A Novel Golgi Retention Signal RPWS for Tumor Suppressor UBIAD1

UBIAD1 plays critical roles in physiology including vitamin K and CoQ10 biosynthesis as well as pathophysiology including dyslipimedia-induced SCD (Schnyder’s corneal dystrophy), Parkinson’s disease, cardiovascular disease and bladder carcinoma. Since the subcellular localization of UBIAD1 varies in different cell types, characterization of the exact subcellular localization of UBIAD1 in specific human disease is vital for understanding its molecular mechanism. As UBIAD1 suppresses bladder carcinoma, we studied its subcellular localization in human bladder carcinoma cell line T24. Since fluorescent images of UBIAD1-EGFP in T24, human prostate cancer cell line PC-3, human embryonic kidney cell line HEK293 and human hepatocyte cell line L02 are similar, these four cell lines were used for present study. Using a combination of fluorescent microscopy and immunohistochemistry, it was found that UBIAD1 localized on the Golgi and endoplasmic reticulum (ER), but not on the plasma membrane, of T24 and HEK293 cells. Using scanning electron microscopy and western blot analysis, we found that UBIAD1 is enriched in the Golgi fraction extracted from the L02 cells, verifying the Golgi localization of UBAID1. Site-directed mutagenesis showed that the RPWS motif, which forms an Arginine finger on the UBIAD1 N terminus, serves as the Golgi retention signal. With both cycloheximide and brefeldin A inhibition assays, it was shown that UBIAD1 may be transported from the endoplasmic reticulum (ER) to the Golgi by a COPII-mediated mechanism. Based upon flow cytometry analysis, it is shown that mutation of the RPWS motif reduced the UBIAD1-induced apoptosis of T24 cells, indicating that the proper Golgi localization of UBIAD1 influences its tumor suppressant activity. This study paves the way for further understanding the molecular mechanism of UBIAD1 in human diseases.UBIAD1在诸多生理过程中发挥关键作用，包括维生素K与辅酶Q10（CoQ10）的生物合成；同时亦参与多种病理过程，如血脂异常诱导的施奈德角膜营养不良（Schnyder’s corneal dystrophy）、帕金森病、心血管疾病以及膀胱癌。由于UBIAD1的亚细胞定位（subcellular localization）在不同细胞类型中存在差异，明确其在特定人类疾病中的精准亚细胞定位，对于解析其分子机制至关重要。鉴于UBIAD1可抑制膀胱癌发生，本研究针对人膀胱癌细胞系T24内的UBIAD1亚细胞定位开展探究。因UBIAD1-增强型绿色荧光蛋白（EGFP）在人前列腺癌细胞系PC-3、人胚肾细胞系HEK293、人肝细胞系L02以及T24细胞中的荧光成像结果高度相似，故选取上述四种细胞系开展本研究。采用荧光显微镜（fluorescent microscopy）与免疫组织化学（immunohistochemistry）联合检测技术，本研究发现UBIAD1定位于T24与HEK293细胞的高尔基体（Golgi）和内质网（endoplasmic reticulum, ER），而非细胞膜。通过扫描电子显微镜（scanning electron microscopy）与蛋白质免疫印迹（western blot）分析，证实UBIAD1在L02细胞提取的高尔基体组分中富集，验证了UBIAD1的高尔基体定位。定点诱变（site-directed mutagenesis）实验显示，位于UBIAD1 N端、可形成精氨酸指（Arginine finger）的RPWS基序（motif），可作为高尔基体滞留信号（Golgi retention signal）。通过环己酰亚胺（cycloheximide）与布雷菲德菌素A（brefeldin A）抑制实验，结果表明UBIAD1可能通过COPII介导的通路从内质网（ER）转运至高尔基体。基于流式细胞术（flow cytometry）分析结果，RPWS基序的突变会削弱UBIAD1诱导的T24细胞凋亡，提示UBIAD1的正常高尔基体定位可影响其抑癌活性。本研究为进一步解析UBIAD1在人类疾病中的分子机制奠定了重要基础。