The human immunodeficiency virus type-1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS) in the world. As there is no cure currently available to treat HIV-1 infections or AIDS, the major focus of drug development efforts has been to target viral replication in an effort to slow down the progression of the infection to AIDS. The aspartyl protease of HIV-1 is an important component in the viral replication cycle and thus, has been an important anti-HIV-1 drug target. Currently there are nine protease inhibitors (PIs) that are being used successfully as a part of highly active antiretroviral therapy (HAART). However, as is with all HIV-1 drug targets, the emergence of drug resistance substitutions within protease is a major obstacle in the use of PIs. Understanding how amino acid substitutions within protease confer drug resistance is key to develop new PIs that are not influenced by resistance mutations. Thus, the primary focus of my dissertation research was to understand the molecular basis for drug resistance caused by some of these resistance substitutions. Until recently, the genetic diversity of the HIV-1 genome was not considered to be important in formulating treatment strategies. However, as the prevalence of HIV-1 continues, the variability of the HIV-1 genome has now been identified as an important factor in how the virus spreads as well as how fast the infection progresses to AIDS. Clinical studies have also revealed that the pathway to protease inhibitor resistance can vary between HIV-1 clades. Therefore, in studying the molecular basis of drug resistance in HIV-1 protease, I have also attempted to understand how genetic variability in HIV-1 protease contributes to PI resistance. In Chapters II, III and Appendix 1, I have examined how clade specific amino acid variations within HIV-1 CRF01_AE and clade C protease affect enzyme structure and activity. Furthermore, I have examined how these sequence variations, which are predominantly outside the active site, contribute to inhibitor resistance in comparison to clade B protease. With the results presented in Chapter II, I was able to show that sequence variations within CRF01_AE protease resulted in structural changes within the protease that might influence enzyme activity. In Chapter III, I focused on how sequence variations in CRF01_AE influence protease activity and inhibitor binding in comparison to clade B protease. Enzyme kinetics data showed that the CRF01-AE had reduced catalytic turnover rates when compared to clade B protease. Binding data also indicated that CRF01_AE protease had an inherent weaker affinity for the PIs nelfinavir (NFV) and darunavir (DRV). In work described in Chapter III, I have also examined the different pathways to NFV resistance seen in CRF01_AE and clade B protease. Using x-ray crystallographic studies I have shown the molecular mechanism by which the two different pathways confer NFV resistance. Furthermore, I provide a rational for why different resistance pathways might emerge in the two clades. In Appendix I, I present results from a parallel study carried out on clade C protease. In Chapter IV, I have examined the role of residue 50 in HIV-1 protease in modulating inhibitor binding. Patients failing amprevavir (APV) and DRV therapy often develop the I50V substitution while the I50L substitution is often observed in patients failing atazanavir (ATV) therapy. This indicates that by making subtle changes at residue 50 the protease is able to confer differential PI resistance. With binding data presented in this chapter I have shown that substitutions at residue 50 change the susceptibility profiles of APV, DRV and ATV. Furthermore, from analyses of protease-inhibitor complexes, I have described structural insights into how substitutions at residue 50 can modulate inhibitor binding. This thesis presents results that reveal mechanistic insights into how a number of resistance substitutions within protease confer drug resistance. The results on non-B clade proteases demonstrate that clade specific sequence variations play a role in modulating enzyme activity and influence the pathway taken to confer PI resistance. Furthermore, the results provide structural insights into how amino acid substitutions outside the active site effectively alter inhibitor binding.

HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART). HIV PIs are extremely effective at suppressing viral load, but have been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease. Recent studies indicate that activation of endoplasmic reticulum (ER) stress is an important cellular mechanism underlying HIV PI-induced dysregulation of lipid metabolism. However, the exact role of ER stress in HIV PI-associated lipodystrophy and dyslipidemia remains to be identified. Hepatocytes and adipocytes are important players in regulating lipid metabolism and the inflammatory state. Dysfunction of these two cell types is closely linked to various metabolic diseases. In this dissertation research, we aimed to define the role of activation of ER stress in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes and further identifty the potential molecular mechanisms. Both cultured and primary mouse adipocytes and hepatocytes were used to examine the effect of individual HIV PIs on ER stress activation and lipid metabolism. The results indicated that HIV PIs differentially activate ER stress through depletion of ER calcium stores, activating the unfolded protein response (UPR). UPR activation further lead to an alteration of cellular differentiation through downstream transcription factor CHOP. At the same time, HIV PIs also altered adipogenesis via differential regulation of the adipogenic transcription factor PPARγ. HIV PI-induced ER stress was closely linked to dysregulation of autophagy activation through CHOP, and upstream ATF-4, signaling pathways. In hepatocytes, the integrase inhibitor raltegravir abrogated HIV PI-induced lipid accumulation by inhibiting ER stress activation and dysregulation of autophagy pathway. Our studies suggest that both ER stress and autophagy are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV HAART-treated patients.

Mesmo com o grande número de estudos relacionados à proteases do subtipo B e de como suas mutações podem interferir na estrutura, na resistência a inibidores e na eficiência catalítica da enzima, existe ainda uma lacuna de como as mudanças polimórficas de proteases de HIV de outros subtipos de HIV-1 interferem nesses fatores. Nesse contexto insere-se esse trabalho, que utilizou proteases de HIV-1 isoladas de pacientes brasileiros HIV-1 infectados com o subtipo F, e outros dois mutantes, sendo que um do subtipo F e outro do subtipo B para ensaios frente a seis inibidores comercialmente disponíveis: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir e saquinavir. Nossos resultados experimentais revelam que os seis inibidores comerciais estudados são significantemente menos ativos para o subtipo F e para as mutantes quando comparados ao subtipo B. Além disso, os valores de vitalidade dessas proteases também são considerados maiores que os obtidos para a proteína selvagem do subtipo B. O acúmulo de mutações comumente detectadas e o polimorfismo natural tornam a protease selvagem do subtipo F cataliticamente suficiente para manter a viabilidade do vírus e garantir alto grau de resistência cruzada frente a todos os inibidores estudados. / Despite years of intense research around the world, HIV continues to represent considerable therapeutical challenge. In order to gain more insights into resistance of polymorphic mutations of existing HIV subtypes toward commercially available pharmaceutics, we studied inhibition of subtypes B and F HIV proteases (PRs) [native and two mutant enzymes clinically identified in Brazilian patients] by six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir). Our results show that all these inhibitors have significantly higher Ki values for the subtype F HIV PR (Fwt) and both mutant enzymes than that for the B subtype HIV PR (Bwt). Furthermore, the biochemical fitnesses of these proteases, or their vitalities, are also considerably higher than that of Bwt. The accumulation of commonly detected resistant mutations in HIV PRs with natural polymorphisms turns Fwt sufficiently catalytically active to guarantee the virus viability and confers it a large degree of cross resistance against all studied inhibitors.

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

The objective of this dissertation is to study the mechanism by which HIV protease inhibitors enter into the central nervous system (CNS) and breast milk of rats, and what effects MDR modulators have on the distribution and metabolism of HIV protease inhibitors. The transporter P-glycoprotein (P-gp) has been shown to limit the distribution of HIV protease inhibitors into the CNS of rodents. This thesis examined the effects of GF120918, an MDR modulator, on the CNS distribution of amprenavir, an HIV protease inhibitor, in rats. GF120918 significantly increased the unbound CNS concentrations of amprenavir without altering the unbound blood concentrations of amprenavir. The results of these studies show that GF120918 can inhibit P-gp at the blood brain barrier (BBB) to increase the unbound CNS concentration of amprenavir and potentially other HIV protease inhibitors. Many first generation MDR modulators inhibited both P-gp transport and CYP3A metabolism. Therefore, a principal goal of this thesis was to determine if GF120918 could selectively inhibit P-gp transport without inhibiting CYP3A metabolism. Using in vitro (human) and in vivo (rat) studies, GF120918 selectively inhibited P-gp at the BBB without inhibiting CYP3A metabolism. The transporter MRP1 has been shown to both transport HIV protease inhibitors and expressed in the CNS. Studies contained in the thesis have shown that mrp1 is not localized to the BBB of rats, therefore, mrp1 is unlikely to play a significant role in the distribution of HIV protease inhibitors into the CNS of rats. The distribution of nelfinavir, an HIV protease inhibitor, into rat breast milk was studied in the thesis as a first approach in understanding the extent to which HIV protease inhibitors can accumulate into milk. The concentration of nelfinavir in rat milk was approximately half that of plasma. P-gp protein expression was detected in lactating rat mammary tissue. However, GF120918 showed no effect on the distribution of nelfinavir into rat milk suggesting that P-gp does not play a significant role in the distribution of HIV protease inhibitors into milk.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed2009/06/10 ). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 127-141).

Mesmo com o grande número de estudos relacionados à proteases do subtipo B e de como suas mutações podem interferir na estrutura, na resistência a inibidores e na eficiência catalítica da enzima, existe ainda uma lacuna de como as mudanças polimórficas de proteases de HIV de outros subtipos de HIV-1 interferem nesses fatores. Nesse contexto insere-se esse trabalho, que utilizou proteases de HIV-1 isoladas de pacientes brasileiros HIV-1 infectados com o subtipo F, e outros dois mutantes, sendo que um do subtipo F e outro do subtipo B para ensaios frente a seis inibidores comercialmente disponíveis: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir e saquinavir. Nossos resultados experimentais revelam que os seis inibidores comerciais estudados são significantemente menos ativos para o subtipo F e para as mutantes quando comparados ao subtipo B. Além disso, os valores de vitalidade dessas proteases também são considerados maiores que os obtidos para a proteína selvagem do subtipo B. O acúmulo de mutações comumente detectadas e o polimorfismo natural tornam a protease selvagem do subtipo F cataliticamente suficiente para manter a viabilidade do vírus e garantir alto grau de resistência cruzada frente a todos os inibidores estudados. / Despite years of intense research around the world, HIV continues to represent considerable therapeutical challenge. In order to gain more insights into resistance of polymorphic mutations of existing HIV subtypes toward commercially available pharmaceutics, we studied inhibition of subtypes B and F HIV proteases (PRs) [native and two mutant enzymes clinically identified in Brazilian patients] by six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir). Our results show that all these inhibitors have significantly higher Ki values for the subtype F HIV PR (Fwt) and both mutant enzymes than that for the B subtype HIV PR (Bwt). Furthermore, the biochemical fitnesses of these proteases, or their vitalities, are also considerably higher than that of Bwt. The accumulation of commonly detected resistant mutations in HIV PRs with natural polymorphisms turns Fwt sufficiently catalytically active to guarantee the virus viability and confers it a large degree of cross resistance against all studied inhibitors.

As the first structural example of this class of proteins, crystals of Pro-RIP and MOD were grown and diffracted to 2.4 and 2.5 A respectively. The structures of the two proteins are solved and found to be highly similar, with main chain RMSD of 0.519. Each protein has two domains. The N-terminal domain consists of five alpha-helices and five-stranded mixed beta-sheet. The conserved active site residues Y94, Y130, E207, R210 and W241, similar to those of other RIPs, are located at the cleft between the N-terminal and C-terminal domains. In Pro-RIP, the 25-amino acid internal inactivation region is found on the surface of the N-terminal domain and consists of a flexible loop followed by a long alpha-helix. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure of maize RIP reveals that the active site is too small to accommodate two glutamate residues and suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. / Pull-down assay indicated that the internal inactivation region diminished the interaction of Pro-RIP with purified ribosomes and ribosomal proteins P0, P1 and P2. Surface plasmon resonance assays showed that Pro-RIP has a slower association rate and faster dissociation rate on intact ribosomes when compared to MOD, resulting 80-fold decrease in binding affinity. These evidences strongly suggested that the reduced ribosome-inactivating activity and cytotoxicity of Pro-RIP is the result of its diminished interaction with the ribosomes. The ribosome binding site of MOD is found to be different from TCS and saporin, which are located between the anti-parallel beta-sheet in the C-terminal domain. In MOD, the positive-charged residues K158, K159, K160 and K161 that were found to be important for ribosome binding are located in the N-terminal domain, underneath the internal inactivation region. / Ribosome-inactivating proteins (RIPs) are rRNA N-glycosidases, which hydrolyze the N-glycosidic bond of A-4324 in 28S rRNA of eukaryotic ribosomes. Based on the number of subunits, RIPs are grouped into three classes. Type I RIPs (e.g. trichosanthin and saporin) are monomeric polypeptide with molecular weights of 25-32 kDa. Type II RIPs (e.g. ricin and cinnamomin) are heterodimeric proteins whose subunits are linked by a disulphide bridge, with molecular weights of 60-65 kDa. Chain A of type II RIPs is the catalytic subunit, while chain B is the lectin subunit, which facilitates the cellular entry of the protein by interacting with carbohydrates on the cell surface. Maize ribosome-inactivating protein is classified as a type III RIP, or an atypical RNA N-glycosidase. It is synthesized and stored in the kernel as a 34 kDa inactive precursor (Pro-RIP). During germination, the precursor undergoes proteolysis to generate a two-chain active RIP (MOD). Previous study has found that the 25-amino acid residues at the acidic internal inactivation region, which are removed during activation of Pro-RIP, is the major control element to suppress its in vitro protein synthesis inhibition activity. / Since the internal inactivation region of Pro-RIP controls the ribosome-inactivating activity and cytotoxicity, it provides an opportunity to engineer an on/off switch forits activity by HIV-1 protease through engineering HIV-1 protease recognition sites into the internal inactivation region of Pro-RIP. A variant that contains two HIV-1 protease recognition sites incorporated to the 25-amino acid internal inactivation region was found to be activated by HIV-1 protease in vitro. This variant entered cells more efficiently than Pro-RIP and was as cytotoxic as MOD. This switch may be applied to other RIPs such as ricin A chain and other protease recognition sequences may be used for increasing the specificity of an RIP toward viral infected cells. / The internal inactivation region of Pro-RIP greatly decreases its cytotoxicity, but not cellular uptake through alpha-2 macroglobulin receptor. On the contrary, the acidic residues within the region hinder fluid-phase endocytosis. Moreover, it is found that the internal inactivation region does not affect sub-cellular localization of the protein - MOD and Pro-RIP locate in the same cellular compartment (nucleus in JAR or cytoplasm in J774A.1 and C8166). / Mak, Nga Sze Amanda. / "July 2007." / Adviser: Shaw Pang Chui. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 216-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.