域，其也存在于几种不相关的蛋白质中，并且可能具有伴侣样活性。我们利用功能蛋白质组学策略来鉴定潜在的AMP 18相互作用物，目的是增加关于其在胃细胞系中的功能作用的知识，为此目的，纯化重组生物素化的AMP 18，并通过应用亲和层析策略与来自人正常胃粘膜的蛋白质提取物温育。采用MALDI-TOF质谱法通过肽质量指纹图谱鉴定相互作用蛋白。相互作用的蛋白质的池包含SLC 26 A3，一种在肠上皮细胞的顶膜中表达的蛋白质，被认为在Cl-吸收和流体稳态中起关键作用。在AMP 18下拉后，还通过在转染的AGS细胞提取物上用抗SLC 26 A3进行蛋白质印迹证实了相互作用。此外，AMP 18和SLC 26 A3之间的相互作用也通过共聚焦显微镜验证，其显示两种蛋白质在质膜水平上的共定位。更重要的是，我们首次发现SLC 26 A3在胃癌中表达下调，AMP转染的胃癌细胞中AMP 18的过表达在转录和翻译水平上上调了SLC 26 A3的表达，后者可能通过激活MAP激酶途径。这些发现有力地表明，AMP 18可能还通过调节SLC 26 A3水平在维持粘膜完整性方面发挥抗炎作用。(C)2015 Elsevier B. V.和法国生物化学与生物分子学会（SFBBM）。All rights reserved.
AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity.In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues.To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl- absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the over expression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level. (C) 2015 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.