Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells
Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells
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DOI：
10.3324/haematol.2018.207787
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发表时间：
2019-09-30
期刊：
影响因子：
10.1
通讯作者：
Bierings, Ruben
中科院分区：
文献类型：
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作者：
Karampini, Ellie;Schillemans, Maaike;Bierings, Ruben
Weibel-Palade 小体是内皮分泌细胞器，含有冯维勒布兰德因子、P-选择素和 CD63。 Weibel-Palade 小体释放冯维勒布兰德因子对于初次止血期间的血小板粘附至关重要。成熟过程中 CD63 等蛋白质向 Weibel-Palade 小体的内体运输依赖于衔接蛋白复合物 3 复合物。 AP3B1 基因（编码接头蛋白复合物 3 beta 1 亚基）的突变会导致 Hermansky-Pudlak 综合征 2，这是一种罕见的遗传性疾病，会导致中性粒细胞减少症和轻度出血素质。这是由于中性粒细胞和血小板中的物质运输到分泌细胞器的缺陷导致异常颗粒形成引起的。这些缺陷对内皮分泌途径的影响很大程度上未知。在这项研究中，我们研究了内皮细胞 Weibel-Palade 体成熟过程中衔接蛋白复合物 3 依赖性机制在蛋白质运输中的作用。使用从具有 AP3B1 复合杂合突变的患者中分离的血液生长内皮细胞建立了 2 型 Hermansky-Pudlak 综合征的离体患者来源的内皮模型。 Hermansky-Pudlak 综合征 2 型内皮细胞和 CRISPR-Cas9 工程改造的 AP3B1(-/-) 内皮细胞含有完全不含 CD63 的 Weibel-Palade 小体，表明内体运输受到破坏。 Hermansky-Pudlak 综合征 2 型内皮细胞的 Ca2+ 介导和 cAMP 介导的胞吐作用受损。全蛋白质组分析表明，除了接头蛋白复合物 3 beta 1 之外，mu 1 亚基和 v-SNARE VAMP8 也被耗尽。在 CRISPR-Cas9 工程改造的 VAMP8(-/-) 内皮细胞中，刺激诱导的血管性血友病因子分泌受到损害。我们的数据表明，Weibel-Palade 小体的接头蛋白复合物 3 依赖性成熟的缺陷通过影响 VAMP8 的募集而损害胞吐作用。
Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 beta 1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1. Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1(-/-) endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 beta 1, also the mu 1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8(-/-) endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.