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Identification and characterization of novel regulators of the tumor suppressor and ubiquitin ligase SCF-FBXW7

Richter, Kai

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Abstract

The evolutionarily conserved protein ubiquitin serves as a posttranslational modification in eukaryotic cells. Ubiquitylation links specific proteins with different effector reactions and pathways, including proteasomal degradation. The canonical ubiquitylation machinery transfers ubiquitin to substrate proteins in a cascade reaction comprising three different enzymes (E1, E2, E3). In human cells, there exist more than 600 E3s, denoted ubiquitin ligases. Ubiquitin ligases provide the ubiquitin system with substrate specificity as they directly interact with the target proteins and mediate the final step of the ubiquitylation reaction. Cullin-RING ubiquitin ligases are the largest subfamily of ubiquitin ligases. The SCF complex is a Cullin-RING ubiquitin ligase that contains a central CUL1 subunit, the RING domain containing protein RBX1, the adaptor protein SKP1 and an F-box protein. The F-box protein is the SCF subunit that recruits the substrate. In humans, there are about 70 F-box proteins. They are interchangeable within the SCF complex and have different substrate specificities. One of the best-characterized F-box proteins is FBXW7. FBXW7 promotes the proteasomal degradation of important oncoproteins, including Cyclin E, MYC, JUN and NOTCH. Accordingly, FBXW7 functions as a tumor suppressor protein. Indeed, FBXW7 is frequently mutated in human cancers. While the downstream effects of FBXW7 are already well-characterized, the upstream regulation of FBXW7 is only poorly understood so far. In order to unravel novel regulation mechanisms for FBXW7, a biochemical screen for FBXW7 interaction partners was performed in the presented study. Immunoprecipitation combined with mass spectrometry identified FBXO45, MYCBP2, XIAP and RAE1 as putative interaction partners of FBXW7. In vivo verification of the putative interactions revealed that FBXO45, MYCBP2, XIAP and RAE1 specifically interact with a negatively charged motif within the N-terminal domain of the FBXW7 α-isoform. Additional interaction studies indicated that FBXW7α, FBXO45, MYCBP2, XIAP and RAE1 are found in a complex. In vitro, only FBXO45 interacted with the N-terminus of FBXW7α, suggesting that FBXO45 is the direct interaction partner of FBXW7α within the complex. FBXO45 and MYCBP2 have been described to form a ubiquitin ligase complex. siRNA-mediated downregulation of FBXO45 and MYCBP2 caused an increase in FBXW7α protein levels, which was specifically observed during mitotic arrest. Furthermore, FBXO45 and MYCBP2 promoted ubiquitylation of FBXW7α. FBXO45 depletion led to a stabilization of FBXW7α in a cycloheximide chase experiment. These findings suggest that FBXO45 and MYCBP2 promote the proteasomal degradation of FBXW7α upon mitotic arrest. FBXW7 is a known regulator of mitotic cell fate. Upon mitotic arrest, FBXW7 promotes mitotic cell death and prevents mitotic slippage. In contrast to FBXW7, this thesis identified FBXO45 and MYCBP2 to promote mitotic slippage and to prevent mitotic cell death. Hence, FBXW7 and FBXO45/MYCBP2 have opposing effects on mitotic cell fate. In conclusion, this thesis describes FBXO45 and MYCBP2 as novel regulators of FBXW7α protein levels during mitotic arrest. Thus, the presented thesis contributes to a better understanding of the regulatory mechanisms underlying the function of the important tumor suppressor protein FBXW7. As FBXW7 contributes to mitotic cell death, the FBXO45/MYCBP2-FBXW7α axis could be a novel target for the improvement of chemotherapy approaches.

Item Type: Dissertation
Supervisor: Hoffmann, Prof. Dr. Ingrid
Date of thesis defense: 26 October 2017
Date Deposited: 23 Nov 2017 11:12
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > German Cancer Research Center (DKFZ)
Subjects: 570 Life sciences
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