Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.

Maintaining genome integrity is indispensible for cells to prevent and limit accruement of deleterious mutations and to promote viable cell growth and proliferation. Cells possess a myriad of mechanisms to detect, prevent and repair incurred cellular damage. Here we discuss various proteins and their accompanying cellular pathways that promote genome stability. We first investigate the NEDD8 protein and its role in promoting homologous recombination repair via multiple Cullin E3 ubiquitin ligases. We provide specific mechanisms through which, UBE2M, an E2 conjugating enzyme, neddylates various Cullin ligases to render them catalytically active to degrade their substrates by the proteasome. We show that CUL1, CUL2 and CUL4 are important in regulating various steps in the DNA damage response. Our data indicates that UBE2M and the neddylation pathway are important for genome stability. Our second topic discusses the role of the USP1- UAF1 deubiquitinating enzyme in promoting homologous recombination. We show that USP1-UAF1 interact with and stabilize RAD51AP1 (RAD51- Associated Protein 1). RAD51AP1 has previously been reported to promote homologous recombination by facilitating recombinase activity of RAD51, an essential protein involved in homologous recombination repair. We show that USP1, UAF1 and RAD51AP1 depletion leads to genome instability. Our data demonstrates the importance of these proteins in promoting genome integrity via homologous recombination.

3-M syndrome (named after three authors who first described the condition) is an autosomal recessive condition characterised by pre- and post-natal growth impairment, facial dysmorphism and radiological features (slender long bones and tall vertebral bodies). It is caused by loss of function mutations in the Cullin 7 (CUL7) and Obscurin-like 1 (OBSL1) genes. CUL7 is a protein involved in ubiquitination (the process of targeted protein degradation) and OBSL1 is a putative cytoskeletal adaptor protein. The mechanisms through which loss of function mutations in OBSL1 or CUL7 lead to growth impairment is unclear but previous work suggests impaired placental function and altered insulin-like growth factor 1 (IGF-1) signaling as possibilities. The overall aim of this study was to elucidate the mechanisms underlying growth impairment in 3-M syndrome. Initially phenotypic data was collected on a cohort of patients and a genotype-phenotype comparison was undertaken. Skin fibroblast cell lines were derived from four patients with 3-M syndrome and used to study growth hormone (GH) and IGF-1 signal transduction, cell proliferation and apoptosis. Subsequently a hypothesis generating approach to identify novel mechanisms underlying 3-M growth impairment was undertaken in whole transcriptome and metabolomic studies. In addition an animal model using morpholino oligonucleotide mediated knock down of OBSL1 in Xenopus tropicalis was developed to study the effects on growth in a non placenting vertebrate to determine if the growth impairment seen in 3-M syndrome is independent of placental function. Cell proliferation was reduced in 3-M fibroblasts while apoptosis was not different from controls. No differences in GH signal transduction were identified but reduced activation of AKT following IGF-1 stimulation was identified in 3-M fibroblast cell lines. IGF2 was identified as the top downregulated probeset in 3-M fibroblasts compared to control in the whole genome transcriptome analysis. Metabolomic changes related to energy metabolism were identified in 3-M syndrome fibroblasts. Knock down of xtOBSL1 using two independent morpholinos resulted in growth impairment at embryonic stage 50, suggesting the growth impairment seen is at least in part independent of placental function. These studies suggest impaired placental function is not a key component of the growth impairment in 3-M syndrome. Impairment of IGF-1 signal transduction and IGF2 silencing are likely to contribute to the growth impairment in 3-M syndrome. The mechanisms relating to this IGF2 silencing require further studies.

Zusammenfassung Ein fehlerhafter Proteinabbau führt in höheren Eukaryoten zu diversen Krankheiten wie z.B. neurodegenerativen Störungen und Krebs. Es ist daher bedeutend die Regulationsmechanismen des Proteinabbaus zu verstehen. Intrazelluläre Proteine werden spezifisch durch das Ubiquitin-Proteasome System abgebaut. Cullin-RING Ligasen, welche durch das ubiquitin-ähnliche Protein Nedd8 aktiviert werden, binden und markieren das Zielprotein mit Ubiquitin. Diese ubiquitinierten Proteine werden durch das 26S Proteasome abgebaut. Die zwei Deneddylasen DenA und COP9 Signalosome (CSN) entfernen Nedd8 von unterschiedlichen Substraten. Diese Arbeit zeigt im Modellorganismus Aspergillus nidulans, dass DenA aus einer Kernfraktion sowie einer dynamischen zytoplasmatischen Subpopulation besteht. Zudem wird (A) das Zusammenspiel zwischen DenA und CSN im Kern untersucht und (B) die bisher unbekannte Phosphatase DipA, welche an der Regulation des zytoplasmatischen DenA und an der Zelldifferenzierung beteiligt ist, charakterisiert. (A) Eine erhöhte DenA Konzentration kann teilweise das Fehlen eines aktiven CSN kompensieren, indem es der Akkumulation an neddylierten Proteinen und damit CSN assoziierten Entwicklungsstörungen entgegenwirkt. Beide pilzlichen Deneddylasen haben somit unterschiedliche aber auch überlappende Funktionen. Zusätzlich zeigt sich, dass die DenA Kernfraktion, welche mit dem CSN interagiert, in der pilzlichen Entwicklung durch fünf benachbarte CSN Untereinheiten destabilisiert wird. Da diese Untereinheiten eine funktionelle Oberfläche bilden ist anzunehmen, dass die Interaktion von DenA mit dieser Oberfläche wichtig für die Stabilitätskontrolle der DenA Kernsubpopulation ist. (B) Zytoplasmatisches DenA wird zusammen mit DipA transportiert und akkumuliert an den Septen. Fehlt DipA erhöht sich die DenA Stabilität. Somit spielt DipA eine wichtige Rolle in der zytoplasmatischen DenA Stabilitätskontrolle. Zusätzlich führt das Fehlen von DipA zu einer erhöhten Septenbildung und Defekten in der lichtabhängigen Zellentwicklung des Pilzes. DipA wird somit, neben der DenA Stabilitätskontrolle, für die Zelldifferenzierung benötigt. Die Stabilität der zwei DenA Subpopulationen wird zusätzlich durch Phosphorylierung reguliert. Während vegetativen Bedingungen wird DenA durch die Phosphorylierung von S243 und S245 stabilisiert, was für die Initiierung der nachfolgenden asexuellen Entwicklung wichtig ist. Anschließend wird DenA durch eine Änderung des Phosphorylierungsmusters destabilisiert und abgebaut. Zusammenfassend zeigt diese Studie Einblicke in komplexe Mechanismen des DenA Proteinabbaus, welche womöglich auch in höheren Eukaryoten relevant sind.

No description available.

A la suite d’un criblage in silico nous avons identifié un nouveau gène codant pour une protéine à domaine BTB-POZ, XBTBD6.<p>Nous avons déterminé que la protéine XBTBD6 est une protéine cytoplasmique. Dans les cellules Hela, CHO, U2OS et COS7 la protéine XBTBD6 est localisée dans des corpuscules cytoplasmiques, localisation similaire à celle des protéines XBTBD3, HBTBD1 et HBTBD2. Nous avons observé que la partie N-terminale de la protéine, contenant le domaine BTB-POZ, est localisée dans la cellule comme la protéine entière ;par contre la partie C-terminale est exclusivement nucléaire. De plus, nous avons observé que XBTBD6 est localisée de façon diffuse dans le cytoplasme des cellules Neuro2A, 9L et 518A2e. Nous avons montré que la protéine XBTBD6 homodimérise et hétérodimérise avec XBTBD3 et XBTBD2 et qu’elle interagit avec l’ubiquitine ligase E3 XCullin 3. L’ensemble de ces interactions nécessite la présence du domaine BTB-POZ. Ces données montrent que les protéines BTBD6, BTBD3, BTBD1 et BTBD2 possèdent des propriétés communes indiquant qu’elles appartiennent à un sous groupe de la famille des protéines à domaine BTB-POZ.<p>Le profil d’expression a été analysé par la technique de protection à la RNAse et par hybridation in situ. Les résultats montrent que ce gène est fortement exprimé dans le système nerveux adulte et embryonnaire. Des expériences de surexpression par micro-injection d’ARNm ont permis de placer le gène XBTBD6 dans la cascade d’activation des gènes proneuraux en aval de XNgnr-1, XNeuroD, Xath3 et Xebf3. Ces résultats montrent que XBTBD6 est un marqueur neuronal chez le xénope. <p>Au cours de l’étude de la fonction du gène XBTBD6, nous avons montré que la surexpression et la perte de fonction de ce gène dans l’embryon de xénope n’induit pas de variation du nombre de neurones dans la plaque neurale. Par contre nous avons observé que la surexpression du gène XBTBD6 dans des cellules Neuro2A en différentiation régule négativement la croissance des neurites.<p>Nous avons élaboré un modèle de fonctionnement biochimique hypothétique où la protéine XBTBD6 fonctionnerait comme protéine adaptatrice dans un complexe d’ubiquitination permettant l’ubiquitination d’une protéine cible. Nous avons recherché les partenaires potentiels de XBTBD6 en utilisant la technique du double hybride en levure mais sans y parvenir.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.