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OTUD5 binds GPX4. 19 / 20
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"Next, we determined the interaction of OTUD5 and GPX4 by co‐immunoprecipitation and mass spectrometry (Figure  4A; Table S2, Supporting Information)."
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"The direct interaction of OTUD5 and GPX4 was further confirmed by GST‐pull down, surface plasmon resonance (SPR) and microscale thermophoresis (MST) (Figure 4B,C; Figure S5E, Supporting Information), and the colocalization of OTUD5 and GPX4 proteins in the NRCMs was verified by immunofluorescence (Figure 4D)."
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"As OTUD5 belongs to the DUBs family and plays a critical role in controlling the Ub‐dependent degradation of various proteins, 4‐HNE may reduce the interaction between OTUD5 and GPX4 to promote GPX4 ubiquitination and subsequent degradation.To test whether OTUD5 affects the protein stability of GPX4, we co‐transfected GPX4 with Flag‐OTUD5 or its enzyme‐inactive mutant C224S into HEK293T cells."
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"Taken together, OTUD5 specifically deubiquitinated and stabilized the protein level of GPX4.Furthermore, we confirmed that GPX4 and OTUD5 could form a complex through reciprocal coimmunoprecipitation, immunofluorescence, duolink proximity ligation in situ assay (PLA), and GST‐pull down assay, and that 4‐HNE could weak this binding (Figure  5A–D; Figure S6E, Supporting Information)."
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"Taken together, these results indicate that OTUD5 depletion results in increased ferroptosis in cardiomyocytes.2.6 4-HNE Affects the Interaction between OTUD5 and GPX4 by Addition and Carbonylation."
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"We used the database to predict potential ubiquitination‐related enzymes targeting GPX4 (Figure S5A, Supporting Information) and found that E3 ligases MIB2, NEDD4L, STUB1, and deubiquitinase OTUD5 could bind to GPX4 in co‐immunoprecipitation experiments (Figure S5B, Supporting Information)."
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"In addition, our results demonstrated that only the binding between OTUD5 and GPX4 was affected under 4‐HNE treatment, while the interactions between E3 ligases (MIB2, NEDD4L, and STUB1) and GPX4 were not changed (Figure S5C, Supporting Information)."
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"In ALDH2 cKO mice, co‐immunoprecipitation assays showed that the levels of 4‐HNE adducted to GPX4 and OTUD5 were increased and the binding between GPX4 and OTUD5 was reduced (Figure S8E,F, Supporting Information)."
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"In mutant‐transfected HEK293T cells, the carbonylation of GPX4 and OTUD5 were significantly abolished (Figure 6G), the ability of 4‐HNE to weaken the interaction of GPX4 and OTUD5 and promote GPX4 ubiquitin–proteasomal degradation was eliminated (Figure 6H)."
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"N‐acetyl cysteine (NAC), an analog of cysteine that competitively binds to 4‐HNE, and amino acid substitution rescued 4‐HNE‐induced cysteine carbonylation of GPX4 and OTUD5, recovered the interaction between GPX4 and OTUD5, alleviated ubiquitination‐dependent degradation of GPX4, and inhibited myocyte ferroptosis."
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"Subsequent reciprocal immunoprecipitation experiments confirmed endogenous interaction between GPX4 and OTUD5, with their interaction being reduced upon H/R induction (Fig. 2e)."
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"Here, the authors demonstrate that the OTUD5 interaction with GPX4 is key in resisting ischemia/reperfusion-induced ferroptosis in renal cells, offering a new strategy for treating acute kidney injury."
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"OTUD5 interacts with GPX4 to stabilize it in response to H/R."
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"Subsequent reciprocal immunoprecipitation experiments confirmed endogenous interaction between GPX4 and OTUD5, with their interaction being reduced upon H/R induction (Fig.  xref )."
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"Next, we confirmed the interaction between OTUD5 and GPX4."
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"OTUD5 interacts with GPX4."
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"Next, we confirmed the interaction between OTUD5 and GPX4."
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"Co‐IP showed that OTUD5 interacts with GPX4 in cells (Figure  xref ), which was further confirmed by their spatial proximity through immunofluorescence (Figure  xref )."
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"Previous studies have suggested that p53 can promote ferroptosis, however, whether OTUD5–GPX4 axis plays a role in this regulatory process remains unclear."
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