Thesis (Ph. D.)--State University of New York at Stony Brook, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Includes bibliographical references. Also available online.

The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.

The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.

Today, it is estimated that 35 million people are living with human immunodeficiency virus (HIV). Since its initial discovery in 1981, researchers and medical providers have worked endless hours to understand the pathology, transmission, and medical management of HIV. In the early days of HIV, life expectancy after diagnosis was 10 years. However, after the development of zidovudine (AZT) in 1987, life expectancy of HIV patients began to slowly increase, albeit still lower than that of the general population. The development of AZT opened the door for more antiretroviral drugs and more drug classes. Now, patients undergo a triple drug regimen to manage HIV. These patients are able to maintain viral suppression and are no longer experiencing opportunistic infection or other AIDS-related conditions. While HIV is medically managed, this is a chronic condition and to-date, not cured. As opposed to opportunistic infections and other AIDS-related conditions, patients are succumbing to non-AIDs related conditions such as renal, neurological, bone disorders, and liver complications. The leading non-AIDs related condition is cardiovascular disease (CVD). Even with viral suppression, HIV infection itself contributes to the pathology and development of atherosclerosis and CVD. It is clear that chronic immune activation, HIV proteins, and dyslipidemia appear to be key factors in CVD development. Since the life expectancy of HIV patients has increased, physicians are now seeing an older generation of HIV patients. Medical providers are shifting focus toward understanding the long-term effects of not just HIV, but antiretroviral therapy (ART) as well. It appears that drug interactions and long-term toxicity augment CVD development. Protease inhibitors (PIs), compared to other ART drug classes, appear to increase the risk of atherosclerosis, especially through dyslipidemia. Due to management of HIV being life-long, compliance is difficult because of high pill burden, drug-drug interactions, and drug side effects. This can result in drug failure leading HIV patients to switch to second-line ART regimens. PIs are a common component of second-line ART regimens. Compared to other ART drugs, PIs have a high genetic barrier to resistance. However, PIs have a low bioavailability requiring high dosage and/or boosting with ritonavir (RTV). Lopinavir (LPV) boosted with RTV (LPV/r) is a favorable PI as it is used in a combination pill and is the most cost effective. However, multiple studies have shown LPV/r correlates more to CVD compared to other PIs. Patients on LPV/r exhibit an increased intima-medial thickening, a hallmark characteristic of atherosclerosis and an increased risk for myocardial infarction. Unfortunately, researchers are greatly conflicted as to why this is and in general why PIs increase the risk of CVD. Future medical treatment for HIV is complex and requires long-term medical management. In recent years, integrase inhibitors (IIs) have exhibited promise to provide better lipid profiles while maintaining viral suppression. However, as this drug class is relatively new and expensive, the financial burden on HIV patients is high. The next step toward addressing the global health issue of HIV is understanding the exact mechanism of how PIs contribute to CVD. This will not only increase the life expectancy of HIV patients, but reduce drug toxicity, non-AIDS related conditions, and increase adherence and viral suppression. It is clear that future research must be focused on understanding the role PIs have in CVD development. Physicians are seeing an older generation of HIV patients, and a vast majority are on second-line regimens. By understanding this relationship, researchers could design alternative drugs to manage CVD risk, by modifying current PIs or designing entirely new drugs.

Viral pathogens have plagued human civilizations since ancient times and continue to pose a serious and constant global threat to not only human health but all facets of life. To date, more than 200 viruses capable of infecting humans have been identified, and the combined efforts of the academic and pharmaceutical sectors have yielded both extensive understanding of the biology and pathology of the viral infections as well as breakthrough interventions against a number of devastating diseases such as those caused by HIV (human immunodeficiency virus) and HCV (hepatitis C virus). In late 2019, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the etiological agent of COVID-19 (coronavirus disease 2019), rapidly spread worldwide, leading to detrimental public health and socioeconomic crises. While the immediate response of the scientific community to the pandemic, which involved investigation of the disease and discovery of several therapeutic options at an unprecedented pace, has been impressive, this recent experience exposes the serious need to continuously fortify our fundamental knowledge of virology and equip our antiviral arsenal in preparation for future outbreaks. Moreover, given the scale of the challenge at hand, it highlights the value in the development and application of experimental approaches that accelerate the rate at which this information is obtained. In this dissertation, we utilize various techniques that allow high-throughput analysis of the SARS-CoV-2 3CL (3-chymotrypsin-like) protease to better understand its functional landscape as a favorable therapeutic target of the virus, and to investigate its response in developing resistance against the clinically used protease inhibitor, nirmatrelvir, at scale. We further expand our efforts to develop a platform for multiplexed drug screening that has the capacity to detect viral protease inhibitors for not only coronaviruses but also other targets across six additional virus families. Using this approach, we are able to rapidly identify broad-acting inhibitors, which are favorable for pandemic preparedness purposes where the exact nature of the future threat is difficult to predict a priori. To perform our studies, we make use of a variety of model systems, from a simple yeast-based system for detecting viral protease activity to the passaging of live virus within cultured human cells. Utilizing our yeast-based reporters, we comprehensively profile the activity landscape of all possible single mutants of the SARS-CoV-2 3CL protease via deep mutational scanning (DMS), uncovering its general malleability while also identifying several immutable regions within the enzyme that can serve as targets for the design of the next generation of protease inhibitors. Among the sites that show tolerance to changes, we predict E166 to be a residue that may confer nirmatrelvir resistance upon mutation based on available structural data which reveal its critical role in the binding of the drug to the active site. We prove this to be true by demonstrating a 265-fold loss in EC50 for the E166V mutant relative to the wild type protease within the recombinant virus. Recognizing that the plasticity of the enzyme could translate to a lower genetic barrier to resistance, we extend our investigation to study the whole virus response to nirmatrelvir at scale via in vitro passaging of SARS-CoV-2 in increasing concentrations of the drug. Upon examining 53 independent viral lineages to explore the ways by which resistance can be acquired, we identify a total of 23 mutations that arise in often non-overlapping combinations, with T21I, P252L, and T304I being the most common precursor mutations within all analyzed mutational trajectories. Validation of select single, double, and triple mutants based on the frequency of their appearance reveals that most single mutations, including the aforementioned founder mutations, confer low-level resistance (~5 – 6 fold) while greater resistance is acquired with the accumulation of additional mutations. Moreover, some mutations, such as T21I and L50F, appear to mediate, through a compensatory mechanism, the acquisition of secondary mutations such as E166V, which alone may confer much greater resistance but also cause significant loss in replicative fitness. Overall, the myriad of solutions that exist for the virus to escape the drug further corroborate the malleability of the SARS-CoV-2 3CL protease as established by our initial DMS study. These findings also establish a foundation for extended analysis of the mechanism of resistance and informed drug design. Lastly, by introducing additional viral proteases into our yeast cellular chassis and labelling each model with a set of unique DNA-barcodes, we develop a method of screening a pool of 40 unique protease targets simultaneously against small molecule libraries. Using this platform, we screen 2,480 structurally diverse compounds, and identify and orthogonally validate a series of broad-acting coronavirus 3CL protease inhibitors with a chromen-2-one structure. Together, the work described in this thesis underline the importance of innovative high-throughput approaches to investigating biology as demonstrated by their application to viral protease research.

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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