麦醇溶蛋白肽 P31-43 定位于内吞小泡，并干扰其成熟。

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.

背景：乳糜泻（CD）既是一种常见疾病（1:100），也是一种由食物引起的疾病的有趣模型。它由对小麦面筋和麦醇溶蛋白的免疫反应组成，已发现这种反应发生在特定的遗传背景下；然而，这种反应仍然只是部分理解。麦醇溶蛋白肽对先天免疫的激活是疾病早期事件的一个重要组成部分。特别是所谓的“毒性”A-麦醇溶蛋白肽 P31-43 诱导包括表皮生长因子受体（EGFR）依赖性肌动蛋白重塑和培养细胞系和 CD 患者肠细胞中的增殖在内的多种多效性效应。这些效应是通过内吞体中 EGFR 降解的延迟和 EGFR 激活的延长来介导的。在本研究中，我们研究了麦醇溶蛋白肽对内吞体囊泡运输和成熟的影响。

方法/主要发现：用荧光染料标记的 P31-43 和对照肽 P57-68 均被发现进入 CaCo-2 细胞，并在脉冲和追踪、延时、实验中与内吞体区室相互作用。在 P57-68 携带的囊泡成熟为晚期内体时，P31-43 定位于携带早期内吞体标记的囊泡上。在延时实验中，无论它们携带的货物如何，P31-43 标记的囊泡的运输都被延迟。此外，在来自培养的十二指肠活检的乳糜泻肠细胞中，P31-43 的运输在早期内吞体囊泡中被延迟。序列相似性搜索显示，P31-43 与 Hrs 非常相似，Hrs 是调节内吞体成熟的关键分子。A-麦醇溶蛋白肽 P31-43 通过 Western blot 和免疫荧光显微镜显示，干扰 Hrs 正确定位到早期内体，从而干扰 P31-43 正确定位到早期内体。

结论：P31-43 和 P57-68 通过内吞作用进入细胞。只有 P31-43 定位于内吞体膜上，并通过干扰 Hrs 介导的成熟到晚期内体来延迟囊泡运输，从而在细胞和肠活检中延迟囊泡运输。因此，在 P31-43 处理的细胞中，受体酪氨酸激酶（RTK）的激活被延长。这一发现可以解释麦醇溶蛋白肽在诱导 CD 活检肠细胞增殖和其他效应中所起的作用。