Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
PLoS One. 2011;6(8):e23188. doi: 10.1371/journal.pone.0023188. Epub 2011 Aug 10.
E-cadherin plays a powerful tumor suppressor role. Germline E-cadherin mutations justify 30% of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations are found in 30% of these families. We found possible to restore in vitro mutant E-cadherin associated to HDGC syndrome by using Chemical Chaperones (CCs). Herein, our aim was to disclose the molecular mechanisms underlying the CCs effects in E-cadherin regulation. Using cells stably expressing WT E-cadherin or two HDGC-associated missense mutations, we show that upon DMSO treatment, not only mutant E-cadherin is restored and stabilized at the plasma membrane (PM), but also Arf6 and PIPKIγ expressions are altered. We show that modulation of Arf6 expression partially mimics the effect of CCs, suggesting that the cellular effects observed upon CCs treatment are mediated by Arf6. Further, we show that E-cadherin expression recovery is specifically linked to Arf6 due to its role on endocytosis and recycling pathways. Finally, we demonstrated that, as DMSO, several others CCs are able to modulate the trafficking machinery through an Arf6 dependent mechanism. Interestingly, the more effective compounds in E-cadherin recovery to PM are those that simultaneously inhibit Arf6 and stimulate PIPKIγ expression and binding to E-cadherin. Here, we present the first evidence of a direct influence of CCs in cellular trafficking machinery and we show that this effect is of crucial importance in the context of juxtamembrane E-cadherin missense mutations associated to HDGC. We propose that this influence should be taken into account when exploring the therapeutic potential of this type of chemicals in genetic diseases associated to protein-misfolding.
E-钙黏蛋白发挥着强大的肿瘤抑制作用。胚系 E-钙黏蛋白突变可解释 30%的遗传性弥漫性胃癌（HDGC），并且在这些家族中发现了 30%的错义突变。我们发现通过使用化学伴侣（CCs）可以在体外恢复与 HDGC 综合征相关的突变 E-钙黏蛋白。在此，我们的目的是揭示 CCs 对 E-钙黏蛋白调节的分子机制。使用稳定表达 WT E-钙黏蛋白或两种与 HDGC 相关的错义突变的细胞，我们表明，在用 DMSO 处理后，不仅突变的 E-钙黏蛋白被恢复并稳定在质膜（PM）上，而且 Arf6 和 PIPKIγ 的表达也发生改变。我们表明，Arf6 表达的调节部分模拟了 CCs 的作用，表明在 CCs 处理后观察到的细胞效应是由 Arf6 介导的。此外，我们表明，E-钙黏蛋白表达的恢复与 Arf6 特异性相关，因为它在胞吞作用和回收途径中起作用。最后，我们证明，与 DMSO 一样，其他几种 CCs 能够通过依赖 Arf6 的机制调节运输机制。有趣的是，在 PM 上恢复 E-钙黏蛋白的更有效化合物是那些同时抑制 Arf6 并刺激 PIPKIγ 表达和与 E-钙黏蛋白结合的化合物。在这里，我们首次证明了 CCs 对细胞运输机制的直接影响，并表明在与 HDGC 相关的近膜 E-钙黏蛋白错义突变的背景下，这种影响至关重要。我们提出，在探索与蛋白质错误折叠相关的遗传疾病中此类化学物质的治疗潜力时，应考虑这种影响。
