Heidelberg, Univ., Diss., 2007. / Online publiziert: 2008.

Stuttgart, Univ., Diss., 2003.

Thèse (Ph. D.)--Université Laval, 2004. / Dans le titre, dans l'expression "glutaminyl-ARNtGln" les lettres Gln sont suscrites, de même que les lettres Asn dans l'expression "asparaginyl-ARNtAsn". Les légendes des ill. se trouvent sur f. en regard, avec pagination continue. Bibliogr.: f. 149-170. Publié aussi en version électronique.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 161 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 114-127).

We employed imaging, electrophysiological, and molecular techniques with the genetically tractable model organism Drosophila melanogaster to unravel fundamental biological and genetic underpinnings regulating synaptic function and plasticity. Using a forward genetic screen, we identified mutations in the Drosophila ortholog of a human WD40 repeat-containing protein termed DDB1 and CUL4 associated factor 12 (DCAF12). We show that DCAF12 likely serves as an adaptor protein for the DDB1-Cul4 E3 ubiquitin ligase complex by recruiting specific target proteins for ubiquitination. DCAF12 is expressed in neurons, muscles, and glia. In mitotically active cells such as muscles, DCAF12 is localized to nuclei and co-localizes in distinct foci with CUL4, suggesting that DCAF12 mediates a nuclear role for the CUL4 E3 ubiquitin ligase complex. In neurons, DCAF12 is localized to both cytoplasmic and nuclear compartments of motor neuron cell bodies, where it colocalizes with Cul4 in nuclei. DCAF12 is also expressed at the periactive zone of presynaptic terminals, but does not distinctly associate with DDB1 or Cul4 at this region. Evoked neurotransmitter release at larval NMJs is significantly reduced in DCAF12 mutants. These defects are rescued by presynaptic expression of wild-type DCAF12, demonstrating that DCAF12 is required presynaptically and serves as an important component of the machinery that facilitates evoked release. In addition, our studies show that DCAF12 is required for the differential expression of glutamate receptor subunits at the larval NMJ through transcriptional and post-translational mechanisms. GluRIID subunit mRNA levels and GluRIIA/C/D subunit protein levels are increased at DCAF12 mutant NMJs. Normal GluRIIA subunit levels can be restored by postsynaptic expression of wild-type DCAF12, but not with a truncated DCAF12 protein lacking a nuclear localization signal (∆NLS-DCAF12). Furthermore, DCAF12 overexpression in muscle nuclei reduces synaptic GluRIIA levels, an effect that can be suppressed by removing a copy of Cul4. These data strongly indicate that DCAF12 in muscle nuclei is required for GluRIIA expression and/or function in a Cul4-dependent manner. Moreover, homozygous DCAF12-GluRIIA double mutants show a strong synthetic lethality phenotype, providing further support for the hypothesis that GluRIIA directly or indirectly requires DCAF12. Mutations in glutamate receptors at larval NMJs trigger a retrograde trans-synaptic signal that leads to a compensatory increase in presynaptic release, which precisely restores the normal efficacy of synaptic transmission and muscle excitation. Reducing the gene dosage of DCAF12 by one gene copy suppresses the initiation and maintenance of GluRIIA-mediated synaptic homeostatic potentiation. This block of synaptic homeostatic potentiation can be rescued by presynaptic expression of DCAF12. In our studies, we determined that DCAF12 is critical for 3 distinct synaptic mechanisms: evoked neurotransmitter release, neurotransmitter reception by regulation of GluR subunit composition, and retrograde synaptic homeostatic signaling. Future research will strive to identify presynaptic and postsynaptic protein targets of DCAF12 and the Cul4 E3 ubiquitin ligase complex and the role of ubiquitination in regulating these synaptic processes.

Philosophiae Doctor - PhD / Retinoblastoma-binding protein 6 (RBBP6) is a multi-domain human protein known to play a role in mRNA splicing, cell cycle control and apoptosis. The protein interacts with tumour suppressor proteins p53 and pRb and recent studies have shown that it plays a role in the ubiquitination of p53 by interacting with Hdm2, the human homologue of mouse double minute protein 2 (Mdm2), in which the RING finger domain plays an essential role. Recently, RBBP6 has been shown to ubiquitinate the mRNA-associated proteinYB-1 through its RING finger domain, causing it to be degraded in the proteasome.RING (Really Interesting New Gene) fingers are small commonly-occurring domains of approximately 70 amino acids in length which coordinate two zinc ions in a cross-brace fashion.They are characterized by a conserved pattern of eight Cysteine or Histidine residues which are involved in coordinating the zinc ions. In terms of this conserved consensus, the RING finger from RBBP6 is expected to coordinate the zinc ions through eight Cysteine residues, making it a “C4C4” RING finger similar to those identified in transcription-associated proteins CNOT4(CCR4-NOT transcription complex, subunit 4) and p44 (interferon-induced protein 44). The amino acid sequence of the domain also shares many similarities with the U-box family of domains, which have an identical three-dimensional structure despite not requiring zinc ions in order to fold. This thesis reports the bacterial expression of a fragment containing the RING finger domain from human RBBP6, and determination of its structure using heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy. Preliminary NMR analysis of the fragment revealed that the domain was folded, but that it was preceded by an unstructured region at the N-terminus. A shortened fragment was therefore expressed and used for structural studies. Isotope-enriched protein samples were generated by growing bacteria in minimal media supplemented with 15NNH4Cl and 13C-glucose and purified using a combination of glutathione agarose affinity chromatography, anion exchange and size exclusion chromatography. A complete set of heteronuclear NMR data was collected at 600 MHz from which almost complete assignment of the backbone, side-chain and aromatic resonances was achieved. By exchange of Zn2+ with 113Cd2+ we managed to confirm that the domain binds two Zn2+ ions, and confirm that they are coordinated in the expected cross-brace manner. Structural data in the form of 2-Dimensional Nuclear Overhauser Enhancement Spectroscopy (2D-NOESY), 15N-separated NOESY and 13Cseparated NOESY spectra were recorded and used to determine the structure using restrained molecular dynamics on the Combined Assignment and Dynamics Algorithm for NMR Applications (CYANA) platform.As expected, the structure contains a triple-stranded β-sheet packing against an α-helix and two zinc-stabilized loops as found in all RING fingers. However, it also contains a C-terminal helix which packs against an N-terminal loop which is similar to that found in many U-box domains.A search using the DALI server revealed that the structure is most similar to the U-box from CHIP (C-terminus of Hsp70-interacting protein), an E3 ligase that cooperates with Hsp70 to degrade unfolded proteins that cannot be refolded. Using NMR we showed that the domain dimerizes with a KD of approximately 200 Μm, which means that it is dimeric at the concentrations used for NMR structure determination. Chemical shift analysis showed the dimerization interface to be very similar to that identified in U-box domains found in C-terminus of Hsp70 interacting proteins (CHIP).The structural similarities reported here between the RING finger from RBBP6 and the U-box family lead us to conclude that RBBP6 may, like CHIP, play a role in protein quality control.

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

Non-homologous end joining (NHEJ) is one of two pathways responsible for the repair of double-strand breaks in eukaryotic cells. The mechanism involves the alignment of broken DNA ends with minimal homology, fill in of short gaps by DNA polymerase(s), and ligation by XRCC4-DNA ligase IV complex. The gap-filling polymerase has not yet been positively identified, but recent biochemical studies have implicated DNA polymerase λ (pol λ), a novel DNA polymerase that has been assigned to the pol X family, in this process. Here we demonstrate that purified pol λ can efficiently catalyze gap-filling synthesis on DNA substrates mimicking NHEJ. By designing two truncated forms of pol λ, we also show that the unique proline-rich region in pol λ plays a role in limiting strand displacement synthesis, a feature that may help its participation in in vivo NHEJ. Moreover, pol λ interacts with XRCC4-DNA ligase IV via its N-terminal BRCT domain and the interaction stimulates the DNA synthesis activity of pol λ. Taken together, these data strongly support that pol λ functions in DNA polymerization events during NHEJ.