IndraLab

Statements

USP3 binds MYD88. 22 / 22
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"USP3 is a key component of the MyD88-USP3 axis, essential for the development of innate immune tolerance [ xref ]."
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"Specifically, using recombinant proteins, we showed that USP3 and MyD88 interacted directly in vitro (Fig  xref )."
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"Our results showed that both USP3 domains could bind to MyD88, but the ZnF domain exhibited a stronger binding ability with MyD88 (Fig  xref )."
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"To determine the physiological relevance of these findings, we stimulated the THP‐1 cells or peripheral blood mononuclear cells (PBMCs) with LPS and found that the interaction between MyD88 and USP3 was induced by LPS treatment and peaked at 60 min after LPS treatment, which correlated with p‐IKK activation (Fig  xref )."
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"Furthermore, to identify the region of USP3 binding to MyD88, we generated two deletion mutants of USP3 containing the zinc‐finger Ub‐binding domain (ZnF‐UBP) (referred to as ZnF) or ubiquitin‐specific protease catalytic domain (UCH)."
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"However, an in vitro binding assay using recombinant MyD88 showed that neither of the individual domains of USP3 could directly interact with non‐ubiquitinated MyD88 purified from E. coli (Fig  xref )."
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"This work identifies a previously unrecognized post‐translational feedback loop in the MyD88–USP3 axis, which is critical for inducing normal “tolerance” innate immune memory."
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"This indicates that the intact structure of USP3 is critical for its direct interaction with MyD88, and the interaction between individual domains of USP3 with MyD88 shown in Fig  xref may be attributed to the ubiquitination of MyD88."
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"Next, we tested whether the individual domains of USP3 interact selectively with specific ubiquitinated domains of MyD88."
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"Our results revealed that the ZnF domain of USP3 strongly interacted with both domains of MyD88, while the UCH domain of USP3 interacted with the TIR domain of MyD88 but not the DD domain (Fig  xref )."
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"Notably, the ZnF domain of USP3 interacted with both ubiquitinated domains of MyD88, whereas the UCH domain of USP3 only interacted with the highly ubiquitinated TIR domain of MyD88."
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"The confocal microscopic analysis also revealed that the USP3 NLS mutant alone exhibited a diffused localization pattern throughout the cytoplasm and nucleus, but co‐expression of USP3 NLS and MyD88 formed a colocalized punctum (Fig  xref )."
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"Collectively, these results further suggest that the MyD88–USP3 axis plays a pivotal role in the formation of innate immune tolerance in vivo , which could protect the host from severe septic shock."
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"However, the individual domains of USP3 can interact with ubiquitinated MyD88, likely through their interaction with ubiquitin chains."
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"Although the ZnF domain of USP3 interacted with both ubiquitinated domains of MyD88, the UCH domain of USP3 only interacted with the highly ubiquitinated TIR domain of MyD88."
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"To determine the physiological relevance of these findings, we stimulated the THP‐1 cells or peripheral blood mononuclear cells (PBMCs) with LPS and found that the interaction between MyD88 and USP3 was induced by LPS treatment and peaked at 60 min after LPS treatment, which correlated with p‐IKK activation (Fig 3G and H)."
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"Collectively, our works reveal a feedback system mediated by the MyD88‐USP3 axis, which is essential for the establishment of innate immune tolerance and the maintenance of immune homeostasis."
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"Furthermore, to identify the region of USP3 binding to MyD88, we generated two deletion mutants of USP3 containing the zinc‐finger Ub‐binding domain (ZnF‐UBP) (referred to as ZnF) or ubiquitin‐specific protease catalytic domain (UCH)."
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"Taken together, our results identify a previously unrecognized post‐translational feedback loop of the MyD88–USP3 axis, which is critical for the establishment of normal endotoxin tolerance (Fig  xref )."
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"We next sought to determine whether USP3 could interact with MyD88."
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"The remaining red blood cells were further removed by ACK buffer, and PBMC cells were cultured in the incubator for 24 h before stimulation with LPS to check the interaction between USP3 and MyD88."
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"Co‐immunoprecipitation (Co‐IP) experiments showed that USP3 interacted with MyD88 (Fig  xref )."
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