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Objective By establishing a copper deficiency cell model to explore whether there are copper-dependent enzymes involved in cardiac hypertrophy caused by copper deficiency. Methods The Ctr1 knockout H9c2 cell strain was established by CRISPR/Cas9 technology; The proteins in wildtype (WT), Ctr1-/-, and copper chelator TTM-treated H9c2 cells were collected, and the protein was screened by copper affinity resin pull down experiment combined with mass spectrometry analysis to find out an enzyme which could interact with copper; The downstream signaling pathway of this enzyme in copper-deficient cells was detected by Western blot. Results The H9c2 cell strain with knockout of Ctr1 was successfully established, and the copper level in this cell strain was significantly reduced (P＜0.05). Mass spectrometry analysis screened out the deubiquitinase OTUB1 that could interact with copper; The level of ubiquitination in copper-deficient H9c2 cells was increased; Under the condition of copper deficiency, the level of OTUB1 target protein DEPTOR decreased, and the mTOR signaling pathway was activated in H9c2 cells. Conclusions The deubiquitinase OTUB1 is involved in the cellular response to copper deficiency, suggesting that it may be involved in the development of copper deficiency-induced cardiac hypertrophy.