IndraLab

Statements

USP4 is ubiquitinated. 22 / 22
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"Ubiquitination of USP4 could physically block its interaction with CtIP or induce conformational change of USP4."
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"We therefore assessed ubiquitylation of GFP-USP4 WT, GFP-USP4 CD, and GFP (assessment of endogenous USP4 ubiquitylation events could more directly address the physiological nature of such modifications but was technically not feasible; data not shown) by co-expressing these with human influenza hemagglutinin-epitope-tagged ubiquitin (HA-Ub) and immunoprecipitating ubiquitylated proteins with an HA antibody in the presence of 1 M NaCl."
26455393
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"However, if Rho52 ubiquitinates USP4 the DUB may be degraded."
19489724
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"Our mapping of USP4 cysteine ubiquitylations and our observation that such ubiquitylations are labile under reducing conditions, highlight how cysteine ubiquitylation and deubiquitylation might occur more generally, at least within the USP-DUB ubiquitin protease family, many of which contain zinc-binding cysteine motifs ( xref )."
26455393
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"To verify that USP4 was indeed ubiquitylated, we prepared GFP-immunoprecipitates from lysates of cells co-expressing HA-ubiquitin together with GFP-USP4 WT or CD."
26455393
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"Together, these findings supported a model in which USP4 ubiquitylations, including those on zinc-binding cysteine residues, are subject to turnover by USP4 catalytic activity."
26455393
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"Surprisingly, it was shown that USP4 is ubiquitylated on multiple residues including cysteine residues and that autodeubiquitylation of these residues by USP4 is required for the interaction with MRN [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"For both cases, the E3 ligases responsible for ubiquitylation of USP4 and NFRKB in the context of HR remain to be identified."
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"More importantly, USP4 ubiquitination can block USP4-CtIP interaction, whereas the USP4CA mutant could not mediate its autodeubiquitination, and sustained ubiquitination of USP4 would block its intera[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"In its inactive ubiquitinated form USP4 cannot interact with these factors but due to auto-de-ubiquitination these modifications are removed, enabling interaction with MRN-CtIP."
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"In contrast, ubiquitylation of GFP-USP4 is not differentially affected by myc-BRAP or its mutant counterpart ( xref B , compare lanes 2 and 9 )."
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"As shown in Figure 7 A, in USP4-depleted 293T cells, we put back wild-type USP4 and USP4CA mutant and then check the USP4 ubiquitination state; we found that the ubiquitination level of the wild-type[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Additional biochemical analyses will be needed to address precisely how USP4 ubiquitylations inhibit its interactions with other proteins."
26455393
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"To assess USP4 ubiquitylation further, we carried out tandem mass spectrometry studies on GFP-USP4 CD or WT, purified from cell lysates via GFP-immunoprecipitations, followed by post-translational modification analysis to identify GlyGly modifications on amino acid residues, representing remains of ubiquitin or ubiquitin-like modifications after trypsin digestion."
26455393
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"Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair."
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"For instance, USP4 can be ubiquitinated at multiple sites such as the cysteine residues."
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"Rho52 in turn can ubiquitinate USP4 ( xref )."
19489724
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"Together with the stringency of the immunoprecipitation conditions we used, these data indicated that USP4 is ubiquitylated and that catalytically dead USP4 contained greater levels of covalently bound ubiquitin than the wild-type protein."
26455393
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"It remains to be established which ubiquitin ligase(s) mediate(s) USP4 ubiquitylation and whether the auto-regulatory paradigm we have established is constitutive or is affected by factors such as cell-cycle status, chromatin structure, or DDR signaling."
26455393
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"Indeed, by mass-spectrometry, we identified various USP4 ubiquitylations that were enhanced upon USP4 catalytic inactivation, including those on cysteine residues within an evolutionarily conserved USP4 zinc-binding motif."
26455393
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"Furthermore, in line with our other findings and previously reported USP4 ubiquitylation events ( xref ), these ubiquitylated species were more prominent with GFP-USP4 CD than with GFP-USP4 WT ( xref C)."
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"In light of this and our other data, we tested whether we could detect USP4 ubiquitylations that were sensitive to β-mercaptoethanol treatment."
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