Studies of the purification, characterization and primary structure of protein inhibitors of trypsin and -amylase from seeds of Job's Tears (Coix lachryma-jobi) were undertaken. The major trypsin inhibitor from seeds of Coix was purified by heat treatment, fractional precipitation with ammonium sulphate, ion-exchange chromatography, gel filtration and preparative reversed-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S- carboxymethylated protein by digestion with trypsin, chymotrypsin and the S.aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong similarity with a number of Bowman-Birk inhibitors from legume and cereal seeds. A protein inhibitor of locust gut ζ-amylase was purified from seeds of Coix using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two major isomers, each a dimer of two identical or closely similar subunits of M(_r) about 26 400. These two isomers also had very similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, B. subtilis. A. oryzae and barley malt. The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform. This showed a high degree of similarity with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean). Endochitinase activity was demonstrated by following the release of radioactivity from [(^3)H] chitin. As far as can be ascertained from the literature this is the first characterization of a plant protein with activity as an enzyme and as an enzyme inhibitor. Preliminary molecular studies were also carried out, including the isolation and in vitro translation of mRNA fractions from developing seeds of Coix.

Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.

Os inibidores de proteases desempenham nas plantas funções como: defesa contra ataque de predadores de sementes, regulação de enzimas endógenas e fontes de proteínas e aminoácidos. Muitos destes inibidores são utilizados em estudos bioquímicos, bem como no tratamento de patologias humanas como inflamação e câncer. Neste trabalho, um inibidor de serinoprotease, presente na semente de Stryphnodendron barbatinan (barbatimão), foi purificado, caracterizado e denominado SbKI. Sementes de barbatimão maduras foram trituradas, até a obtenção de uma farinha, e esta foi suspensa em PBS, pH 7,4 (1 :5 m/v), sob agitação por 14 horas a 4°C. O extrato foi centrifugado, filtrado e tratado com PVPP, sendo denominado EB, o qual apresentou inibição da coagulação sanguínea e da atividade de algumas serinoproteases. O inibidor SbKI foi purificado utilizando-se três procedimentos cromatográficos: cromatografia de exclusão molecular (Superdex-75, 10/30), troca iônica (Mono-S HR, 5/5), ambas acopladas em um sistema &TA Purifier e fase reversa (C-18, Waters 250 x 4,6mm) acoplada a um sistema HPLC. Em cada etapa de purificação a presença do inibidor foi monitorada pelos testes de atividade inibitória da tripsina e da coagulação, ambos in vitro. SDS-PAGE, sob condições redutoras, mostrou que o inibidor é formado por duas cadeias polipeptídicas (cadeia pesada e leve) unida por ligação dissulfeto. As cadeias foram separadas pela cromatografia de &se reversa após serem reduzidas e alquiladas. Suas seqüências N-terminais foram determinadas pela degradação de Edman, em seqüenciador automatizado, apresentando alta identidade seqüencial com inibidores do tipo Kunitz de outras leguminosas. A determinação da massa/molecular do inibidor e de suas cadeias isoladas, foram determinadas por espectroscopia de massa (LCtESI-MS system) mostrando massas moleculares de 19.570Da7 15530Da e 4040Da, respectivamente. A espectroscopia de dicroísmo circular (CD) revelou que o inibidor é formado predominantemente por elementos beta e estruturas desordenadas. SbKI foi estável a variações de pHs (2-12) e temperaturas extremas e a temperatura de transição foi calculada em 73,3\" C. A determinação das constantes de inibição (KI) foi realizada para as serinoproteases tripsina (KI = 5,5 nM) e calicreína plasmática (KI = 1,l nM). / Proteinase inhibitors perform many beneficia1 roles in plants such as defense against the attack of seed predators, regulation of endogenous enzymes and sources of proteins and amino acids. Many inhibitors are used in biochemistry research, as well as human pathology treatment such as inflammation and cancer. In this work, a serino proteinase inhibitor found in Stryphnodendron barbatiman seeds (barbatimão) was purified, characterized and denoted SbKI. Mature barbatimão seeds were ground and suspended in PBS pH 7.4 (15 wlv) and stirred for 14 hours at 4OC. The suspension was centrifuged, filtered and treated with PVPP and denoted EB. This EB inhibited blood coagulation and some serine proteinases activities. The inhibitor SbKI was purified by three chromatography step: molecular exclusion (on Supredex-75, 10/30), ion exchange (on Mono-S, 5/5), both connected to AKTA Purifíer System and reversed phase (on C-18, Waters 250 x 4.6 mm) connected to HPLC System. In each purification step the presence of inhibitor was monitored, in vitro, by trypsin and coagulation inhibitory activity. SDS-PAGE, reduced conditions, showed two polypeptide chains (heavy and light chains) linked by one disulphide bridge. The chains were separated by reversed phase chromatography aíter reduced and alquilated. The N-terminal sequence were performed on automated protein sequencer by Edman degradation and showed homology with Kunitz type inhibitors from Leguminosae. Molecular weight of inhibitor and its chains were determined by mass spectrometry (LC/ESI-MS System) and showed molecular weight of 19.570Da, 15.530Da and 4040Da, respectively. Circular dichroism spectroscopy showed SbKI is constituted predominantly by P elements and unordered structures. SbKI was stable over extreme ranges of pH (2-12) and temperature and the transition temperature 73.3\"C investigated by CD and fluorescence emission spectroscopies. Inhibition constants (Ki) were determined by typsin (Ki = 5.5 nM) and human plasmatic kallikrein (Ki = 1.1 mM)

Os inibidores de proteases desempenham nas plantas funções como: defesa contra ataque de predadores de sementes, regulação de enzimas endógenas e fontes de proteínas e aminoácidos. Muitos destes inibidores são utilizados em estudos bioquímicos, bem como no tratamento de patologias humanas como inflamação e câncer. Neste trabalho, um inibidor de serinoprotease, presente na semente de Stryphnodendron barbatinan (barbatimão), foi purificado, caracterizado e denominado SbKI. Sementes de barbatimão maduras foram trituradas, até a obtenção de uma farinha, e esta foi suspensa em PBS, pH 7,4 (1 :5 m/v), sob agitação por 14 horas a 4°C. O extrato foi centrifugado, filtrado e tratado com PVPP, sendo denominado EB, o qual apresentou inibição da coagulação sanguínea e da atividade de algumas serinoproteases. O inibidor SbKI foi purificado utilizando-se três procedimentos cromatográficos: cromatografia de exclusão molecular (Superdex-75, 10/30), troca iônica (Mono-S HR, 5/5), ambas acopladas em um sistema &TA Purifier e fase reversa (C-18, Waters 250 x 4,6mm) acoplada a um sistema HPLC. Em cada etapa de purificação a presença do inibidor foi monitorada pelos testes de atividade inibitória da tripsina e da coagulação, ambos in vitro. SDS-PAGE, sob condições redutoras, mostrou que o inibidor é formado por duas cadeias polipeptídicas (cadeia pesada e leve) unida por ligação dissulfeto. As cadeias foram separadas pela cromatografia de &se reversa após serem reduzidas e alquiladas. Suas seqüências N-terminais foram determinadas pela degradação de Edman, em seqüenciador automatizado, apresentando alta identidade seqüencial com inibidores do tipo Kunitz de outras leguminosas. A determinação da massa/molecular do inibidor e de suas cadeias isoladas, foram determinadas por espectroscopia de massa (LCtESI-MS system) mostrando massas moleculares de 19.570Da7 15530Da e 4040Da, respectivamente. A espectroscopia de dicroísmo circular (CD) revelou que o inibidor é formado predominantemente por elementos beta e estruturas desordenadas. SbKI foi estável a variações de pHs (2-12) e temperaturas extremas e a temperatura de transição foi calculada em 73,3\" C. A determinação das constantes de inibição (KI) foi realizada para as serinoproteases tripsina (KI = 5,5 nM) e calicreína plasmática (KI = 1,l nM). / Proteinase inhibitors perform many beneficia1 roles in plants such as defense against the attack of seed predators, regulation of endogenous enzymes and sources of proteins and amino acids. Many inhibitors are used in biochemistry research, as well as human pathology treatment such as inflammation and cancer. In this work, a serino proteinase inhibitor found in Stryphnodendron barbatiman seeds (barbatimão) was purified, characterized and denoted SbKI. Mature barbatimão seeds were ground and suspended in PBS pH 7.4 (15 wlv) and stirred for 14 hours at 4OC. The suspension was centrifuged, filtered and treated with PVPP and denoted EB. This EB inhibited blood coagulation and some serine proteinases activities. The inhibitor SbKI was purified by three chromatography step: molecular exclusion (on Supredex-75, 10/30), ion exchange (on Mono-S, 5/5), both connected to AKTA Purifíer System and reversed phase (on C-18, Waters 250 x 4.6 mm) connected to HPLC System. In each purification step the presence of inhibitor was monitored, in vitro, by trypsin and coagulation inhibitory activity. SDS-PAGE, reduced conditions, showed two polypeptide chains (heavy and light chains) linked by one disulphide bridge. The chains were separated by reversed phase chromatography aíter reduced and alquilated. The N-terminal sequence were performed on automated protein sequencer by Edman degradation and showed homology with Kunitz type inhibitors from Leguminosae. Molecular weight of inhibitor and its chains were determined by mass spectrometry (LC/ESI-MS System) and showed molecular weight of 19.570Da, 15.530Da and 4040Da, respectively. Circular dichroism spectroscopy showed SbKI is constituted predominantly by P elements and unordered structures. SbKI was stable over extreme ranges of pH (2-12) and temperature and the transition temperature 73.3\"C investigated by CD and fluorescence emission spectroscopies. Inhibition constants (Ki) were determined by typsin (Ki = 5.5 nM) and human plasmatic kallikrein (Ki = 1.1 mM)

Ma Kit Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 189-206). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of Contents --- p.iii / Abstract --- p.xi / 摘要 --- p.xiv / Abbreviation List --- p.xv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ubiquitination --- p.1 / Chapter 1.1.1 --- Ubiquitin --- p.1 / Chapter 1.1.2 --- Ubiquitin Pathway --- p.3 / Chapter 1.1.3 --- Functions of Ubiquitination --- p.5 / Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8 / Chapter 1.2 --- SUMO Proteins --- p.10 / Chapter 1.2.1 --- SUMO Isoforms --- p.10 / Chapter 1.2.2 --- SUMO Structure --- p.11 / Chapter 1.3 --- Sumoylation --- p.14 / Chapter 1.3.1 --- Functions of Sumoylation --- p.14 / Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15 / Chapter 1.3.1.2 --- Function of Sumoylation on Transcription Factors / Chapter 1.3.1.3 --- Specific Function of SUMO-2/3 Conjugation / Chapter 1.3.2 --- Sumoylation Pathway --- p.19 / Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24 / Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23 / Chapter 1.4.2 --- Structure of PI AS --- p.23 / Chapter 1.4.3 --- Function of PI AS --- p.27 / Chapter 1.5 --- Aims of Study --- p.29 / Chapter Chapter 2 --- Materials & Methods --- p.30 / Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30 / Chapter 2.1.1 --- Primer Design --- p.30 / Chapter 2.1.2 --- Semi-quantitative PCR --- p.31 / Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31 / Chapter 2.1.2.2 --- PCR --- p.32 / Chapter 2.2 --- DNA Cloning --- p.34 / Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34 / Chapter 2.2.1.1 --- Primer Design --- p.34 / Chapter 2.2.1.2 --- PCR --- p.36 / Chapter 2.2.1.3 --- Purification of PCR Product --- p.37 / Chapter 2.2.2 --- Restriction Digestion --- p.37 / Chapter 2.2.3 --- Ligation --- p.40 / Chapter 2.2.4 --- Transformation --- p.40 / Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40 / Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41 / Chapter 2.2.5 --- Plasmid Preparation --- p.42 / Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43 / Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43 / Chapter 2.3 --- Subcellular Localization Study --- p.45 / Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45 / Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46 / Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46 / Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47 / Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48 / Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48 / Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49 / Chapter 2.3.6 --- Western Blotting --- p.49 / Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49 / Chapter 2.3.6.2 --- Protein Extraction --- p.50 / Chapter 2.3.6.3 --- Protein Quantification --- p.51 / Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51 / Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52 / Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54 / Chapter 2.4.1 --- Sample Preparation --- p.54 / Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54 / Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55 / Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55 / Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57 / Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57 / Chapter 2.4.3.2 --- Silver Staining --- p.58 / Chapter 2.4.4 --- Image Analysis --- p.59 / Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60 / Chapter 2.4.5.1 --- Sample Preparation --- p.60 / Chapter 2.4.5.2 --- Data Acquisition --- p.62 / Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62 / Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63 / Chapter 2.5.1 --- RT-PCR Analysis --- p.63 / Chapter 2.5.1.1 --- RNA Extraction --- p.63 / Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64 / Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64 / Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65 / Chapter 2.5.2 --- Western Blotting --- p.66 / Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67 / Chapter 2.6.1 --- Preparation of Competent Cells --- p.67 / Chapter 2.6.2 --- Small Scale Expression --- p.67 / Chapter 2.6.2.1 --- Transformation --- p.67 / Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68 / Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70 / Chapter 2.6.3.1 --- Transformation --- p.70 / Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70 / Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71 / Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71 / Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73 / Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73 / Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73 / Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74 / Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74 / Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74 / Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75 / Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76 / Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76 / Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77 / Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77 / Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77 / Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78 / Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78 / Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79 / Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79 / Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80 / Chapter 2.6.7.1 --- Transformation --- p.80 / Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80 / Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80 / Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80 / Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81 / Chapter Chapter 3 --- Results --- p.82 / Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82 / Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82 / Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82 / Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83 / Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83 / Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83 / Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84 / Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84 / Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90 / Chapter 3.2.1 --- Overexpression Confirmation --- p.90 / Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91 / Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94 / Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94 / Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95 / Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95 / Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96 / Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97 / Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112 / Chapter 3.3.1 --- Protein Expression Profiles after Transient Transfection / Chapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113 / Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124 / Chapter 3.4.1 --- RT-PCR Analyses --- p.124 / Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124 / Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125 / Chapter 3.4.2 --- Western Blotting --- p.128 / Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128 / Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129 / Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129 / Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133 / Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133 / Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135 / Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137 / Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137 / Chapter 3.5.3.2 --- Cleavage after Elution --- p.137 / Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138 / Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138 / Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141 / Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141 / Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141 / Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143 / Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147 / Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148 / Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148 / Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153 / Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153 / Chapter 3.5.6.2 --- Purification by Heparin --- p.153 / Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154 / Chapter Chapter 4 --- Discussion --- p.157 / Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157 / Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157 / Chapter 4.1.2 --- Importance of Sumoylation --- p.158 / Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159 / Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160 / Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162 / Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162 / Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163 / Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164 / Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165 / Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167 / Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169 / Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169 / Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170 / Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172 / Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172 / Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174 / Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175 / Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176 / Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178 / Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178 / Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180 / Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180 / Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181 / Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182 / Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182 / Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183 / Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184 / Chapter 4.5 --- Conclusion --- p.185 / Chapter 4.6 --- Future Perspectives --- p.187 / Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187 / Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187 / Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188 / References --- p.189 / Appendix --- p.207

Influenza virus A and B are common pathogens that cause respiratory disease in humans. Recently, a highly virulent H5N1 subtype avian influenza virus caused disease outbreaks in poultry around the world. Drug resistant type A viruses rapidly emerged, and the recent H5N1 viruses were reported to be resistant to all current antiviral drugs. There is an urgent need for the development of new antiviral drugs target against both influenza A and B viruses. This dissertation describes work to identify small molecule inhibitors of influenza protein NS1 by a high throughput fluorescence polarization assay. The N-terminal GST fusion of NS1A (residue 1-215) and NS1B (residue 1-145) were chosen to be the NS1A and NS1B targets respectively for HT screening. In developing the assay, the concentrations of fluorophore and protein, and chemical additives were optimized. A total of 17,969 single chemicals from four compound libraries were screened using the optimized assay. Six true hits with dose-response activity were identified. Four of them show an IC₅₀ less than 1 [micromolar]. In addition, one compound, EGCG, has proven to reduce influenza virus replication in a cell based assay, presumably by interacting with the RNA binding domain of NS1. High throughput, computer based, virtual screenings were also performed using four docking programs. In terms of enrichment rate, ICM was the best program for virtual screening inhibitors against NS1-RBD. The compound ZINC0096886 was identified as an inhibitor showing an IC₅₀ around 19 [micromolars] against NS1A, and 13.8 [micromolars] against NS1B. In addition, the crystallographic structures of the NS1A effector domain (wild type, W187A, and W187Y mutants) of influenza A/Udorn/72 virus are presented. A hypothetical model of the intact NS1 dimer is also presented. Unlike the wild type dimer, the W187Y mutant behaved as a monomer in solution, but still was able to binding its target protein, CPSF30, with wild type binding affinity. This mutant may be a better target for the development of new antiviral drugs, as the CPSF30 binding pocket is more accessible to potential inhibitors. The structural information of those proteins would be very helpful for virtual screening and rational lead optimization. / text