The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC
The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC
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DOI：
10.1038/ejhg.2012.159
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发表时间：
2013-03-01
影响因子：
5.2
通讯作者：
Seruca, Raquel
中科院分区：
文献类型：
--
作者：
Figueiredo, Joana;Soederberg, Ola;Seruca, Raquel
在遗传性弥漫性胃癌（HDGC）中，CDH 1生殖系基因改变是30%病例的致病事件。在20%的HDGC家族中，CDH 1种系突变是错义型的，突变携带者构成遗传咨询和监测方面的问题。为了获得错义突变的致病相关性，我们以前开发了一种体外方法来功能性地表征它们。与表达野生型（WT）蛋白的细胞相比，致病性E-钙粘蛋白错义突变体不能聚集并变得更具侵袭性。在此，我们的目的是开发一种互补的方法来解开E-cadherin错义突变的致病意义。我们使用了稳定表达WT E-钙粘蛋白和7个HDGC相关突变（5个细胞内和2个细胞外）的细胞，并通过邻近连接试验（PLA）研究了这些突变体如何与E-钙粘蛋白功能和运输的基本调节因子结合。我们将注意力集中在与p120、β-连环蛋白、PIPKI γ和Hakai的相互作用上。我们发现，细胞质E-钙粘蛋白突变影响一个或多个结合伴侣的相互作用，损害E-钙粘蛋白在质膜上的稳定性，并可能影响粘附复合物的能力。在目前的工作中，我们证明了E-cadherin及其结合伙伴之间的相互作用的研究，使用PLA，是一种简单，快速，定量和高度可重复的技术，可以应用于常规实验室，以验证HDGC诊断的E-cadherin错义突变体的致病性，特别是那些位于蛋白质的胞内结构域。欧洲人类遗传学杂志（2013）21，301-309; doi：10.1038/ejhg.2012.159; 2012年8月1日在线发表
In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, beta-catenin, PIPKI gamma and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein. European Journal of Human Genetics (2013) 21, 301-309; doi:10.1038/ejhg.2012.159; published online 1 August 2012