Studying the consequences of removing a protein from a cell tells us a lot about the role that protein plays in the normal cellular function. Thus, the project initiated with the generation of a number of cell lines lacking the satellite proteins we are interested in. This was achieved by employment of the CRISPR/Cas9 genome editing system. This technique leads to the disruption of the targeted genes, meaning the protein of interested is no longer expressed. In total, the projected utilized 6 such cell lines lacking satellite proteins. Additionally, an alternative method of protein knockdown, siRNA, was also used. While these techniques were employed and optimized a number of pharmacological inhibitors, that impact a range of cellular processes, were tested for their effect on satellites. Strikingly, it was observed that inhibition of the proteasome had a profound effect on the appearance of satellites within the cell. The proteasome is a complex that the cell uses to recycle the components that make-up proteins. Therefore, the proteasome is crucial to regulating the concentration of proteins within the cell and preventing the toxic accumulation of damaged proteins. Inhibition of the proteasome leads to the collection of non-degradable proteins into a structure around the centrosome, called the aggresome. We found that satellites became entangled within the aggresome following proteasome inhibition. Further, aggresome formation was blocked in lines lacking satellite proteins. This means that satellites themselves are essential to the formation of the aggresome, which is an important observation for understanding how protein degradation is regulated. This allowed us to build a model in which satellites contribute to the movement of proteins to the aggresome. By closely examining the role of individual satellite proteins in aggresome formation, we have observed that some of the proteins are required earlier in the aggresome pathway, namely when proteins first start to aggregate within the cytoplasm. Knockdown of these proteins also prevented aggresome-like structures induced via alternative methods. This raises the tantalizing possibility that satellites play a role in protein aggregation in diseases that are characterized by the excessive accumulation of protein aggregates. We started to explore this by looking at aggregation of the protein that becomes dysregulated in Huntington’s disease. Satellites associate with these aggregates and when disrupted reduce the size to which the aggregates can grow. These results will be exploited through collaborations we have established with research groups that are experts in neurodegenerative diseases, which are all protein aggregation disorders. Together our findings present a new model for the regulation of protein levels in cells, linking centriolar satellites to a novel activity in cellular regulation and provide insight into how satellites might contribute to disease states. These observations have been disseminated to our colleagues and collaborators through meetings and seminars, and our fields by oral presentation at international conferences. A manuscript describing this work will submitted in the coming months. The public has been made aware of our research through events both in Canada and Ireland.
