PDF(Pubmed)
Abstract：
Retinoic acid receptor-related orphan receptor alpha (RORα), a candidate tumor suppressor, is prevalently downregulated or lost in malignant breast cancer cells. However, the mechanisms of how RORα expression is regulated in breast epithelial cells remain incompletely understood. Protein arginine N-methyltransferase 5 (PRMT5), a type II methyltransferase catalyzing the symmetric methylation of the amino acid arginine in target proteins, was reported to regulate protein stability. To study whether and how PRMT5 regulates RORα, we examined the direct interaction between RORα and PRMT5 by immunoprecipitation and GST pull-down assays. The results showed that PRMT5 directly bound to RORα, and PRMT5 mainly symmetrically dimethylated the DNA-binding domain (DBD) but not the ligand-binding domain (LBD) of RORα. To investigate whether RORα protein stability is regulated by PRMT5, we transfected HEK293FT cells with RORα and PRMT5-expressing or PRMT5-silencing (shPRMT5) vectors and then examined RORα protein stability by a cycloheximide chase assay. The results showed that PRMT5 increased RORα protein stability, while silencing PRMT5 accelerated RORα protein degradation. In PRMT5-silenced mammary epithelial cells, RORα protein expression was decreased, accompanied by an enhanced epithelial-mesenchymal transition morphology and cell invasion and migration abilities. In PRMT5-overexpressed mammary epithelial cells, RORα protein was accumulated, and cell invasion was suppressed. These findings revealed a novel mechanism by which PRMT5 regulates RORα protein stability.
摘要：
维甲酸受体相关孤儿受体α（RORα），候选肿瘤抑制因子,在恶性乳腺癌细胞中普遍下调或丢失。然而,乳腺上皮细胞中RORα表达的调控机制尚不完全清楚。蛋白质精氨酸N-甲基转移酶5（PRMT5），一种II型甲基转移酶，催化靶蛋白中氨基酸精氨酸的对称甲基化，据报道可以调节蛋白质的稳定性。为了研究PRMT5是否以及如何调节RORα，我们通过免疫沉淀和GST下拉法检测了RORα和PRMT5之间的直接相互作用。结果表明，PRMT5直接与RORα结合，和PRMT5主要对称地二甲基化RORα的DNA结合域（DBD），而不是配体结合域（LBD）。为了研究RORα蛋白稳定性是否受PRMT5调节，我们用RORα和PRMT5表达或PRMT5沉默（shPRMT5）载体转染HEK293FT细胞，然后通过环己酰亚胺追踪测定法检查RORα蛋白稳定性。结果表明，PRMT5增加了RORα蛋白的稳定性，而沉默PRMT5加速RORα蛋白降解。在PRMT5沉默的乳腺上皮细胞中，RORα蛋白表达降低,伴随着增强的上皮-间质转化形态和细胞侵袭和迁移能力。在PRMT5过表达的乳腺上皮细胞中，RORα蛋白积累，细胞侵袭被抑制。这些发现揭示了PRMT5调节RORα蛋白稳定性的新机制。
