探討LRH-1核輸入訊號及sumoylation在細胞中運輸扮演的角色

Regulation of LRH-1 cellular trafficking: Role of Nuclear localization signals and sumoylation

楊豐名(Feng-Ming Yang)

指導教授 ： 胡孟君

國立臺灣大學/醫學院/生理學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.02742

摘要

核受器同源體-1 (Liver receptor homolog-1; LRH-1；NR5A2)為孤兒核受器家族的成員之一，會穩定的表現在細胞核中調控基因表現。本篇研究中，我們在LRH-1上確認了兩段具有功能的核輸入訊號(nuclear localization signals, NLSs)，稱為NLS1與NLS2。NLS1位在DNA binding domain，胺基酸序列範圍在117-169並涵蓋了此區的第二個zinc finger。突變結果顯示，zinc finger結構及其兩側的鹼性胺基酸對NLS1核輸入功能具有重要性。NLS2的胺基酸序列範圍為169-204，位於Ftz-F1 box上，符合典型的bipartite NLS。單一存在的NLS1或NLS2都具有將LRH-1運送到細胞核的能力，且胞外實驗顯示兩者分別與importin α 或importin β有交互作用，說明了LRH-1主要是透過importin α/β的機制運送進入細胞核。此外，NLSs上重要的三組鹼性胺基酸的突變也會影響LRH-1的DNA鍵結和轉錄之功能。
LRH-1在細胞核的分布會受到SUMO修飾作用的影響。SUMO鍵結會引導LRH-1至核小體，而降低其轉錄活性。胞外實驗顯示，lysine289會與SUMO-1產生鍵結。然而，在COS-7細胞中lysine173或289的突變，會降低SUMO-1的鍵結作用，亦會阻止LRH-1位移到核小體，顯示lysine173和289為SUMO-1修飾作用的兩個重要位置。在分離的大鼠granulosa 細胞中，五個單一lysine的突變並不影響LRH-1位移至核小體，需同時有三到五個lysines的突變位置，才能阻止此作用。這些結果顯示，LRH-1具有多個SUMO-1鍵結區，在不同的細胞會造成不同的修飾作用。在大鼠granulosa細胞中，forskolin和cholera toxin的處理能減低LRH-1至核小體的位移，進一步的分析顯示，forskolin會影響sumoylation的動態平衡，可能因此降低SUMO的作用而阻斷LRH-1位移到核小體。
總結來說，本篇論文說明，LRH-1的轉錄作用會受到過核輸入系統和SUMO修飾作用所調控。

關鍵字

核受器同源體-1 ； 核輸入訊號 ； SUMO-1基因 ； 大鼠granulosa 細胞 ； 核小體

並列摘要

Liver receptor homolog-1 (LRH-1; NR5A2), is an orphan nuclear receptor that is localized to the nucleus for the regulation of gene expression. In this study, I characterized two functional nuclear localization signals (NLSs) in LRH-1. The first motif, NLS1 (residues 117-168), overlaps the second zinc finger in the DNA binding domain. Mutagenesis analysis showed that the zinc finger structure and two basic clusters on either side of the zinc finger loop are critical for nuclear localization directed by NLS1. The motif NLS2 (residues 169-204) is located in the Ftz-F1 box that contains a bipartite signal. Either NLS1 or NLS2 alone was sufficient to target full-length LRH-1 to the nucleus. Both NLS1 and NLS2 interacted with importin α and importin β in vitro, suggesting that importins mediate the nuclear transport of LRH-1. Three crucial basic clusters in the NLSs are also involved in the DNA binding and transcriptional activities of LRH-1.
I further showed that nuclear localization of LRH-1 was regulated by SUMO modification. SUMO conjugation targets LRH-1 to the transcriptionally inactive nuclear bodies. Lysine 289 is the major site for SUMO-1 conjugation in vitro. However, in COS-7 cells, mutation of either K173 or K289 prevented translocation of LRH-1 into nuclear bodies and reduced the conjugation by SUMO-1, suggesting that K289 and K173 are two important sites involved in SUMO-1 modification. In granulosa cells, three or more altered lysine residues were required for nucleoplasm retention. This result suggests that multiple lysine residues are targets for SUMO conjugation in vivo and that granulosa cells are more sensitive to SUMO-1-mediated LRH-1 localization to nuclear bodies. Nuclear body localization of LRH-1 was suppressed by forskolin and cholera toxin. Forskolin treatment could change the dynamic process of sumoylation and repress LRH-1 targeting to nuclear speckles in rat granulosa cells. Take together, the result of this study suggest that transcription function of LRH-1 is maintained via the nucleocytoplasmic transport system and regulated by sumoylation.

並列關鍵字

LRH-1 ； nuclear localization signals ； SUMO-1 ； rat granulosa cell ； nuclear body

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