Charcterization of ice plant E2 ubiquitin-conjugating enzymes UBC1 and UBC2 and their specificities to E3 ligase McCPN1

泛素(ubiquitin)為一個具高度保留性的蛋白質，它會被共價鍵結在受質蛋白上，這個過程稱為ubiquitination，由三個酵素參與連續催化而成。首先是泛素活化酶(ubiquitin-activating enzyme, E1)水解ATP活化泛素；再經由泛素結合酶(ubiquitin- conjugating enzyme, UBC, E2)與泛素結合形成中間產物；最後由泛素連接酶(ubiquitin ligase, E3)辨識受質蛋白，並催化泛素與受質形成共價鍵結。是生物體內相當重要的蛋白後修飾作用，參與真核生物許多重要生理反應，如蛋白降解、生物逆境及生長的調控等從耐鹽模式植物冰花(Mesembryanthemum crystallinum L.)所鑑定的一個RING-type E3 ligase McCOPIN1 (McCPN1)，已知具有E3 ligase的活性且參與冰花對鹽逆境的適應，但是其參與的ubiquitination途徑機制未明。本實驗室之前已鑑定出兩個冰花E2基因McUBC1與McUBC2，其中McUBC1在鹽處理後之癒傷組織中表現量會上升，但所獲得之UBC1基因並非全長。因此本論文主要目標即在確認McUBC1與McUBC2是否能與E3 McCPN1共同催化protein ubiquitination的作用，進而對於此一途徑參與冰花耐鹽性有進一步的了解。 首先以RACE鑑定出McUBC1完整orf全長為462 bp，可轉譯出153個胺基酸的蛋白；之後比對McUBC1和McUBC2蛋白的胺基酸序列，發現McUBC1與McUBC2相同度為43%，相似度為77%，親緣分析顯示各屬於不同之UBC次家族。利用大腸桿菌蛋白表現系統，得到純化的McUBC1-(His)6與McUBC2-(His)6，以in vitro ubiquitination assay分析E2活性，結果發現McUBC2-(His)6能與McCPN1共同催化ubiquitination的反應，顯示McUBC2-McCPN1是一組具有功能的E2-E3組合。反之McUBC1-(His)6或是McUBC1/UEV2 heterodimer E2均無法催化ubiquitination的反應。以RT-PCR分析鹽處理冰花葉部及小苗McUBC1及McUBC2基因表現量之變化，結果顯示鹽處理對McUBC1與McUBC2表現量影響不顯著，兩基因在冰花小苗或成熟植株階段，均會持續表現。綜合上述之結果，發現鹽處理不會影響McUBC1與McUBC2的基因表現；且McUBC2會透過與McCPN1共同催化ubiquitination反應，參與冰花耐鹽機制的調控，使冰花能在鹽逆境下正常生長。Ubiquitin is a highly conserved small protein. Substrate proteins can be attached with a single or multiple ubiquitins, a process called ubiquitination, which is carried out by a set of three enzymes. Ubiquitin-activating enzyme (E1) consumes ATP to activate ubiquitin, then the activated ubiquitin is spontaneously transferred to ubiquitin-conjugating enzyme (E2) followed by transfer of ubiquitin to substrate protein catalyzed by ubiquitin ligase (E3). Ubiquitination is involved in a number of cellular processes, such as protein degradation by proteasome, stress response, and development. A RING-type E3 McCOPIN1 (McCPN1) was identified from halophyte ice plant (Mesembryanthemum crystallinum L.) and was implicated in participation of the salt stress response in ice plant. The involvement of McCPN1 in stress-related protein ubiquitination remains unknown. Two ice plant E2 McUBC1 and McUBC2 were previously identified in ice plant. The expression of McUBC1 is upregulated in salt-stressed ice plant callus, but we only have partial sequence of McUBC1. The main theme of this thesis is to analyze McUBC1 and McUBC2’s E2 activity, along with their specificity to the E3 ligase McCPN1. RACE was carried out to obtain the full length McUBC1. Full length of McUBC1orf consists of 462 bp, encoding a 153 amino acids protein. Sequence alignment shows 43% identity and 77% similarity between McUBC1 and McUBC2. Phylogenic analysis suggests McUBC1 and McUBC2 belong to different UBC subgroups. McUBC1-(His)6 and McUBC2-(His)6 recombinant proteins was expressed in Escherichai coli and in vitro ubiquitination assay was performed to detect E2 activity of purified McUBC1-(His)6 and McUBC2-(His)6. The results showed McUBC2 and McCPN1 can form a proper E2/E3 complex to carry out ubiquitination. In contrast McUBC1/UEV2 heterodimer E2 failed carry out ubiquitnation with McCPN1. The expression of McUBC1 and McUBC2 under salt stress was further examined through RT-PCR, and their expression levels in seedlings and leaves did not show variation with the treatment of salt stress. In conclusion, McUBC1 and McUBC2 are constitutively expressed in ice plant. McUBC2 may participate in salt stress response through forming the E2/E3 complex with McCPN1, which in turn ubiquinate salt stress-related proteins leading adaptation to the salinity environment.目次 摘要………………………………………………………………………………i Abstract…………………………………………………………………………ii 目次……………………………………………………………………………iv 圖表目次…………………………………………………………………………v 壹、前人研究……………………………………………………………………1 一、植物逆境與鹽害………………………………………………………1 二、耐鹽模式植物冰花……………………………………………………1 三、冰花耐鹽相關蛋白……………………………………………………2 四、Ubiquitin cycle對蛋白的修飾作用……………………………………3 五、Ubiquitin-conjugating enzyme (UBC)蛋白家族………………………5 六、特定E2參與生物對環境逆境的反應………………………………6 七、實驗目的與策略………………………………………………………8 貳、材料與方法…………………………………………………………………9 一、RACE (rapid amplification of cDNA ends)……………………………9 二、菌種與質體……………………………………………………………9 三、構築構築E.coli大量表現載體…………………………………………10 四、蛋白表現與純化………………………………………………………13 五、SDS-PAGE (sodium dodecyl sulfate polyacrylamide electrophoresis)13 六、蛋白質身分鑑定………………………………………………………14 七、In vitro ubiquitination assay……………………………………………15 八、西方點墨法(western blot)……………………………………………16 九、RT-PCR (reverse transcription-PCR)…………………………………17 參、結果…………………………………………………………………………19 肆、討論…………………………………………………………………………25 伍、參考文獻……………………………………………………………………29 陸、附錄…………………………………………………………………………5

RING-type ubiquitin ligase McCPN1 catalyzes UBC8-dependent protein ubiquitination and interacts with Argonaute 4 in halophyte ice plant

RING-type copines are a small family of plant-specific RING-type ubiquitin ligases. They contain an N-terminal myristoylation site for membrane anchoring, a central copine domain for substrate recognition, and a C-terminal RING domain for E2 docking. RING-type copine McCPN1 (copine1) from halophyte ice plant (Mesembryanthemum crystallinum L.) was previously identified from a salt-induced cDNA library. In this work, we characterize the activity, expression, and localization of McCPN1 in ice plant. An in vitro ubiquitination assay of McCPN1 was performed using two ice plant UBCs, McUBC1 and McUBC2, characterized from the same salt-induced cDNA library. The results showed that McUBC2, a member of the UBC8 family, stimulated the autoubiquitination activity of McCPN1, while McUBC1, a homolog of the UBC35 family, did not. The results indicate that McCPN1 has selective E2-dependent E3 ligase activity. We found that McCPN1 localizes primarily on the plasma membrane and in the nucleus of plant cells. Under salt stress, the accumulation of McCPN1 in the roots increases. A yeast two-hybrid screen was used to search for potential McCPN1-interacting partners using a library constructed from salt-stressed ice plants. Screening with full-length McCPN1 identified several independent clones containing partial Argonaute 4 (AGO4) sequence. Subsequent agro-infiltration, protoplast two-hybrid analysis, and bimolecular fluorescence complementation assay confirmed that McCPN1 and AGO4 interacted in vivo in the nucleus of plant cells. The possible involvement of a catalyzed degradation of AGO4 by McCPN1 in response to salt stress is discussed