Dooley Sarah A, Kolobova Elena, Burman Andreanna, Kaji Izumi, Digrazia Jessica R, Stubler Rachel, Goldstein Anna, Packirisamy Charulekha, Coutts Alexander W, Saqui-Salces Milena, Gao Nan, Engevik Melinda A, Shub Mitchell D, Goldenring James R, Engevik Amy C
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina.
Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville, Tennessee; Epithelial Biology Center, Vanderbilt University Medical Center, Nashville, Tennessee.
Gastroenterology. 2025 Jan;168(1):84-98.e9. doi: 10.1053/j.gastro.2024.09.007. Epub 2024 Sep 18.
BACKGROUND & AIMS: The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes.
We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited.
In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug.
Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.
外源性物质流出泵P-糖蛋白在胃肠道的顶端膜上高度表达，在那里它调节细胞内底物的水平。P-糖蛋白在疾病中会发生改变，但调节P-糖蛋白水平的机制仍在探索中。分子马达肌球蛋白Vb（Myo5b）将多种货物运输到肠上皮细胞的顶端膜。我们假设Myo5b负责将P-糖蛋白递送到肠细胞的顶端膜。
我们使用了多种缺乏功能性Myo5b或肌球蛋白结合伴侣Rab11a的小鼠模型来分析P-糖蛋白的定位。分析猪和人类组织以确定MYO5B突变情况下P-糖蛋白的定位。使用肠道类器官来检查P-糖蛋白的运输，并在抑制MYO5时测定P-糖蛋白的功能。
在缺乏Myo5b或结合伴侣Rab11a的小鼠中，P-糖蛋白运输不当，在肠细胞刷状缘中的存在减少。对缺乏功能性Myo5b的猪模型和来自一名Myo5b失活突变患者的人类活检组织进行免疫染色，也显示肠道P-糖蛋白的定位发生了改变。表达无运动能力的MYO5B尾域的人类肠道类器官与P-糖蛋白共定位，证实P-糖蛋白在人类肠细胞中由MYO5B运输。在人类肠道细胞系和类器官中抑制MYO5导致P-糖蛋白能力下降。此外，在人类结肠癌细胞中抑制MYO5会降低P-糖蛋白活性，并增加对化疗药物的细胞死亡反应。
总体而言，这些数据表明Myo5b是P-糖蛋白顶端递送所必需的。
