核糖體抑活蛋白 (RIPs) 屬於糖苷酶的一種，能從23S或28S核糖體核糖核酸中的sarcin-ricin環(sarcin-ricin loop, SRL)移除一個特定的腺嘌呤，引致核糖體失效。由於核糖體蛋白協助RIP到達SRL，因此它們對RIP的核糖體特認性是極大的重要。雖然各RIPs的份子結構及催化活動非常相似，它們的核糖體特認性和效力存著很大的迥異。此外，現時還未能找出只有少數RIPs能同時抑制原核和真核生物的核糖體的原因。我們試圖從玉米核糖體抑活蛋白 (Maize RIP) 和真核生物的核糖體以及志賀毒素 (Shiga toxin) 和原核生物的核糖體的相互作用的研究中去解釋以上的現象。 / 我們發現Maize RIP提供一個前所未見的區域與核糖體蛋白P2結合，並展示RIPs的結構大大限制了它們與核糖體蛋白的相互作用的性質和強度，從而影響RIPs在核糖體上的效力。另外，我們發現志賀毒素跟細菌的核糖體的相互作用比跟真核生物核糖體的相互作用弱，並可能跟細菌核糖體蛋白L7/L10有交聯。我們在蓖麻毒蛋白 (Ricin) 的碳端 (C-terminus) 加上人類免疫缺陷病毒-(HIV-1) 蛋白酶特認的肽以增加 ricin 對HIV-1蛋白酶的特認性，並希望此研究結果有助於應用相類的策略到其他RIPs上。 / Ribosome-inactivating proteins (RIPs) are N-glycosidases that inactivate ribosome by removing a specific adenine from the sarcin-ricin loop (SRL) of 23S or 28S ribosomal RNA. Ribosomal proteins are critical for determining the ribosome specificity of RIPs as they assist RIPs to get access to the SRL. Ribosome specificity and potency of RIPs are highly varied although their tertiary structures and catalytic depurination are highly alike. Moreover, it is still unsolved why only a few RIPs acquiring the ability to inhibit both prokaryotic and eukaryotic ribosomes. We attempted to elucidate the phenomena by investigating the interactions of maize RIP with eukaryotic ribosome and shiga toxin with prokaryotic ribosome. / Here we showed maize RIP presents a novel docking site to interact with ribosomal protein P2 and demonstrated the structure of RIPs imposes a large constraint on the nature and strength of the interaction with ribosomal protein which in turn affect the potency of RIPs on the ribosome. Shiga toxin was found to interact with prokaryotic ribosome weaker than the eukaryotic ribosome and crosslinked to the bacterial ribosomal protein L7/L10. Additionally, we increased the HIV-1 specificity of ricin A chain by incorporating the HIV-1 protease specific peptide to the C-terminus of the toxin and hope our findings would help to extend similar scheme to other RIPs in the future. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Yuen-Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 146-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p. iv - viii / Chapter Chapter One --- Introduction of ribosome-inactivating proteins / Chapter 1.1 --- Nomenclature and distribution of ribosome-inactivating proteins --- p.1 / Chapter 1.2 --- Enzymatic activity of ribosome-inactivating proteins and their biological role --- p.2 / Chapter 1.3 --- Structure and catalytic centre of ribosome-inactivating proteins --- p.3 / Chapter 1.4 --- Ribosome specificity of RIPs and their interaction with ribosome --- p.5 / Chapter 1.5 --- Cytotoxicity and antiviral activity of ribosome-inactivating proteins --- p.6 / Chapter 1.6 --- Antiviral activity of RIPs --- p.9 / Chapter 1.7 --- Cellular trafficking of ribosome-inactivating proteins --- p.10 / Chapter 1.8 --- Application and therapeutic use of ribosome-inactivating proteins --- p.10 / Chapter 1.9 --- Evolution of RIPs --- p.11 / Chapter 1.10 --- Other activities of RIPs --- p.12 / Chapter Chapter Two --- Characterization of the interaction between RIPs and rat liver ribosome and its correlation with the potency of RIPs / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Nature of interaction between RIPs and eukaryotic ribosome --- p.12 / Chapter 2.1.2 --- RIPs interact with specific ribosomal proteins --- p.15 / Chapter 2.1.3 --- RIPs demonstrate different specificity towards ribosomes --- p.16 / Chapter 2.1.4 --- Introduction of maize RIP --- p.20 / Chapter 2.1.5 --- Interaction between maize RIP and ribosome --- p.22 / Chapter 2.2 --- Objectives and significance --- p.22 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Cloning and site-directed mutagenesis of RIPs --- p.23 / Chapter 2.3.2 --- Protein expression and purification --- p.23-26 / Chapter 2.3.2.1 --- Maize RIP and variants / Chapter 2.3.2.2 --- His-myc-MOD and His-MOD / Chapter 2.3.2.3 --- Trichosanthin (TCS) / Chapter 2.3.2.4 --- Shiga toxin chain A [E167AE170A] (StxA) / Chapter 2.3.2.5 --- Ricin chain A (RTA) / Chapter 2.3.2.6 --- Pokeweed antiviral protein (PAP) / Chapter 2.3.2.7 --- C-terminal His-tagged MOD, TCS and RTA / Chapter 2.3.2.8 --- His-SUMO-protease / Chapter 2.3.2.9 --- P2 and its variants / Chapter 2.3.2.10 --- Protein concentration and storage / Chapter 2.3.3 --- Purification of rat liver ribosome --- p.26 / Chapter 2.3.4 --- In vitro pull-down assay with ribosome --- p.27 / Chapter 2.3.5 --- On-resin crosslinking and mass spectrometry --- p.27 / Chapter 2.3.6 --- Crosslinking assay and western blotting --- p.28 / Chapter 2.3.7 --- In vitro pull-down assay with P2 --- p.29 / Chapter 2.3.8 --- In vitro pull-down assay with P2 and its variants --- p.29 / Chapter 2.3.9 --- Surface Plasmon Resonance --- p.29 / Chapter 2.3.10 --- N-glycosidase activity assay and quantitative PCR --- p.30 / Chapter 2.3.11 --- Cytotoxicity on 293T --- p.31 / Chapter 2.3.12 --- Cellular uptake of RIPs and western blotting --- p.32 / Chapter 2.4 --- Results / Chapter 2.4.1 --- In vitro pull-down assay with ribosome --- p.32 / Chapter 2.4.2 --- On-resin crosslinking and mass spectrometry of crosslinked proteins --- p.37 / Chapter 2.4.3 --- Crosslinking assay and western blotting --- p.40 / Chapter 2.4.4 --- In vitro pull-down assay with P2 --- p.43 / Chapter 2.4.5 --- Sensorgram of binding between P2 and Maize RIP variants --- p.44 / Chapter 2.4.6 --- N-glycosidase activity of maize RIP variants --- p.45 / Chapter 2.4.7 --- Cytotoxicity of maize RIP variants --- p.48 / Chapter 2.4.8 --- In vitro pull-down assay with P2 and its variants --- p.49 / Chapter 2.4.9 --- Surface Plasmon Resonance of P2 and various RIPs --- p.52 / Chapter 2.4.10 --- N-glycosidase activity assay and quantitative PCR --- p.55 / Chapter 2.4.11 --- Cytotoxicity of RIPs to 293T --- p.57 / Chapter 2.5 --- Discussion --- p.59 / Chapter 2.6 --- Conclusion --- p.72 / Chapter Chapter Three --- Identifying prokaryotic ribosomal protein(s) interacting with shiga toxin / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Background of shiga toxin --- p.74 / Chapter 3.1.2 --- Trafficking and activation of shiga toxin --- p.75 / Chapter 3.1.3 --- Intoxication by Shiga toxin --- p.76 / Chapter 3.1.4 --- Dual specificity on ribosome --- p.77 / Chapter 3.2 --- Objectives and significance --- p.78 / Chapter 3.3 --- Materials and methods / Chapter 3.3.1 --- Cloning of Shiga toxin and ribosomal proteins --- p.79 / Chapter 3.3.2 --- Expression and purification --- p.79-80 / Chapter 3.3.2.1 --- His-SUMO StxA, His-StxA, and His-StxA [E167Q] / Chapter 3.3.2.2 --- Ribosomal proteins / Chapter 3.3.3 --- Isolation of E. coli ribosome and rat liver ribosome --- p.80 / Chapter 3.3.4 --- Pull-down assay of prokaryotic and eukaryotic ribosome --- p.81 / Chapter 3.3.5 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.81 / Chapter 3.3.6 --- Pull-down assay of StxA with HepG2 and C41 lysate --- p.82 / Chapter 3.3.7 --- Two-dimensional electrophoresis --- p.82 / Chapter 3.3.8 --- Mass spectrometric analysis of pull-down assay --- p.83 / Chapter 3.3.9 --- Crosslinking of StxA with r-proteins --- p.84 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Cloning of wild-type shiga toxin --- p.84 / Chapter 3.4.2 --- Pull-down with prokaryotic and eukaryotic ribosome --- p.85 / Chapter 3.4.3 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.88 / Chapter 3.3.4 --- Pull-down assay of StxA with HepG2 and C41 lysates --- p.90 / Chapter 3.4.5 --- Crosslinking of StxA with r-proteins --- p.97 / Chapter 3.5 --- Discussion and conclusion --- p.99 / Chapter Chapter Four --- Engineering ricin A chain for increasing its specificity toward Human Immunodeficiency Virus (HIV) / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.1.1 --- Human immunodeficiency virus --- p.104 / Chapter 4.1.2 --- Current drugs for HIV --- p.105 / Chapter 4.1.3 --- Anti-HIV mechanism of RIPs --- p.105 / Chapter 4.1.4 --- Engineering cytotoxic protein into HIV-1 specific toxin --- p.107 / Chapter 4.2 --- Objectives and significance --- p.109 / Chapter 4.3 --- Materials and methods / Chapter 4.3.1 --- Design and cloning of RTA HIV-1 specific variants --- p.109 / Chapter 4.3.2 --- Cloning, expression and purification of ricin variants --- p.112 / Chapter 4.3.3 --- Purification of HIV-1 protease --- p.112 / Chapter 4.3.4 --- HIV-1 protease induced cleavage of RTA variants --- p.113 / Chapter 4.3.5 --- Cytotoxicity on 293T and JAR --- p.114 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Purity check of RTA variants --- p.114 / Chapter 4.4.2 --- HIV-1 protease induced cleavage of RTA variants --- p.115 / Chapter 4.4.3 --- Cytotoxicity on 293T and JAR --- p.119 / Chapter 4.5 --- Discussion --- p.124 / Chapter 4.6 --- Conclusion --- p.126 / Concluding remarks and future prospect --- p.127 / Appendices / Appendix 1 --- p.128 - 132 / Appendix 2 --- p.133 - 134 / Appendix 3 --- p.135 - 138 / Appendix 4 --- p.139 - 145 / Bibliography --- p.146 - 159
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328030 |
| Date | January 2012 |
| Contributors | Wong, Yuen-Ting, Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (viii, 159 leaves) : ill. (chiefly col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
Page generated in 0.0034 seconds