| dc.description.abstract | The proteasome activator 28 (PA28αβ) regulates both the influx of substrates through the terminal rings of the proteolytic core and the proteolytic activity of the core particle (CP). Functional docking of PA28αβ onto the CP involves 1) the physical interaction between the two complexes, and 2) the activation of the proteolytic activity of the CP. Physical docking requires the insertion of the last carboxyl-terminal tyrosine of PA28α into the outer rings of the CP. Another factor involved in docking is a phosphorylated amino acid in PA28αβ, as the dephosphorylation of this complex abolishes stimulating abilities. As phosphorylation patterns of PA28αβ change within the cell due to stimulation, the question arose if this change is used as a regulatory mechanism for docking of it onto the CP. Therefore, this study aimed to set up a cellular assay to characterize the docking behaviour of PA28αβ in murine Ana-1 macrophages. Experiments were performed using stable, monoclonal cell lines of WT, and PA28β-deficient murine macrophages, obtained by utilizing the CRISPR/Cas9 gene-editing system and were performed in both unstimulated and interferon-gamma stimulated cells. First, the expression of both PA28αβ subunits was determined via quantitative western blot and flow cytometry analysis. Secondly, Ana-1 cells were treated with the CRISPR/Cas9 gene-editing system. In the first way, in both wildtype and ΔPA28β Ana-1 macrophages an affinity tag was inserted at the N-terminus of the Psme1 gene, encoding for PA28α, and this was validated using flow cytometry analysis. The second way, via utilizing error-prone DNA repair, the efficiency and efficacy of single-guide RNAs (sgRNAs) were investigated. Subsequently, genotyping was performed to validate if the designed single-guide RNAs (sgRNAs) targeted the Cas9 enzyme to the right cutting site in Cas9-treated Ana-1 macrophages. Lastly, an immunoprecipitation protocol was partly optimised with the intention to isolate PA28αβ out of the cell to study its docking behaviour. I found that PA28β is upregulated in response to stimulation, whereas PA28α levels remain unaffected. However, the question arose about how successful stimulation of the cells had been, as the major histocompatibility complex class I levels remained unaffected too. The insertion of the affinity tag was successful, but any result was abolished after a freeze-and-thaw cycle. Lastly, immunoprecipitation remained unsuccessful and still needs several optimisation steps. Unravelling the highly complex interconnection of pathways influencing proteins' fate from synthesis to degradation is fundamental to improving our understanding of the interplay between cellular proteostasis and the immune system before we are able to specifically target it during therapy. | |
