mLRH-1 泛素化作用之探討

Abstract

肝受器同源體-1 (Liver receptor homolog-1; LRH-1) 為轉錄因子，屬於核受器 NR5A 家族的成員之一，主要表現於肝臟、小腸、胰臟、脂肪前驅細胞與卵巢。本實驗室先前發現 mLRH-1 在細胞中表現的蛋白質量不高，推測 mLRH-1 可能經由蛋白酶體降解路徑分解，而影響到 mLRH-1 蛋白質的穩定性，但是目前對於 mLRH-1 蛋白質穩定性的調控機制尚未釐清。我們將 mLRH-1 與突變型泛素 (UbK0) 共同表現於 293 細胞中，發現 mLRH-1 的蛋白質表現量會顯著增加，說明 mLRH-1 可能有泛素化反應，而影響蛋白質的穩定性。為了了解 mLRH-1 是否會與泛素鍵結，我們將帶有不同標籤的 mLRH-1 與帶有 HA 標籤的泛素 (HA-Ub) 共同表現於 293 細胞中，並利用共同免疫沉澱法與西方墨點法進行分析。結果發現 Flag-mLRH-1、Myc-mLRH-1 和 Myc-CA-mLRH-1 蛋白質皆會與泛素鏈 (poly-ubiquitin chain) 鍵結。另外，給與蛋白酶體抑制劑 MG-132 並不影響 mLRH-1 與泛素所形成的蛋白質複合體的量，我們推測 mLRH-1 與泛素形成鍵結後可能與蛋白酶體降解路徑無關。 當我們刪除 mLRH-1 蛋白質的羧基端後，mLRH-1 與泛素所形成的蛋白質複合體的量會顯著減少，說明 mLRH-1 羧基端的胺基酸序列可能為進行泛素化作用的主要區域。我們建立了持續在 293 細胞中表現 Myc-CA-mLRH-1 融合蛋白質的 stable clones，並且確定這些 stable clones 皆具有 mLRH-1 的活性。我們發現在 stable clone 中 Myc-CA-mLRH-1 蛋白質會與細胞內的內生性泛素鍵結而形成泛素鏈。綜合所有實驗結果，我們的結論是在哺乳類細胞中 mLRH-1 蛋白質會進行泛素化作用，並且可能是透過 mLRH-1 蛋白質的羧基端與泛素鏈形成鍵結。

Liver receptor homolog-1 (LRH-1; NR5A2) is a transcriptional factor and belongs to the nuclear receptor 5A subfamily. LRH-1 is predominantly expressed in liver, intestine, exocrine pancreas, preadipocytes and ovary. Our previous studies indicated that the expression of mLRH-1 protein in transfected cells is low, suggesting that mLRH-1 could be degraded via proteasome pathway. However, the regulation of mLRH-1 protein stability is largely unknown. In the present study, overexpression of mLRH-1 and mutant type ubiquitin (UbK0) in 293 cells leaded to increased amount of mLRH-1 protein. It is suggested that mLRH-1 might be ubiquitinized, which could affect the mLRH-1 protein stability. We overexpressed mLRH-1 and HA-conjugated ubiquitin (HA-Ub) in 293 cells, co-immunoprecipitation and western blot demonstrated that Flag-mLRH-1, Myc-mLRH-1 and Myc-CA-mLRH-1 bind poly-ubiquitin chain in mammalian cells. Treatment of cells with proteasome inhibitor MG-132 had no effect on the amount of ubiquitin-conjugated mLRH-1 protein complexes, suggesting that the complexes might not related to the proteasomal degradation pathway. Deletions of the C-terminus of mLRH-1 decrease the amount of ubiquitin-conjugated mLRH-1 protein complexes. The results indicated that the ubiquitin binding site might locate at the C-terminus of mLRH-1. We established stable clones which overexpress Myc-CA-mLRH-1 protein in 293 cells. The activity of CYP11A1 promoter was significantly stimulated in these stable clones compared to the control cells. In addition, we found that mLRH-1 can bind endogenous poly-ubiquitin chain in stable clones. Our results revealed that mLRH-1 poly-ubiquitination may through the C-terminus of mLRH-1 in mammalian cells.