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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張世宗(Shih-Chung Chang) | |
dc.contributor.author | Jou-Han Chen | en |
dc.contributor.author | 陳柔含 | zh_TW |
dc.date.accessioned | 2021-06-16T23:25:37Z | - |
dc.date.available | 2015-08-10 | |
dc.date.copyright | 2012-08-10 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-07-31 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65112 | - |
dc.description.abstract | 泛素 (ubiquitin) 為一參與蛋白質轉譯後修飾的小分子蛋白質,透過其C端的Gly和目標蛋白質的Lys形成異胜肽鍵。真核生物亦具有數種類泛素蛋白質,立體結構和作用機制皆與泛素類似,其中又以NEDD8 (neuronal precursor cell expressed developmentally downregulated protein 8) 和泛素的相似度最高,但其修飾後對目標蛋白質所產生的影響卻與泛素有很大的差異。泛素需由去泛素化酵素 (deubiquitinase) 將C端di-glycine後的胺基酸片段切除以成為成熟形式,才能發揮蛋白質修飾的功能。目前已知去泛素化酵素中有些只對泛素進行作用,有些則對泛素和NEDD8都有活性,具有雙重專一性。本研究的目的即是探討去泛素化酵素中的USP (ubiquitin-specific protease) 和UCH (ubiquitin C-terminal hydrolase) 家族辨認泛素和 NEDD8的機制。
本研究透過分析泛素和USP2催化區的立體結構以及比較泛素和NEDD8胺基酸序列的差異,選定泛素的第4、12、14、51、72號胺基酸作為點突變的目標,將泛素和NEDD8 上的這些位置互換為相對應的胺基酸,並且製備成C 端帶有His tag 的泛素和NEDD8 突變株,用以比較這些位點對USP2催化效率的影響。結果發現,USP2對泛素F4K、T12E、T14E的水解效率接有明顯下降,因其突變會阻礙泛素和USP2催化區的交互作用;而72號則是扮演輔助角色,因該位置可和USP2產生靜電吸引以穩定催化反應,且T12E/T14E/R72A的抑制效果比T12E/T14E更為顯著;但51號胺基酸因突變前後所產生的差異不大,認為可能不是USP2所辨認的位置。透過比對USP家族成員的催化區序列,發現其與泛素第4、12、14、72號胺基酸進行交互作用的胺基酸皆具有保守性,因此這些位點應是USP家族所共通用來辨認泛素和NEDD8的關鍵胺基酸。另一方面,已知UCHL3除了會與泛素作用之外,對NEDD8也有很強的水解能力,但是對於UCHL1的特性研究則仍不完全清楚。本研究發現UCHL1與UCHL3對泛素和NEDD8皆有活性,證明UCHL1為具有雙重專一性的酵素。 | zh_TW |
dc.description.abstract | Ubiquitination is a posttranslational modification resulted from the attachment of ubiquitin’s C-terminal glycine to lysine residues on target proteins. There are several ubiquitin-like proteins sharing similar protein structure and modification pathway with ubiquitin. NEDD8 (neuronal precursor cell expressed developmentally downregulated protein 8) is its closest relative. Before conjugation, ubiquitin needs to be processed by deubibiquitinases to expose the C-terminal di-glycine motif. Most of deubiquitinases are specific for ubiquitin, whereas some are reported with NEDD8 cross-reactivity. The purpose of this work is to study the substrate recognition mechanism of USP and UCH by which they can discriminate between ubiquitin and NEDD8.
By examining the crystal structure of ubiquitin-USP2 binding complex and the amino acid sequence of ubiquitin and NEDD8, residues 4, 12, 14, 51 and 72 of ubiquitin were mutated to corresponding residues in NEDD8 and vice versa. By fusing ubiquitin and NEDD8 with C-terminal His tag, hydrolysis of the substrate by USP2 was analyzed by western blotting using the anti-His antibody. The data showed that mutations at F4K, T12E and T14E of ubiquitin partially inhibited its hydrolysis by USP2, due to the steric hindrance and electric repulsion between the mutated residues and USP2. Residue 72, on the other hand, may act as a helper since it interacts with USP2 and further inhibited hydrolysis in T12E/T14E/R72A ubiquitin mutant than T12E/T14E mutant. However, the E51N mutation of ubiquitin showed minor effects on catalytic inhibition, therefore it may not serve as a recognition site for USP2. Moreover, amino acids interacting with residues 4, 12, 14 and 72 of ubiquitin are conserved among USP members, suggesting a general recognition mechanism in the USP protein family. In this work, the experimental result also showed that UCHL1 contains dual specificity for processing ubiquitin and NEDD8. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T23:25:37Z (GMT). No. of bitstreams: 1 ntu-101-R99b22015-1.pdf: 11788717 bytes, checksum: aec37b785d981046afae818ba86661ea (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 摘要 i
Abstract ii 縮寫表 iii 第一章 緒論 1 1.1泛素與類泛素蛋白質 1 1.1.1 泛素修飾系統 2 1.1.2 NEDD8修飾系統 3 1.2 去泛素化酵素 4 1.2.1 USPs 4 1.2.2 UCHs 4 1.2.3 OTUs 5 1.2.4 Josephins 5 1.2.5 JAMMs 5 1.3 去泛素化酵素之基質專一性 6 1.4 研究動機 6 第二章 材料與方法 8 2.1 實驗材料 8 2.1.1 人類細胞 8 2.1.2 大腸桿菌 8 2.1.3 原核表現系統載體 8 2.2 蛋白質表現質體之製備 8 2.2.1 泛素表現質體 8 2.2.2 NEDD8表現質體 9 2.2.3 去泛素化酵素表現質體 9 2.3 重組蛋白質之表現 9 2.4 重組蛋白質之純化 10 2.5 去泛素化酵素之基質專一性分析 10 2.6 胺基酸序列分析 10 2.7 USP2與泛素之結構分析 11 第三章 研究結果 12 3.1 泛素、NEDD8、去泛素化酵素之表現質體建立 12 3.1.1泛素之表現質體建立 12 3.1.2 NEDD8之表現質體建立 12 3.1.3 去泛素化酵素之表現質體建立 12 3.2 泛素、NEDD8、去泛素化酵素之重組蛋白質表現與純化 13 3.2.1泛素、NEDD8重組蛋白質表現與純化 13 3.2.2 去泛素化酵素重組蛋白質表現與純化 13 3.3 去泛素化酵素對泛素和NEDD8之基質專一性分析 13 3.3.1 USP2之基質專一性分析 13 第四章 討論 15 4.1 USP2對泛素與NEDD8之辨識機制 15 4.2 USP家族之基質專一性 16 4.3 UCH家族之基質專一性 16 參考文獻 18 圖與表 22 附錄 36 | |
dc.language.iso | zh-TW | |
dc.title | 去泛素化酵素對泛素及 NEDD8 之基質專一性探討 | zh_TW |
dc.title | The Molecular Determinants for Ubiquitin and NEDD8 Discrimination by Human Deubiquitinases | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊榮輝(Rong-Huay Juang),張麗冠(Li-Kwan Chang),陳威戎(Wei-Jung Chen),林翰佳(Han-Jia Lin) | |
dc.subject.keyword | 泛素,NEDD8,deubiquitinase,USP,UCH, | zh_TW |
dc.subject.keyword | ubiquitin,NEDD8,deubiquitinas,USP,UCH, | en |
dc.relation.page | 48 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-07-31 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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