mLRH-1 泛素化作用之探討

Identification and characterization of mLRH-1 ubiquitination

黃守賢(Shou-Hsien Huang)

指導教授 ： 胡孟君

國立臺灣大學/醫學院/生理學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00907

摘要

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。

關鍵字

肝受器同源體-1 ； 泛素化作用 ； 泛素鏈

並列摘要

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.

並列關鍵字

Liver receptor homolog-1 ； ubiquitination ； poly-ubiquitin chain

參考文獻

Annicotte JS, Fayard E, Swift GH, Selander L, Edlund H, Tanaka T, Kodama T, Schoonjans K, Auwerx J. 2003. Pancreatic-duodenal homeobox 1 regulates expression of liver receptor homolog 1 during pancreas development. Mol Cell Biol 23:6713-6724.

Asaoka Y, Kanai F, Ichimura T, Tateishi K, Tanaka Y, Ohta M, Seto M, Tada M, Ijichi H, Ikenoue T, Kawabe T, Isobe T, Yaffe MB, Omata M. 2009. Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3. J Biol Chem 285:4185-4194.

Becker-Andre M, Andre E, DeLamarter JF. 1993. Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences. Biochem Biophys Res Commun 194:1371-1379.

Bhoj VG, Chen ZJ. 2009. Ubiquitylation in innate and adaptive immunity. Nature 458:430-437.

Bloom J, Amador V, Bartolini F, DeMartino G, Pagano M. 2003. Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell 115:71-82.