Biochemical and functional analysis of the FBXO7 interactome
- Biochemische und funktionelle Analyse des FBXO7 Interaktoms
Crnkovic, Vanessa Stefanie; Stegmüller, Judith (Thesis advisor); Müller-Newen, Gerhard (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2022
The protein FBXO7 is a critical subunit of the SCF-complex. Being an E3 ubiquitin ligase complex, it is an important component of the ubiquitin proteasome system (UPS), catalyzing the ubiquitination of substrates for proteasomal degradation. Mutations in the FBXO7 protein lead to functional impairment of this protein and thereby to a so-called Parkinsonian-Pyramidal Syndrome (PPS). Several proteins have been found to be associated with FBXO7 but for many the functional relationship remains to be elucidated. In my dissertation, Biochemical and functional analysis of the FBXO7 interactome, I chose the three subunits BAG6, GET4 and UBL4A, of the BAG6-complex, to investigate whether or not any of these subunits is a potential substrate of FBXO7. Furthermore, I used the CRISPR/Cas9 technology for FBXO7-specific gene editing to generate a useful tool for future FBXO7-dedicated research. Using western blot analysis, I discovered that in the presence of the functional E3 ligase, only GET4 shows a significant smear indicative of ubiquitination. Carrying out cell-based ubiquitination assays, confirmed that the observed pattern was a modification of the protein by ubiquitin. In this way, I identified the protein GET4 as a potential new substrate of FBXO7 and on the other hand excluded the other two subunits of the BAG6 complex, since they did not show any signs of specific ubiquitination pattern in the presence of the functional E3 ubiquitin ligase. As a first step for my second aim, I generated CRISPR/Cas9 plasmids targeting human FBXO7. Carrying out western blot analysis, validated that the newly generated plasmids affect FBXO7 protein levels. If this change in expression would also trigger the functional impairment of the FBXO7 protein, was investigated via proteasome activity assays using clonal cell lines for more valid results. The knockout cell lines showed, compared to FBXO7 wild type, a significant reduction in proteasome activity, proving that loss of FBXO7 leads to an impairment of the proteasome activity.Thus, I have demonstrated that the CRISPR/Cas9 system is a highly efficient but also simple technology to precisely modify specific target regions within a gene, and that FBXO7 is required for the proper functioning of the proteasome.
- RWTH PUBLICATIONS: RWTH-2022-02515