The generation and characterization of a mouse embryonic stem cell line with <i>Psmb9</i> immunoproteasome gene knockout

Immunoproteasomes, a unique type of proteasome complex, play a critical role in antigen presentation and cellular homeostasis. Unlike the constitutive 20S proteasome, the catalytic subunits β1, β2, and β5 in the immunoproteasome are replaced by inducible isoforms: β1i (LMP2), β2i (MECL-1), and β5i (LMP7). The expression of the genes encoding these subunits (Psmb9, Psmb10, and Psmb8) is activated by cytokines, primarily interferon-γ (IFNγ). Although it has been demonstrated more and more convincingly that immunoproteasomes are expressed in embryonic stem cells (ESCs), their involvement in maintaining pluripotency, promoting self-renewal, and regulating differentiation processes remains unexplored. This study implemented CRISPR/Cas9 technology to generate a <i>Psmb9</i> gene knockout (Psmb9KO) mouse ESC line. The resulting cells exhibited a normal karyotype and morphology, maintained normal proliferation rates, and retained the capacity to form teratomas containing derivatives of all three germ layers. However, the differentiation induced by retinoic acid (RA) and IFNγ caused an accumulation of Mecl-1 precursors in Psmb9KO cells, suggesting modifications in immunoproteasome assembly. Furthermore, an increase in the caspase-like activity of immunoproteasomes was detected, suggesting the integration of a constitutive β1-subunit into the complex in place of Lmp2. The findings underscore the adaptability of the ubiquitin-proteasome system in maintaining cellular proteostasis by compensatory mechanisms that counteract the lack of Lmp2. The Psmb9KO line can serve as a valuable model for examining the function of immunoproteasomes in proteostasis regulation during early mammalian embryogenesis differentiation.