Identification of Proteins Selectively Digested by Immunoproteasome in the Absence of PA28α Subunit
The PA28 alpha subunit is a protein that is a subunit of the 11S regulator of the immunoproteasome. This study attempts to identify select substrates of the Immunoproteasome by utilizing western blots. The Immunoproteasome targets a select group of proteins under stress or inflammation to alter cellular physiology, metabolism, and function and identifying the select substrates of the Immunoproteasome in absence of one of the subunits will greatly advance our knowledge on how cells response to stress or inflammation. HCT116 cells were developed where the gene responsible for the PA-28 α subunit, the PSME1 gene had been knocked out by CRISPR-Cas9 method. These cells were used along with wild type cells as control to prepare cell lysate. An analysis was done on the abundance of a number of candidate proteins. An analysis was done on a number of proteins to see differences in cell concentrations of the proteins in the knockout and wild type cells. These proteins included transcription factors and metabolic enzymes that play crucial roles in maintaining cellular homeostasis, in particular some proteins involved in the process of autophagy. To test this, western blots were used.to compare changes. Nrf1, Rictor, and c-Myc decreased whereas P62 was shown to increase. The absence of PA28 alpha seems to promote cell proliferation and oncogenesis.