Dissertations / Theses on the topic 'Nedd4 Family E3 Ligases'

Nedd4L is a HECT-type E3 ubiquitin ligase (it covalently binds ubiquitin molecules before transferring them to the final substrate). Ubiquitination is a posttranslational modification (PTM) that labels proteins for a variety of fates, the most relevant one being proteasome-mediated degradation. Nedd4L is responsible for the regulation of the turnover of the sodium channel ß-ENaC as well as Smad2/3, mediator proteins of the signalling pathway activated by TGF-ß-like cytokines. It also targets the TGF-ß receptor itself. Defects in its function have been related to hereditary hypertension (Liddle’s syndrome), and could be relevant in certain sorts of cancer and metastasis. CDK8/9 and GSK3-ß are two kinases that regulate the phosphorylation of the Smads, enabling them to carry out their function in cooperation with transcription factors and other partner proteins. At the same time, they label the Smads for their recognition by ubiquitin ligases. This provides the cell with a mechanism to give a transient response to the cytokines of the TGF-ß type. In order to identify the residues and the phosphorylation patterns that are relevant for the interactions of the Smads with both the transcription factors and the ubiquitin ligases, we have prepared a set of phosphopeptides corresponding to the sequences of Smad1 and Smad3. Like all other members of the Nedd4 family, Nedd4L has a multi-domain architecture of the type C2-WW-HECT. Several ligases of the family exist in a latent conformation established through inter-domain contacts that occlude the catalytic site in the HECT domain, involving either the C2 domain (Smurf1, Smurf2, WWP2, Nedd4, Nedd4L) or the central segment where the WW domains are located (Itch). Certain cellular events displace these contacts, inducing the transition to the active conformation. In the case of Nedd4L, increases of the intracellular levels of Ca2+ activate the ligase. The hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) delivers into the cytosol the inositol 1,4,5-triphosphate (IP3), a second messenger that mobilizes the intracellular Ca2+ reserves. The C2 domain of Nedd4L interacts both with Ca2+ ions and with IP3. Using a structural and biophysical approach based on Nuclear Magnetic Resonance (NMR) we have described the specific interactions between the HECT and C2 domains that inhibit the catalytic function. Ca2+ binds the C2 domain with high affinity using the same binding surface and compromises these contacts. In addition, it mediates the interaction with IP3. These results provide the structural fundament for the activation and the relocation to the plasma membrane of Nedd4L mediated by Ca2+. The HECT domain has a highly conserved PY site (HECT-PY). The PY motifs are the sequences recognized by WW domains. Central to this recognition is the coordination of the tyrosine residue in the PY motif by the WW domain. In the crystallographic structure of the Nedd4L HECT domain the tyrosine residue of the HECT-PY motif appears buried in the hydrophobic core and not accessible for binding. It has been shown that the WW domains of Nedd4L recognize the HECT-PY motif of the ligase only after the unfolding of the HECT domain. We raised the hypothesis that the recognition of the HECT-PY motif by one of Nedd4L WW domains may play a role in the auto-ubiquitination mechanism of the ligase. Our data confirm that only when the fold of the HECT domain is partially damaged, the PY site is accessible for being recognized by the WW domains. We present the NMR solution structure of the complex between the WW3 domain and the HECT-PY motif. The site is protected in functional Nedd4L molecules, which are able to recognize it in damaged molecules and label them with ubiquitin for degradation.
Nedd4L és una E3 ubiquitín lligasa responsable de la regulació de la vida mitja del canal de sodi ß-ENaC i de Smad2/3, proteïnes mediadores de la ruta de senyalització activada per citocines TGF-ß. Defectes en la seva funció han estat relacionats amb la hipertensió hereditària (Síndrome de Liddle), i podrien ser rellevants en determinats tipus de càncer i metàstasi. CDK8/9 i GSK3-ß són dues quinases que regulen l’estat de fosforilació de les Smads, habilitant-les per dur a terme llur funció en cooperació amb factors de transcripció al mateix temps que les marquen per ser reconegudes per ubiquitín lligases. Amb l’objectiu d’identificar els residus i els patrons de fosforilació rellevants hem preparat un set de fosfopèptids que corresponen a les seqüències de Smad1/3. Nedd4L presenta una arquitectura multi-domini C2-WW-HECT. Diverses lligases de la família de Nedd4 existeixen en una conformació latent en què contactes inter-domini oclouen el lloc catalític en el domini HECT, involucrant bé el domini C2 (Smurf1/2, WWP2, Nedd4, Nedd4L) o la zona central amb els dominis WW (Itch). Certs esdeveniments cel•lulars desplacen aquests contactes, induint la transició a la conformació activa. L’increment dels nivells intracel•lulars de Ca2+ activa Nedd4L. La hidròlisi del fosfolípid de membrana PIP2 allibera l’IP3 provocant aquest increment. El domini C2 de Nedd4L interacciona tant amb el Ca2+ com amb l’IP3. Utilitzant l’RMN hem descrit els contactes HECT-C2 en la conformació latent i hem observat que el Ca2+ s’uneix al domini C2 amb alta afinitat utilitzant el mateix lloc d’unió, a més d’afavorir la interacció amb l’IP3. Així, hem aportat el fonament estructural per a l’activació i re­localització a la membrana cel•lular de Nedd4L. El domini HECT presenta un lloc PY altament conservat (HECT-PY). Els motius PY són reconeguts pels dominis WW. Proposem que el reconeixement del motiu HECT-PY per part d’un dels dominis WW de Nedd4L estigui implicat en l’auto-ubiquitinació. Hem observat que només quan el plegament del domini HECT està compromès, el lloc PY és accessible. Presentem l’estructura per RMN del complex WW3-HECT-PY. El motiu està protegit en molècules funcionals de Nedd4L, capaces de reconèixer-lo en molècules danyades i ubiquitinar-les.

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

Nervenzellen sind hochspezialisierte Zellen, die an Synapsen miteinander verbunden sind, was die Übertragung von neuronalen Informationen erlaubt. Die Entwicklung von Synapsen und die Informationsverarbeitung und Gedächtnisbildung bei reifen Synapsen erfordert eine dynamische Umorganisation von neuronalen Netzwerken. Das beinhaltet die Bildung und Entfernung von Synapsen, Umsatz von synaptischen Proteinen und die Veränderung und Anpassung von synaptischer Erregungsübertragung. U. a. Ubiquitinierung, als regulatorische, posttranslationale Modifikation von Proteinen, könnte eine entscheidende Rolle für solche komplexe, synaptische Umorganisationen spielen. Nedd4-1, eine HECT-Typ E3 Ubiquitin Ligase, reguliert und fördert die Entwicklung von Nervenzellfortsätzen durch die Ubiquitinierung von Rap2. Um die Bedeutung von Nedd4-abhänginger Ubiquitinierung im entwickelten Gehirn zu untersuchen, wurden Mausmodelle generiert und analysiert, in denen Nedd4-1 und dessen nächstes Homolog Nedd4-2, speziell in Nervenzellen ausgeschaltet wurde. Ich habe herausgefunden, dass Nedd4-1 und Nedd4-2 wichtige regulatorische Proteine für die neuronale Morphogenese und die synaptische Plastizität, insbesondere die Aufrechterhaltung von LTP, darstellen. Desweiteren habe ich festgestellt, dass Synaptopodin (SYNPO), ein Prolin-reiches, Aktin-assoziiertes Protein, von Nedd4-1 und Nedd4-2 in vitro ubiquitiniert wird. Dieses Ergebnis deutet daraufhin, dass SYNPO in dem Mechanismus eine Rolle spielt, durch den Nedd4-1 und Nedd4-2 LTP aufrechterhalten. Diese Studie wirft ein neues Licht auf die funktionelle Rolle von Nedd4-abhänginger Ubiquitinierung bei höheren Funktionen des Gehirns von Säugetieren sowie der neuronalen Entwicklung.

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

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.