內質網胺肽酶之表現與純化及ERAP1內質網滯留訊息之鑑定

Abstract

泛素-26S蛋白解體系統能將蛋白質降解成2-25個胺基酸之胜肽。大部分的胜肽藉由細胞質中的胜肽酶快速將其水解成胺基酸。然而在高等的脊椎動物中，有少部分的胜肽會逃脫此途徑，且被運輸至內質網中，並與第一型主要組織適應性複合體結合而表現在細胞表面。ERAP1 (endoplasmic reticulum aminopeptidase 1) 和ERAP2 (endoplasmic reticulum aminopeptidase 2) 為含有鋅並具有HEXXHX18E motif及GAMEN motif之內質網胺肽酶，並且其活性不受puromycin的抑制，而表現量皆可被 γ-干擾素所誘導。ERAP1和ERAP2能處理N端具多餘胺基酸之抗原胜肽前驅物，並使成為成熟之抗原胜肽，以結合至第一型主要組織相容性複合體。 由於ERAP1沒有KDEL內質網回收訊號，為了探討ERAP1停留在內質網之重要序列，本研究成功建立數個截短之ERAP1片段，並且利用共軛焦雷射顯微鏡觀察其在HeLa cell之表現。結果顯示即使將 ERAP1截短至剩下N端150個胺基酸長度時，它仍然會停留在內質網中；所以推測與 ERAP1交互作用的未知蛋白質就是和N端150個胺基酸進行交互作用。本論文中亦用此150個胺基酸與GST所形成之融合蛋白，與由大鼠所分離出之內質網樣本混合，進而鑑定與ERAP1交互作用之未知蛋白質。為了研究ERAP1、ERAP2和P-LAP (Placental leucine aminopeptidase) 在細胞內之交互作用，分別將ERAP1、ERAP2和P-LAP以昆蟲細胞表現系統進行大量表現，並且成功地得到高純度的ERAP1重組蛋白質，而且桿狀病毒感染Sf-9之最佳稀釋倍率為1:100、感染天數為5天。但運用同樣的系統卻無法表現出ERAP2和P-LAP。除此之外，以大腸桿菌表現ERAP2和P-LAP時，會形成許多降解片段，且降低培養表現溫度或更換表現宿主皆無法改善降解現象。若將ERAP2 N端1-335胺基酸切除，即可成功在大腸桿菌中表現，但是此截短之ERAP2並不具有胺肽酶之酵素活性。

The display of endogenous antigenic peptides or viral proteins by major histocompatibility complex (MHC) class I molecules is important and essential for immune surveillance and the removal of abnormal or infected cells. The 26S proteasome–ubiquitin system is able to degrade proteins to peptides of 2−25 residues long. Nearly all of these peptides are hydrolyzed quickly to amino acids by cytosolic peptidases. However, in higher vertebrates, a small fraction escapes complete degradation and is transported into the endoplasmic reticulum (ER), where it binds to the MHC class I molecules and is exported to the cell surface. Aminopeptidase(s) in the ER, such as ERAP1 and ERAP2, assist the generation of mature MHC class I epitopes from the larger peptides produced by the proteasome in the cytosol. ERAP1 and ERAP2 share a zinc-binding site, HEXXHX18E, and the GAMEN catalytic motif essential for enzymatic activities, which are not inhibited by puromycin. They are both interferon-γ-induced aminopeptidases localized in the ER that process certain viral proteins or damaged transient proteins. They can also trim N-extended antigenic precursors into antigenic peptides for MHC class I presentation. The purified ERAP1 trimmed peptides that are 10 residues or longer, but spared 8- or 9-residue long peptides, which is the length required to bind to the MHC class I molecules. The absence of retention signal KDEL in ERAP1while it is in the ER prompted us to make several truncated ERAP1 constructs to identify the important region that is responsible for retention in the ER. We found that in E.coli system, the N-terminal sequence of ERAP1 may interfere with its expression and folding. Since the expression level of truncated ERAP1 was very low in E. coli expression system, we analyzed the N-terminal sequence of ERAP1 and discovered the existence of many rare codons. For the localization of truncated ERAP1 and its interacting proteins, we transfected the truncated constructs into HeLa cells and then observed their localization under confocal microscopy. The results showed that even when shortened to only 150-residue long fragment, ERAP1 still remain in the ER. Therefore, we hypothesize that it is these 150 amino acids with which interact with the unknown ERAP1-interacting proteins. In the thesis, we used the fusion protein of the 150-residue fragment and GST combined with the ER extract isolated from rat liver to identify the unknown interacting proteins. To investigate the interaction between ERAP1, ERAP2 and P-LAP in vivo, we used insect cell expression system to express the three proteins. We successfully obtained the ERAP1 recombinant protein with high purity. Unfortunately, we were unable to express ERAP2 and P-LAP using the same expression system. Moreover, when expressing ERAP2 and P-LAP in E.coli, they easily formed many degraded fragments. This phenomenon cannot be improved neither by reducing the temperature or changing the express host. We assumed that the appearance of the degraded fragments is due to the complicated sequence at the N-terminus. The removal of the first 335 amino acids from ERAP2’s N-terminus led to successful expression in E.coli. Regrettably, ERAP2 in this form does not retain any aminopeptidase activity.