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Abstract <p><b>Objective:</b> The immunoproteasome, a specialized form of the proteasome that can be induced under inflammatory conditions, is constitutively expressed in immune cells and features the catalytically active proteasome subunits β1, β2, and β5 replaced by Lmp2/β1i, Mecl-1/β2i, and Lmp7/β5i. A well-described function of immunoproteasomes is to generate peptides for MHC-I presentation. A growing body of evidence indicates that the immunoproteasomes are also expressed in embryonic stem cells (ESCs), though its role in self-renewal and differentiation of cells remains unclear. <b>Material and methods:</b> To address this issue, we generated mouse ESCs with CRISPR/Cas9-mediated knockout (KO) of <i>Psmb8</i> gene encoding the immunoproteasome subunit Lmp7/β5i. <b>Results and discussion:</b> The mutant cells exhibited morphology, growth, and viability comparable to those of control ESCs. The <i>Psmb8</i> mutants maintained key pluripotency markers expression (Oct4, Nanog), and showed the capacity to differentiate into derivatives of all three embryonic germ layers in vivo. The<i> Psmb8</i> KO ESCs also showed increased Lmp2 levels in their undifferentiated state, while RA-induced differentiation and IFNγ treatment led to the accumulation of Mecl-1 precursor, implying altered subunit incorporation. Additionally, Lmp7-deficiency resulted in an increased expression of the standard proteasome subunit β5, likely promoting the formation of proteasomes incorporating this subunit. <b>Conclusions:</b> These findings suggest that under normal conditions, standard proteasomes are sufficient to effectively maintain protein degradation in mouse ESCs independently of immunoproteasomes. Further analysis of the <i>Psmb8</i> KO mouse ESC lines should help to elucidate whether this is the case under inflammatory or stress conditions.</p>

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Establishment of Lmp7-Deficient Mouse Embryonic Stem Cell Lines

  • 期刊论文

  • 发表日期：

    2025年2月10日

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  • U. I. Podenkova,
  • D. V. Kriger,
  • A. V. Kuznetsov,
  • A. A. Kuzmin,
  • L.A. Koltunova,
  • A. Yevreiskaya,
  • N. D. Aksenov,
  • N. P. Fokin,
  • A. V. Selenina,
  • A. N. Tomilin,
  • A. S. Tsimokha

摘要

Abstract

Objective: The immunoproteasome, a specialized form of the proteasome that can be induced under inflammatory conditions, is constitutively expressed in immune cells and features the catalytically active proteasome subunits β1, β2, and β5 replaced by Lmp2/β1i, Mecl-1/β2i, and Lmp7/β5i. A well-described function of immunoproteasomes is to generate peptides for MHC-I presentation. A growing body of evidence indicates that the immunoproteasomes are also expressed in embryonic stem cells (ESCs), though its role in self-renewal and differentiation of cells remains unclear. Material and methods: To address this issue, we generated mouse ESCs with CRISPR/Cas9-mediated knockout (KO) of Psmb8 gene encoding the immunoproteasome subunit Lmp7/β5i. Results and discussion: The mutant cells exhibited morphology, growth, and viability comparable to those of control ESCs. The Psmb8 mutants maintained key pluripotency markers expression (Oct4, Nanog), and showed the capacity to differentiate into derivatives of all three embryonic germ layers in vivo. The Psmb8 KO ESCs also showed increased Lmp2 levels in their undifferentiated state, while RA-induced differentiation and IFNγ treatment led to the accumulation of Mecl-1 precursor, implying altered subunit incorporation. Additionally, Lmp7-deficiency resulted in an increased expression of the standard proteasome subunit β5, likely promoting the formation of proteasomes incorporating this subunit. Conclusions: These findings suggest that under normal conditions, standard proteasomes are sufficient to effectively maintain protein degradation in mouse ESCs independently of immunoproteasomes. Further analysis of the Psmb8 KO mouse ESC lines should help to elucidate whether this is the case under inflammatory or stress conditions.