Targeting the immunoproteasome: Analyzing the biological effects of specific versus pan inhibition

Background: The immunoproteasome is expressed constitutively in immune cells alongside standard proteasomes and forms multiple mixed types of proteasome complexes. These mixed 20S proteasomes may have distinct proteolytic activities depending on their subunit composition and may thereby regulate immune cell function. Using site-specific versus pan-inhibitors of the immunoproteasome, we here aim to dissect the biological functions of the three active sites of the immunoproteasome for monocyte cell function. Understanding these activities in full detail will enable defined immunoproteasome inhibition as a therapeutic strategy.

Methods: Peripheral blood monocytic cells (PBMCs) from healthy donors and the monocytic human cell line THP-1 were treated with the site-specific (LU002i and KZR-504) or the pan-immunoproteasome inhibitors LU005i and ONX-0914 in a dose and time-dependent manner. Specific inhibition of the catalytic sites was biochemically assessed. Cytokine secretion was screened by MSD ELISA. Proteomics data were obtained after acute inhibition of THP-1 cells for 4 hours and combined with RNA sequencing after 24 hours. Functional assays based on omics data were performed including flow cytometry analysis of the cell cycle, cell-surface adhesion markers, cell adhesion and migration assays, phagocytosis, and vesicle secretion.

Results: We here show for the first time that the single-site-specific immunoproteasome inhibitors do not induce prominent transcriptional alterations upon short and acute inhibition. In contrast, pan-inhibitors of the immunoproteasome induced transcriptional changes of DNA, RNA, and cell cycle-related pathways that were accompanied by cell cycle arrest. On the protein level, however, all inhibitors resulted in striking alterations of alternative mRNA splicing and in the secretory compartment of THP-1 cells after only 4 hours of inhibiotion. Inhibition of the MECL1 site specifically reduced the THP1 monocytes' adhesion ability and CD11a adhesion marker surface expression.

Conclusion: Our findings uncover unforeseen impacts of both pan- and single-site-specific immunoproteasome inhibitors, indicating rapid changes in vesicle-associated activities such as the secretion of cytokines and phagocytosis, in addition to compromising the adhesion ability. A detailed comprehension of these effects is essential before immunoproteasome inhibitors can be therapeutically applied in the treatment of patients with chronic inflammation and autoimmune disease.