By identifying and studying novel regulators, the studies described in this thesis give substantive insights into the molecular mechanisms and different levels of control of TGF-_/BMP, IL-1_ and... Show moreBy identifying and studying novel regulators, the studies described in this thesis give substantive insights into the molecular mechanisms and different levels of control of TGF-_/BMP, IL-1_ and Wnt signaling pathways. Crucially, our work for the first time demonstrated the monoubiquitination of an I-SMAD by an E2-E3 hybrid, and added an important unraveled mechanism of how monoubiquitination could affect TGF-_/BMP signaling. We found that UBE2O participates in IL-1R/TLR4-mediated NF-_B activation via a different mechanism than in BMP signaling. Future studies of ubiquitin enzymes will benefit from our model in chapter 3, which shows that UBE2O disrupts the interaction between TRAF6 and its upstream adapter MyD88 to limit polyubiquitination of TRAF6. Our studies in this thesis also added a new DUB to the list of deubiquitinases that can regulate IL-1R/TLR4 signaling, emphasizing the importance of controlling ubiquitination status in regulating NF-_B activation. Also, we reported a new co-receptor for Wnt3a-induced signaling, suggesting compensating roles for co-receptors in the control of Wnt signaling. Therefore, the studies in this thesis yield more perception in understanding how TGF-_/BMP, IL-1R/TLR4 and Wnt signaling pathways can be regulated depending on the cell type, cell localization and cell state. Show less
In this thesis the evolutionary background, function and localization of the domesticated transposase DAYSLEEPER are described. We found that DAYSLEEPER-like genes can be found in angiosperms, but... Show moreIn this thesis the evolutionary background, function and localization of the domesticated transposase DAYSLEEPER are described. We found that DAYSLEEPER-like genes can be found in angiosperms, but not in lower plants. We also found that DAYSLEEPER interacts with several proteins and is probably involved in regulating protein degradation through involvement in ubiquitin-mediated protein degradation. Show less
Repair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC... Show moreRepair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC-NER) and the UV Damage Endonuclease repair pathway (UVDE) have been studied. Central to TC-NER is the protein Cockayne Syndrome protein A (CSA). Its biological importance can be seen in that mutations in CSA cause the human, serious disorder Cockayne Syndrome. This thesis describes structural and biochemical studies of this protein, which give insights into its substrate-binding and into how mutations in this protein cause the disease Cockayne Syndrome. Biochemical and structural studies of UVDE show the identity and role of its post-translational modification, a carboxylation. A cocrystal structure of UVDE with 6-4PP DNA shows how UVDE can recognize UV damaged DNA. Show less
The VirB/D4 Type four secretion system (T4SS) is a bacterial multiprotein complex that spans the bacterial envelope, which mediates the translocation of T-DNA and effector virulence proteins into... Show moreThe VirB/D4 Type four secretion system (T4SS) is a bacterial multiprotein complex that spans the bacterial envelope, which mediates the translocation of T-DNA and effector virulence proteins into recipient cell. My research revealed that the Agrobacterium VirE3 and VirD2 proteins are effector proteins that are translocated by the Agrobacterium T4SS into host cells. The translocated effector protein VirF carries an N-terminal F-box domain. My research focused on the identification of the host proteins interacting with VirF. The capacity of VirF to interact in vivo with ASK1 and CUL1, indicates that VirF plays a role in planta as part of a SCF complex, most likely mediating the ubiquitination of proteins targeted for degradation via the proteasome. Five other protein interactors of VirF (PIFs) were identified using the yeast 2-hybrid system. These VirF interactors are involved in different cellular processes, such as in metabolic processes, and in defense and stress responses. This suggests that Agrobacterium may translocate an effector virulence protein into plant host cells, which mimics the function of an eukaryotic F-box protein (FBP) and thus can direct the host SCF E3 Ub-ligase to the relevant plant targets. Show less
Krijger, P.H.L.; Berk, P.C.M. van den; Wit, N.; Langerak, P.; Jansen, J.G.; Reynaud, C.A.; ... ; Jacobs, H. 2011
Gap junctions are groups of transmembrane channels that connect the cytoplasms of adjacent cells to mediate the diffusion of small molecules, such as ions, metabolites, second messengers and small... Show moreGap junctions are groups of transmembrane channels that connect the cytoplasms of adjacent cells to mediate the diffusion of small molecules, such as ions, metabolites, second messengers and small peptides. The building blocks of gap junctions are connexin proteins. The most ubiquitous and best studied connexin is connexin43 (Cx43). Cx43 is also known as the heart connexin. In the heart, gap junctional communication between cardiomyocytes ensures efficient electrical coupling and hence the synchronous propagation of action potentials. Misregulation of Cx43 expression, localisation and channel gating may lead to severe cardiac dysfunction. Cx43-based cell-cell coupling is rapidly disrupted following stimulation of certain G protein-coupled receptors (GPCRs). The studies presented in this thesis focus on the quest to unravel the signalling pathways leading to the transient inhibition of Cx43-based communication by GPCR activation. Our results suggest a model in which GPCR agonists inhibit Cx43-based gap junctional communication in two phases. Initial closure of the Cx43 channels si mediated by depletion of the phospholipid PIP2 from the plasma membrane, followed by internalisation of the gap junctions due to ubiquitination of Cx43 by the ubiquitin ligase Nedd4. New insights into the mechanisms underlying Cx43 gap junction closure may lead to new treatments of heart failure. Show less
