IndraLab

Statements

STAT2 binds USP18. 66 / 73
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"Whilst this STAT2:USP18 interaction has been shown to be essential for negative regulation of type I IFN signaling in vitro ( xref ), its significance in vivo has not previously been examined."
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"This structural insight may be relevant to efforts to therapeutically interfere with the STAT2:USP18 interaction in order to promote the antiviral action of IFNs."
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"Based on our previous report 21 and the results presented above, USP18 interacts with both IFNAR2 and STAT2."
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"Together, these results establish that the interaction between USP18 and STAT2 mediates the inhibitory effect of USP18 on type I IFN receptor assembly and signaling."
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"Consistent with our suspicion that this might impair the STAT2:USP18 interaction through electrostatic or steric hindrance, co-immunoprecipitation experiments in U6A cells stably expressing WT or STAT2 R148W demonstrated a significant reduction of USP18 pull-down with STAT2 R148W compared to WT ( xref ), providing a molecular mechanism for the USP18 insensitivity of patient cells."
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"These results established that the interaction of USP18 and STAT2 is responsible for recruitment of USP18 to IFNAR2 and is critical for the negative effect of USP18 on type I IFN induced JAK1 phosphorylation (XREF_FIG), which is upstream of type I IFN induced STAT1 activation."
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"To explore whether the STAT2-USP18 interaction is important for the effect of USP18 on ligand binding and ternary complex formation, ligand binding assays in U6A cells were performed."
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"In absence of STAT2 binding of USP18 was weakened such as no significant recruitment of USP18 to micropatterned IFNAR2 was detectable in this experimental system."
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"To quantify the interaction between STAT2 and USP18 in live cells, a cell micropatterning approach for spatially controlled immobilization of bait proteins in the plasma membrane via the HaloTag xref was employed ( xref )."
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"Together, these different approaches clearly established that STAT2 directly binds to USP18."
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"Furthermore, the precise residue(s) of STAT2 that bind USP18 were unresolved, although this interaction had been localized to a region including the coiled-coil (CCD) and/or DNA-binding domain(s) of STAT2 ( xref )."
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"USP18 interacts with STAT2."
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"These results established that USP18 independently interacts with IFNAR2 and STAT2."
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"STAT2 directly interacts with USP18 and thus mediates its recruitment to IFNAR2."
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"For both, CC-DB and CC fragments of STAT2 a significant support of USP18-mediated negative regulation of ternary complex formation, confirming the relevance of the STAT2-USP18 interaction for inhibiting IFNAR assembly."
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"To quantify the interaction between STAT2 and USP18 in live cells, a cell micropatterning approach for spatially controlled immobilization of bait proteins in the plasma membrane via the HaloTag 33 was employed (XREF_SUPPLEMENTARY)."
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"The mutation leads to a sustained type I IFN response due to ineffective binding of the mutated STAT2 to USP18, an essential step in the negative autofeedback loop in which USP18 sterically hinders the binding of JAK1 to IFNAR1 ( xref )."
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"USP18 decreased IFNalpha binding to U6A cells only in the presence of full length STAT2 but not STAT2DeltaCC/DB (XREF_FIG), supporting the notion that interaction of STAT2 and USP18 is important for the type I IFN ligand-receptor binding."
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"The recruitment of STAT2 and USP18 to IFNAR2, C-terminally truncated at position 375, was strongly reduced (XREF_FIG and XREF_SUPPLEMENTARY)."
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"Therefore, we aimed to explore the specific mechanism of signal inhibition by the STAT2-USP18 negative feedback interaction."
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"Whilst disruption to the STAT2 R148W :USP18 interaction was the most plausible explanation for the clinical and molecular phenotype, we also considered the contribution of alternative regulatory functions of STAT2."
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"We therefore conclude that the N- and C-terminal regions (aa 36-51 and 317-371) of USP18 play important roles in the interaction with IFNAR2, and the adjacent regions (aa 51-112 and 303-312) are critical for USP18 binding to STAT2."
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"Co-immunoprecipitation showed interaction between exogenously expressed USP18 and STAT2 ( xref )."
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"Co-localization of STAT2 and USP18 together with micropatterned IFNAR2 confirmed constitutive interaction of both STAT2 and USP18, with IFNAR2 (XREF_SUPPLEMENTARY)."
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"Furthermore, a pull-down analysis revealed the direct binding of biochemically purified STAT2 and USP18 ( xref )."
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"STAT2 directly interacts with USP18 and thus mediates its recruitment to IFNAR2."
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"We next examined IFNAR2 independent interaction of STAT2 and USP18 in IFNAR2 deficient U5A cells by using cell micropatterning."
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"To examine the role of the STAT2-USP18 interaction in negative feedback regulation of IFN signaling, we expressed in U6A cells an empty vector control, full length STAT2, or deletion mutants of STAT2."
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"The N- and C-terminal regions of USP18 bind to STAT2 and IFNAR2 and are important for inhibiting the IFN response."
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"In 2017, Arimoto et al. [ xref ] provided evidence that, in addition to IFNAR2, USP18 directly interacts with STAT2 and that all three proteins colocalise in cells."
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"Taken together, our results demonstrate that, by interfering with the USP18-STAT2 interaction, USP18-mediated inhibition of the type I IFN signaling can be suppressed."
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"Taken together, these findings indicate that aa 36-51 and 313-371 of USP18 are critical for the USP18-IFNAR2 interaction and that aa 51-112 and 303-312 of USP18 are important for the USP18-STAT2 interaction."
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"In 2017, Arimoto et al. [58] provided evidence that, in addition to IFNAR2, USP18 directly interacts with STAT2 and that all three proteins colocalise in cells."
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"It is also notable that those molecular defects that result in a failure of negative regulation of IFNAR signaling (i.e. STAT2 R148W and USP18 -/- ) lead to more serious and extensive systemic inflammatory disease than do defects of excessive IFNα/β production ( xref ), suggesting that the STAT2:USP18 axis acts to limit an immunopathogenic response towards both physiological ( xref ) and pathological ( xref ) levels of IFNα/β."
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"When co-immunoprecipitation assays were conducted with cell lysates containing FLAG-tagged USP18 and Myc-tagged STAT2, aa 1-112, 51-242, 1-242, and 243-312, but not aa 113-242 and 313-372 of USP18 interacted with STAT2, suggesting that aa 51-112 and 243-312 of USP18 are two important regions for the STAT2-USP18 interaction ( xref )."
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"Furthermore, using USP18 and additional mutants of STAT2, immunoprecipitation of either USP18 ( xref ) or STAT2 ( xref ) revealed that the CC-only or DB-only-deleted STAT2 were able to associate with USP18."
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"Co-localization of STAT2 and USP18 together with micropatterned IFNAR2 confirmed constitutive interaction of both STAT2 and USP18, with IFNAR2 ( xref )."
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"Furthermore, using 2fTGH, MDA-MB-231, and KT-1 cells, we confirmed the interaction of endogenous USP18 and STAT2 by co-immunoprecipitation assays (XREF_FIG)."
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"Since the STAT2-USP18 interaction is not affected by this mutation ( xref ), USP18 expression still reduced phosphorylation of STAT1 in STAT2 Y690F-expressing U6A cells stimulated with IFNα ( xref )."
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"Further analysis revealed that aa 303-312 of USP18 interacted with STAT2 ( xref )."
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"Indeed, structural modelling highlights at least 3 further residues critical to mediating a STAT2-USP18 interaction, in which mutations might be expected to result in defective negative feedback regulation of IFN-I signalling."
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"USP18 decreased IFNα binding to U6A cells only in the presence of full length STAT2 but not STAT2ΔCC/DB ( xref ), supporting the notion that interaction of STAT2 and USP18 is important for the type I IFN ligand-receptor binding."
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"This was consistent with prior findings [50] and implied a defect of STAT2-dependent recruitment of USP18 to IFNAR2."
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"Targeted disruption of the STAT2-USP18 interaction enhances IFN-dependent response."
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"Together, these different approaches clearly established that STAT2 directly binds to USP18."
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"Our studies demonstrated impaired interaction between STAT2-R148W and USP18, as measured by coimmunoprecipitation in U6A cells stably expressing WT or STAT2-R148W and treated with IFNα to induce USP18 expression [56] (Fig. 3)."
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"Design of modulators for controlling the USP18-STAT2 interaction could therefore yield the ability to enhance or diminish type I and type III IFN responses in therapeutic settings."
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"USP18 interacts with STAT2."
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"Together, these results establish that the interaction between USP18 and STAT2 mediates the inhibitory effect of USP18 on type I IFN receptor assembly and signaling."
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"To functionally access this improper STAT2-USP18 homing, we tested the negative regulatory function of USP18/STAT2 R148Q by its capacity to prevent continual signaling."
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"Therefore, we next examined whether a peptide comprising the STAT2 CC and DB domains can block the USP18-STAT2 interaction and thus maintain IFN signaling responses in presence of USP18."
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"Both coiled-coil (CC) and DNA binding (DB) domains of STAT2 are involved in STAT2 interaction with USP18."
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"However, the protein with only CC and DB domains of STAT2 significantly interacted with USP18 ( xref )."
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"Likewise, direct interaction of STAT2 and USP18 was detected in a targeted yeast two-hybrid assay (XREF_FIG)."
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"We therefore conclude that the N- and C-terminal regions (aa 36-51 and 317-371) of USP18 play important roles in the interaction with IFNAR2, and the adjacent regions (aa 51-112 and 303-312) are critical for USP18 binding to STAT2."
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"These results established that the interaction of USP18 and STAT2 is responsible for recruitment of USP18 to IFNAR2 and is critical for the negative effect of USP18 on type I IFN-induced JAK1 phosphorylation ( xref ), which is upstream of type I IFN-induced STAT1 activation."
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"Binding of STAT2 and USP18 to IFNAR2 is synergistic, in line with the previous observation that the STAT2-IFNAR2 interaction was strengthened by USP18 33."
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"Likewise, direct interaction of STAT2 and USP18 was detected in a targeted yeast two-hybrid assay ( xref )."
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"Since we identified the critical region for the USP18 interaction with STAT2 ( xref ), we also examined whether a peptide comprising USP18 aa 302-313 could have a similar effect as STAT2 CC/DB domains."
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"Furthermore, using 2fTGH, MDA-MB-231, and KT-1 cells, we confirmed the interaction of endogenous USP18 and STAT2 by co-immunoprecipitation assays ( xref )."
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"Since USP18 was induced normally in patient cells ( xref ) as well as in vivo ( xref ), our data implied that STAT2 R148W impedes the proper interaction of STAT2 with USP18, compromising its regulatory function ( xref )."
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"USP18 binds STAT2 and IFNAR2, displacing JAK1 from the cytoplasmic domain of IFNAR2 and inducing a conformational change in the IFN-IFNAR1-IFNAR2 complex, leading to impaired signal transduction (left)."
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"(D) The suggested surface areas with which USP18 binds to STAT2 and IFNAR2 are depicted in blue and green, respectively."
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"Molecularly, STAT2 R148Q associates with USP18 but fails to appropriately shuttle USP18 to IFNAR2, while STAT2 R148W was reported to associate poorly with USP18."
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"Although expression of STAT2 CC/DB can successfully disrupt the STAT2-USP18 negative feedback interaction, it may also compete with wild-type STAT2 in the formation of the ISGF3 complex and in the binding to ISRE promoter regions upon nuclear translocation."
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"STAT2-USP18 interaction regulates ternary complex assembly of the type I IFN receptor."
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