LRH-1 離胺酸 329 參與泛素-蛋白酶體依賴之 蛋白質水解途徑

Abstract

Liver receptor homolog-1 (LRH-1 ; NR5A2) 隸屬孤兒核受器成員之一，對於發育、固醇類荷爾蒙生成以及脂肪代謝相當重要。然而，現今對於LRH-1 蛋白質水解的調控以及機制尚未釐清。我們利用蛋白酶體抑制劑 MG-132 處理 24 小時，發現 LRH-1 蛋白質量明顯地增多。當細胞以 cycloheximide 抑制其蛋白質生成時，發現 LRH-1 會迅速地被分解。然而，當有 MG-132 存在時即會明顯減低 LRH-1 降解的速度。當 LRH-1 和具有 HA 標籤之泛素 (HA-Ub) 共同表現於細胞，發現 LRH-1 會受到聚泛素 (polyubiquitin) 的修飾。這些結果說明 LRH-1 會經由泛素-蛋白酶體路徑而降解。若將 LRH-1 羧基端刪除，蛋白質的量會隨著羧基端的縮減而增加，MG-132 的效應則明顯降低，僅在全長 LRH-1 觀察到顯著增加，此外，泛素結合的量也隨著羧基端的刪除而減少。利用定點突變聚合酶連鎖反應將特定的離胺酸轉變成精胺酸 (K240R、K289R、K329R、K340R， 以及 K357R)，相似於原生型 LRH-1 的結果，MG-132 會顯著促進 K240R、K289R、K340R，以及 K357R 蛋白質的量，但 K329R 蛋白質增加之幅度明顯下降。共同免疫沉澱 (Co-IP) 結果顯示 LRH-1 K329R 與泛素結合的量明顯低於原生型 LRH-1。處理 cycloheximide 研究 LRH-1 K329R 蛋白質半衰期，觀察到半衰期延長為 1.96 小時，而 MG-132 無法有效抑制其蛋白質水解。利用啟動子轉錄活性分析顯示 LRH-1 K329R 轉錄活性約為原生型 LRH-1 的 2.6 倍。綜合結果說明 LRH-1 K329R 與泛素結合減少，蛋白質半衰期延長，蛋白質穩定性增加，使其具有更高之轉錄活性，證實 LRH-1 離胺酸 329 為一重要位點，參與泛素-蛋白酶體之調控。

The orphan nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) is essential for development, steroidogenesis, and metabolism. Little is known about the mechanism(s) of the regulation of LRH-1 degradation. We demonstrated that LRH-1 protein level is significantly increased after treatment of proteasome inhibitor MG-132 for 24 hours. Incubation with cycloheximide to inhibit protein synthesis, found LRH-1 was degraded rapidly. However, the degradation rate of LRH-1 was significantly reduced in the presence of MG132. Coexpression of LRH-1 with ubiquitin (HA-Ub) revealed that LRH-1 was conjugated with polyubiquitin. These results demonstrated that ubiquitin-proteasome pathway is involved in the degradation of LRH-1. C-terminal deletion fragments of LRH-1 protein were sequentially increased in length following the augmentation of C-terminal truncation. The effect of MG-132 on LRH-1 was significantly reduced by C-terminal deletion; we merely observed the enormously increased protein level on full-length LRH-1. Moreover, ubiquitin conjugation decreases as LRH-1 C-terminal deletion expand. By carrying out site-directed mutagenesis to convert certain lysine residue to arginine residue (K240R, K289R, K329R, K340R, and K357R), we revealed K240R, K289R, K340R, and K357R protein levels were increased after treantment with MG-132. Furthermore, co-immunoprecipitation (Co-IP) result showed LRH-1 K329R possessed less ubiquitin conjugation than wild-type LRH-1. Incubation with cycloheximide to evaluate the protein half-life of LRH-1 K329R, we observed its half-life extends to 1.96 hours; however, MG-132 could not further inhibit protein degradation. Using promoter-transactivation assays, we found that transcription activity of LRH-1 K329R is 2.6 fold to wild-type LRH-1. In conclusion, the results show that LRH-1 K329R possesses less polyubiquitin conjugations, owns longer protein half-life, and has higher trans-activation activity than wild-type LRH-1. Our results unveil that residue lysine 329 of LRH-1 is indeed essential for proteasome-dependent degradation.