IndraLab

Statements

USP46 binds E6. 36 / 36
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"To determine if the stabilization of Cdt2 by E6-USP46 alters Cdt2 substrates in cervical cancers, we checked the expression of Set8 in cancer biopsies."
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"Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20."
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"In conclusion, we find that the HPV E6 protein uses the E6-USP46-Cdt2-Set8 pathway to effect epigenetic and gene expression changes in HPV-induced cervical cancers and cell line ( xref ), that the E6 substitutes for the critical role of UAF1 in activating the USP46 deubiquitinase, and that specific regions of Cdt2 are necessary for its stabilization by E6-USP46, ruling out non-specific modes by which Cdt2 might have been deubiquitinated and stabilized."
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"Collectively, these results suggest that the high risk E6 proteins, but not the low risk E6 proteins, selectively interact with USP46 in cells in situ ."
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"Recently, it was demonstrated that HR HPV-16, -18 and -31 E6 bind to USP46, but that no interactions were seen with the LR HPV types [ xref ]."
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"Thus in vitro , E6 interacts with USP46 and promotes the latter’s interaction with Cdt2."
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"The activation of recombinant USP46 deubiquitinase by recombinant HPV-E6 suggests that E6-USP46 complex also stabilizes the conformational changes in the catalytic center necessary for activation of the deubiquitinase."
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"USP46 is enzymatically inactive by itself and requires cofactors like UAF1 and WDR20 for its activity, but co-immunoprecipitation experiments suggested that the E6:USP46 complex does not contain UAF1 or WDR20 [ xref , xref , xref ]."
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"These findings set the stage for screening of inhibitors of USP46 (particularly E6:USP46) that could be of therapeutic value in HPV-induced cancers."
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"Therefore, a specific modification on either protein or a cellular or viral chaperone may be necessary for the formation of the complex, but once formed, such an E6:USP46 complex is stable and catalytically active."
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"In our previous paper, we have demonstrated the interaction of Cdt2 with the E6:USP46 complex and the stabilization of Cdt2 by the complex, but the Cdt2 sequences required for the interaction/stabilization were not identified."
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"The evident decrease of Set8, H4K20-me1 & H4K20-me3 in cervical cancers ( xref A,B) suggests an important role of E6-USP46 mediated Set8 degradation in oncogenic progression."
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"It was noteworthy, that contrary to what we see in cells, where USP46:E6 association is easily detected, only a minor fraction of the recombinant USP46 and E6 was found to associate with each other in vitro ."
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"Thus, we identified at least three sequence stretches that are important for E6-USP46 mediated stabilization of Cdt2: 50–100, 398–500 and 500–550 amino acids, with redundancy between the N terminal 50–100 and the C terminal 500–550 signals, while the middle signal (398–500) cannot seem to stabilize without at least one of the two other signals."
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"Our results show E6 and USP46 form an exclusive complex that excludes UAF1 or WDR20 and is recruited to Cdt2 to deubiquitinate the latter."
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"We propose that the E6-USP46 interaction promotes the tripartite E6-USP46-Cdt2 complex either due to allosteric changes in USP46, or because Cdt2 interacts simultaneously with sites on E6 and USP46 ( xref )."
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"Thus, HPV E6 binds directly to USP46, and this facilitates the interaction of USP46 with its potential substrate Cdt2."
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"E6-USP46 Mediated Set8 Downregulation Is Required for Induction of EGFR and a Subset of Its Target Genes."
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"Stabilization of Cdt2 by E6:USP46 needs three prerequisites: (a) interaction of Cdt2 with E6:USP46, (b) recognition of Cdt2 by its E3 ubiquitin ligases and (c) the presence on Cdt2 of sites of polyubiquitination."
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"We and others have shown that Cdt2 is degraded by ubiquitination by specific E3 ubiquitin ligases [ xref , xref ], making it plausible that the deubiquitination of Cdt2 by E6:USP46 leads to the stabilization of Cdt2."
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"The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2."
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"Purification of the His-E6:USP46 complex on Ni-NTA column revealed that only a minor pool of the expressed USP46 bound to E6 and could be seen, and that too only upon concentrating the pulled-down material 30X (the USP46 band denoted by * in xref B)."
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"A critical observation in our previous paper was that the E6:USP46 complex lacked UAF1 or WDR20 [ xref ]."
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"The activity of purified E6-USP46 complex was 0.78 FU/min compared to 0.83 FU/min for USP46-UAF1 complex ( xref C)."
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"Thus E6 and USP46 interacted with each other directly."
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"Multiple Domains of Cdt2 Protein Are Required for Its stabilization by E6-USP46 Complex."
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"We identified Cdt2, an oncogenic protein to be the target of the E6-USP46 complex."
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"They also show the specificity of the interaction of E6 with USP46, because a closely related deubiquitinase, USP12, fails to enter into the E6-Cdt2 complex and is not responsible for stabilizing Cdt2."
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"We reported that HPV-E6 recruits USP46, a human deubiquitinase (DUB) to form an E6-USP46 complex, which targets proteins for stabilization by deubiquitination [ xref ]."
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"In our previous report we demonstrated that the E6:USP46 complex did not contain UAF1 or WDR20."
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"We find that E6-USP46 contributes to the activation of EGFR by inducing epigenetic changes on the DNA by degrading the Set8 protein."
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"To test whether E6 and USP46 can interact with each other independent of other cellular proteins, a column with bacterial GST-16E6 was incubated with bacterially produced His-USP46."
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"Finally, are there specific parts of the Cdt2 protein that are critical for the stabilization by E6: USP46, and if so, do they indicate that Cdt2 has to be associated with CRL4 or with substrates of CRL4 for Cdt2 to be stabilized by E6:USP46?"
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"E6-USP46 Mediated Stabilization of Cdt2 Leads to Set8 Degradation and Loss of H4K20 Methylation in HPV Positive Cancers."
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"His-USP46 was retained on the column suggesting that E6 and USP46 can directly interact with one another ( xref , lane 2)."
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"The purified E6:USP46 complex (where all the USP46 had to be associated with E6) was concentrated and applied to in-vitro deubiquitination of Ub-AMC substrate."
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