Characterization of the PIAS family (protein inhibitors of activated STATs) of the sumoylation E3 ligases.

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.iTable of Contents --- p.iiiAbstract --- p.xi摘要 --- p.xivAbbreviation List --- p.xvList of Figures --- p.xviiList of Tables --- p.xxiiiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Ubiquitination --- p.1Chapter 1.1.1 --- Ubiquitin --- p.1Chapter 1.1.2 --- Ubiquitin Pathway --- p.3Chapter 1.1.3 --- Functions of Ubiquitination --- p.5Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8Chapter 1.2 --- SUMO Proteins --- p.10Chapter 1.2.1 --- SUMO Isoforms --- p.10Chapter 1.2.2 --- SUMO Structure --- p.11Chapter 1.3 --- Sumoylation --- p.14Chapter 1.3.1 --- Functions of Sumoylation --- p.14Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15Chapter 1.3.1.2 --- Function of Sumoylation on Transcription FactorsChapter 1.3.1.3 --- Specific Function of SUMO-2/3 ConjugationChapter 1.3.2 --- Sumoylation Pathway --- p.19Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23Chapter 1.4.2 --- Structure of PI AS --- p.23Chapter 1.4.3 --- Function of PI AS --- p.27Chapter 1.5 --- Aims of Study --- p.29Chapter Chapter 2 --- Materials & Methods --- p.30Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30Chapter 2.1.1 --- Primer Design --- p.30Chapter 2.1.2 --- Semi-quantitative PCR --- p.31Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31Chapter 2.1.2.2 --- PCR --- p.32Chapter 2.2 --- DNA Cloning --- p.34Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34Chapter 2.2.1.1 --- Primer Design --- p.34Chapter 2.2.1.2 --- PCR --- p.36Chapter 2.2.1.3 --- Purification of PCR Product --- p.37Chapter 2.2.2 --- Restriction Digestion --- p.37Chapter 2.2.3 --- Ligation --- p.40Chapter 2.2.4 --- Transformation --- p.40Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41Chapter 2.2.5 --- Plasmid Preparation --- p.42Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43Chapter 2.3 --- Subcellular Localization Study --- p.45Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49Chapter 2.3.6 --- Western Blotting --- p.49Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49Chapter 2.3.6.2 --- Protein Extraction --- p.50Chapter 2.3.6.3 --- Protein Quantification --- p.51Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54Chapter 2.4.1 --- Sample Preparation --- p.54Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57Chapter 2.4.3.2 --- Silver Staining --- p.58Chapter 2.4.4 --- Image Analysis --- p.59Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60Chapter 2.4.5.1 --- Sample Preparation --- p.60Chapter 2.4.5.2 --- Data Acquisition --- p.62Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63Chapter 2.5.1 --- RT-PCR Analysis --- p.63Chapter 2.5.1.1 --- RNA Extraction --- p.63Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65Chapter 2.5.2 --- Western Blotting --- p.66Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67Chapter 2.6.1 --- Preparation of Competent Cells --- p.67Chapter 2.6.2 --- Small Scale Expression --- p.67Chapter 2.6.2.1 --- Transformation --- p.67Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70Chapter 2.6.3.1 --- Transformation --- p.70Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80Chapter 2.6.7.1 --- Transformation --- p.80Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81Chapter Chapter 3 --- Results --- p.82Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90Chapter 3.2.1 --- Overexpression Confirmation --- p.90Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112Chapter 3.3.1 --- Protein Expression Profiles after Transient TransfectionChapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124Chapter 3.4.1 --- RT-PCR Analyses --- p.124Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125Chapter 3.4.2 --- Western Blotting --- p.128Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137Chapter 3.5.3.2 --- Cleavage after Elution --- p.137Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153Chapter 3.5.6.2 --- Purification by Heparin --- p.153Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154Chapter Chapter 4 --- Discussion --- p.157Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157Chapter 4.1.2 --- Importance of Sumoylation --- p.158Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184Chapter 4.5 --- Conclusion --- p.185Chapter 4.6 --- Future Perspectives --- p.187Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188References --- p.189Appendix --- p.20