Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

No description available.

No description available.

The yeast Fus1p SH3 domain recognizes non-PXXP motif targets. This is contrary to most SH3 domains, which recognize PXXP-containing sequences. In this thesis, I characterized atypical binding of the Fus1p SH3 domain and provide insight into atypical SH3 domain interactions. I demonstrated that the Fus1p SH3 domain binds R(S/T)(S/T)SL-containing peptides derived from yeast proteins, Bnr1p and Ste5p. Through mutagenesis studies and comparisons to other SH3 domains, I showed that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, I created a version of the Fus1p SH3 domain that binds a PXXP-containing peptide with extremely high affinity. Based on my data and analysis, I clearly delineated two distinct surfaces comprising the typical SH3 domain binding interface, and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3 domain-mediated interaction. I demonstrated that the Fus1p SH3 domain also binds Bni1p and Pea2p through yeast two-hybrid experiments, which do not contain either PXXP or R(S/T)(S/T)SL motifs. Through mutagenesis studies and two-hybrid experiments, I showed that the Fus1p SH3 domain utilizes a binding surface comprised of two sub-surfaces to interact with Bni1p and Pea2p. The sub-surfaces include the same portion of the binding surface in typical SH3 domains utilized in R(S/T)(S/T)SL binding of the Fus1p SH3 domain, and a novel binding site. I also showed that the protein binding surface of the Fus1p SH3 domain has a role(s) for efficient mating. Based on my findings in this thesis, I propose that dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of sub-binding sites within the binding surface.

E2A is a transcription factor that plays a particularly critical role in lymphopoiesis. The chromosomal translocation 1;19, disrupts the E2A gene and results in the expression of the fusion oncoprotein E2A-PBX1, which is implicated in acute lymphoblastic leukemia. Both E2A and E2A-PBX1 contain two activation domains, AD1 and AD2, which comprise conserved ΦxxΦΦ motifs where Φ denotes a hydrophobic amino acid. These domains function to recruit transcriptional co-activators and repressors, including the histone acetyl transferase CREB binding protein (CBP) and its paralog p300. The PCET motif within E2A AD1 interacts with the KIX domain of CBP/p300, the disruption of which abrogates the transcriptional activation by E2A and the transformative properties of E2A-PBX1. The generation of a peptide-based inhibitor targeting the PCET:KIX interaction would serve useful in further assessing the role of E2A and E2A-PBX1 in lymphopoiesis and leukemogenesis. An interaction between E2A AD2 and the KIX domain has also been recently identified, and the TAZ domains of CBP/p300 have been shown to interact with several transcription factors that contain ΦxxΦΦ motifs. Thus the design of an inhibitor of the E2A:CBP/p300 interaction requires the full complement of interactions between E2A and the various domains of CBP/p300 to be elucidated. Here, we have used nuclear magnetic resonance (NMR) spectroscopy to determine that AD2 interacts with KIX at the same site as PCET, which indicates that the E2A:KIX interaction can be disrupted by targeting a single binding site. Using an iterative synthetic peptide microarray approach, a peptide with the sequence DKELQDLLDFSLQY was derived from PCET to interact with KIX with higher affinity than the wild type sequence. This peptide now serves as a lead molecule for further development as an inhibitor of the E2A:CBP/p300 interaction. Fluorescence anisotropy, peptide microarray technology, and isothermal titration calorimetry were employed to characterize interactions between both TAZ domains of CBP/p300 and the PCET motif and AD2 of E2A. Alanine substitution of residues within PCET demonstrated that the ΦxxΦΦ motif is a key mediator of these interactions, analogous to the PCET:KIX interaction. These findings now inform future work to establish possible physiological roles for the E2A:TAZ1 and E2A:TAZ2 interactions. / Thesis (Master, Biochemistry) -- Queen's University, 2013-08-06 13:52:28.248

YB-1 est un régulateur important de l’expression des gènes dans les cellules eucaryotes. En plus de son rôle dans la transcription, YB-1 joue un rôle clé dans la traduction et la stabilisation des ARN messagers. Nous avons identifié plusieurs nouveaux partenaires de la protéine YB-1 par chromatographie d’affinité à partir de différents extraits tissulaires. Parmi ces partenaires, nous avons démontré que YB-1 interagit avec la tubuline et les microtubules et stimule fortement l'assemblage des microtubules in vitro. Les microtubules assemblés en présence de YB-1 ont une ultrastructure normale, et les données montrent que YB-1 recouvre probablement la surface extérieure des microtubules. De la même façon YB-1 stimule aussi l'assemblage de la tubuline-MAP qui est plus proche des complexes protéiques qui existent dans la cellule, et de la tubuline clivée par subtilisine ce qui suggère que son interaction avec la tubuline ne relève pas seulement d’effets électrostatiques. Nous avons enfin découvert que la tubuline interfère avec la formation des complexes ARNm:YB-1. Ces résultats suggèrent que YB-1 peut réguler l'assemblage des microtubules in vivo et que son interaction avec la tubuline peut contribuer à la régulation de la traduction des ARN messagers. En effet, in vivo, la traduction des mRNPs dépend de l’état de saturation de l’ARN messager par YB-1. Nous avons montré ici que lorsque le rapport YB-1:ARNm est faible, les complexes mRNPs possèdent des structures non-compactes, alors que les mRNPs saturés sont compacts. Ce changement structural est observé de façon parallèle à l'inhibition de la traduction des ARN messagers lorsqu’ils passent des polysomes (traduits) aux mRNPs libres (non traduits). De façon intéressante, nous avons découvert que les mRNPs saturés se lient aux microtubules via des interactions protéine:protéine et ont tendance à former des agrégats sur la surface des microtubules. Cette dernière propriété pourrait contribuer à la formation de granules de stress et à la localisation des mRNPs dans le cytoplasme. Finalement, un modèle de diffusion facilité a été développé pour expliquer l'assemblage des microtubules orchestré par les polyamines naturelles (telles que YB-1 qui sont positivement chargées dans la cellules). L’ensemble de ces données contribuent à une meilleure compréhension de processus biologiques fondamentaux concernant l’assemblage de la tubuline en microtubules et le trafic des ARN dans la cellule. Ils pourraient avoir un intérêt pour développer de nouveaux médicaments qui ciblent les microtubules. / YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. We identify several novels YB-1 protein partners by affinity chromatography of different tissue extracts. We observed that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. Microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure where YB-1 probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Additionally, we demonstrated that tubulin interferes with mRNA:YB-1 complexes. These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. The translational status of mRNPs in vivo depends on amount of YB-1 associated with mRNA. We show here that at low YB-1:mRNA ratios mRNP complexes possess an incompact structures, whereas saturated mRNPs are compact. This structural change corresponds to translation inhibition when mRNA moves from polysomal (translatable) to free (untranslatable) mRNPs. Saturated mRNPs bind to microtubules via protein:protein interactions and tend to self-aggregate on microtubule surface. This property could contribute to stress granule formation, mRNPs traffic and localization of translation apparatus within cytoplasm. Finally, the facilitated diffusion model was developed to explain enhancement of microtubule assembly by positively charged natural polyamines in living cells. Altogether our data contribute to the understanding of fundamental biological processes.

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Chan chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-95). / Abstracts in English and Chinese. / Dedication --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Abbreviations --- p.vi / List of Figures --- p.ix / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1. --- Thesis synopsis --- p.1 / Chapter 1.2. --- hNP220 protein --- p.1 / Chapter 1.2.1. --- Domain organization --- p.1 / Chapter 1.2.2. --- Known splice variants --- p.5 / Chapter 1.2.3. --- Subcellular localization --- p.7 / Chapter 1.2.4. --- Proposed roles in transcriptional activation and RNA processing --- p.7 / Chapter 1.2.5. --- Interaction between C-terminal of hNP220 and FHL2 --- p.9 / Chapter 1.3. --- Hypothesis --- p.12 / Chapter 1.4. --- Principles of key methods --- p.14 / Chapter 1.4.1. --- RLM-RACE --- p.14 / Chapter 1.4.2. --- CytoTrap® two-hybrid system --- p.15 / Chapter CHAPTER 2 --- Materials and Methods --- p.18 / Chapter 2.1. --- Cloning protocol --- p.18 / Chapter 2.1.1. --- Amplification of DNA fragment --- p.18 / Chapter 2.1.2. --- Purification of PCR product --- p.19 / Chapter 2.1.3. --- Restriction endonuclease digestion --- p.20 / Chapter 2.1.4. --- Dephosphorylation of cloning vector 5'-termini --- p.20 / Chapter 2.1.5. --- Insert/vector ligation --- p.20 / Chapter 2.1.6. --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.21 / Chapter 2.1.7. --- Transformation of ligation product into chemically competent bacterial cells --- p.22 / Chapter 2.1.8. --- Small-scale preparation of bacterial plasmid DNA --- p.22 / Chapter 2.1.9. --- Screening for recombinant clone --- p.24 / Chapter 2.1.10. --- Dideoxy DNA sequencing --- p.24 / Chapter 2.1.11. --- Midi-scale preparation of recombinant plasmid DNA --- p.25 / Chapter 2.2. --- Determination of the transcription start site (TSS) of hNP220 gene --- p.27 / Chapter 2.2.1. --- RNA ligase-mediated rapid amplification of cDNA 5'-end (5-RLM-RACE) --- p.27 / Chapter 2.3. --- Isolation and identification of the third splice variant of HNP220 (hNP220ε) --- p.29 / Chapter 2.3.1. --- PCR from human heart/testis cDNAs pool --- p.29 / Chapter 2.3.2. --- RT-PCR --- p.29 / Chapter 2.3.3. --- Northern hybridization --- p.30 / Chapter 2.4. --- Human tissue distribution of hNP220 --- p.31 / Chapter 2.4.1. --- RT-PCR --- p.31 / Chapter 2.4.2. --- Northern hybridization --- p.31 / Chapter 2.5. --- Visualization of the subcellular localization patterns of GFP-tagged hNP220ε in HepG2 cell line --- p.32 / Chapter 2.5.1. --- Cloning of hNP220a and hNP220s into vector pEGFP-Cl --- p.32 / Chapter 2.5.2. --- Transfection of GFP fusion constructs into HepG2 cell line --- p.32 / Chapter 2.5.3. --- Epi-fluorescence microscopy --- p.33 / Chapter 2.6. --- Identification of the protein-protein interaction between hNP220ε and MAPRE1 --- p.34 / Chapter 2.6.1. --- CytoTrap® XR HeLa Cell cDNA Library screening --- p.34 / Chapter 2.6.1.1. --- Cloning of hNP220ε into yeast two-hybrid bait vector pSos --- p.34 / Chapter 2.6.1.2. --- Preparation of cdc25Ha & cdc25Hα yeast competent cells --- p.34 / Chapter 2.6.1.3. --- Autonomous activation study of bait fusion construct pSos-hNP220ε --- p.36 / Chapter 2.6.1.4. --- Cotransformation of pSos-hNP220ε and CytoTrap® XR HeLa Cell cDNA Library --- p.36 / Chapter 2.6.1.5. --- Verification of interaction by yeast mating --- p.38 / Chapter 2.6.1.5.1. --- Generation of yeast plasmid segregant for mating --- p.38 / Chapter 2.6.1.5.2. --- Yeast mating in 96-well plate --- p.39 / Chapter 2.6.1.6. --- Identification of putative interaction partner --- p.39 / Chapter CHAPTER 3 --- Results --- p.42 / Chapter 3.1. --- Transcription start site of the HNP220 gene is located 312 nucleotides upstream the initiation codon --- p.42 / Chapter 3.2. --- Third splice variant of hNP220 gene hNP220s) is identified --- p.44 / Chapter 3.3. --- In silico analysis of hNP220ε --- p.54 / Chapter 3.4. --- hNP220a and hNP220s are ubiquitously expressed in human fetal and adult tissues --- p.65 / Chapter 3.5. --- hNP220ε shows a punctate subnuclear localization pattern in HepG2 cell line --- p.67 / Chapter 3.6. --- hNP220ε interacts with MAPRE1 --- p.69 / Chapter CHAPTER 4 --- Discussion --- p.71 / Chapter 4.1. --- "Identification of hNP220s, the third splice variant of hNP220 gene" --- p.71 / Chapter 4.2. --- Biological resemblance between hNP220α (hNP220) and hNP220ε --- p.73 / Chapter 4.3. --- Protein-protein interaction between hNP220ε and MAPRE1 --- p.74 / Chapter 4.3.1. --- MAPRE1 protein --- p.77 / Chapter 4.3.2. --- Wnt signaling pathway --- p.78 / Chapter 4.4. --- Potential roles of hNP220 in the regulation of chromosome stability and oncogenesis --- p.82 / Chapter 4.5. --- Summary --- p.85 / Chapter 4.6. --- Concluding questions --- p.86 / Chapter 4.7. --- Future work --- p.87 / References --- p.89 / Appendix --- p.96

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