IndraLab

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reach
"In this case, STAT2 was able to bind USP18, but USP18 interaction with IFNAR was impaired, leading to prolonged type I IFN signaling (49)."

sparser
"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."

sparser
"In this case, the mutation, p.(R148Q), retained USP18-binding capacity, but the STAT2-USP18 dimer could not traffic to IFNAR2 (so as to displace JAK1), also resulting in enhanced IFN-I signalling."

sparser
"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."

sparser
"3D modelling of the STAT2-USP18 heterodimer showed both the amino acid residues, A219 and R148, to localize to the interface between STAT2 and USP18 (Fig.  xref c), suggesting that the A219, like the R148, residue might play an essential role in mediating a STAT2-USP18 protein interaction."

reach
"MDA-MB-468 cells contain a single nucleotide polymorphism splice variant in STAT2, which may alter Type I IFN signal transduction and/or the scaffold interaction between USP18 and STAT2 required for IFNAR repression."

sparser
"Therefore, we aimed to explore the specific mechanism of signal inhibition by the STAT2-USP18 negative feedback interaction."

reach
"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."

reach
"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."

reach
"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."

sparser
"Since USP18, itself an ISG product, was induced in p.(A219V) cells to a higher amount (Fig.  xref d, e), we reasoned that it was a defective STAT2-USP18 binding, rather than a lack of USP18, that was responsible for the excessive IFN-I signalling."

reach
"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."

sparser
"Collectively, the above data suggest a model in which the homozygous STAT2 p.(A219V) mutation disrupts the interaction of STAT2 with USP18, leading to a failure of negative feedback regulation of IFN-I signalling and prolonged IFNAR activation and enhanced ISG expression (Fig.  xref )."

sparser
"In silico deep mutagenesis of the predicted STAT2-USP18 interface highlighted a further 3 amino acid residues (E144, D151, and R223) as critical to mediating this interaction (Fig. xref ), all clustering in the STAT2 CCD domain in close proximity to the described mutant A219 and R148 residues."

sparser
"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."

sparser
"Despite the described difference in USP18-binding capacity, in both cases, IFN-I signalling was prolonged due to loss of negative IFN-I regulation by STAT2-USP18."

sparser
"This is supported by USP18 binding to STAT2, which abrogates JAK1 binding to IFNAR2 [57] ."

sparser
"R148 and A219 are located within the CCD domain of STAT2, critical to the interaction of STAT2 with USP18 and thus inhibition of IFN-I signalling [ xref ], with in silico deep mutagenesis of the predicted STAT2-USP18 interface highlighting a further 3 amino acid residues (E144, D151, and R223) as potentially critical to this interaction, and in which mutations might be expected to result in prolonged IFN-I signalling due to loss of negative regulation by STAT2-USP18."

sparser
"Of note, defective negative feedback regulation of IFN-I signalling has also been reported in the case of a USP18 mutation, USP18-I60N, resulting in a type I interferonopathy phenotype due to an impaired interaction of USP18-I60N and STAT2 [ xref ]."

sparser
"Independent of its isopeptidase activity on ISG15 [ xref , xref ], USP18 interacts with STAT2 to facilitate its recruitment to IFNAR2, where it can inhibit receptor dimerization by interfering with cytoplasmic interactions between IFNAR subunits [ xref , xref , xref ]."

sparser
"However, the protein with only CC and DB domains of STAT2 significantly interacted with USP18 ( xref )."

sparser
"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."

sparser
"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."

sparser
"While both mutations resulted in excessive Type I interferon signaling and similar phenotypes, mechanistically the R148W mutation caused a defect of STAT2-USP18 binding [ 41 ] while R148Q bound STAT2 [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"

sparser
"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 )."

sparser
"Further analysis revealed that aa 303-312 of USP18 interacted with STAT2 ( xref )."

sparser
"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."

sparser
"Molecularly, STAT2 R148Q associates with USP18 but fails to appropriately shuttle USP18 to IFNAR2, while STAT2 R148W was reported to associate poorly with USP18."

reach
"Although the precise molecular mechanisms underlying the non-catalytic function of USP18 remain incompletely understood, data suggest that there is a direct interaction between USP18 and STAT2, which is crucial for recruiting USP18 to the plasma membrane, where it competes with janus kinase 1 (JAK1) for binding to IFNAR2."

reach
"3D modelling of the STAT2-USP18 heterodimer showed both the amino acid residues, A219 and R148, to localize to the interface between STAT2 and USP18 (Fig. 2c), suggesting that the A219, like the R148, residue might play an essential role in mediating a STAT2-USP18 protein interaction."

sparser
"Interestingly, USP18 interacts with STAT2 via its coiled-coil and DNA binding domains."

sparser
"We then hypothesized that the interaction of USP18 with STAT2 competes with NS5 binding, which in turn could result in a lower efficiency of virus evasion mechanism."

sparser
"Both coiled-coil (CC) and DNA binding (DB) domains of STAT2 are involved in STAT2 interaction with USP18."

sparser
"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."

sparser
"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 )."

sparser
"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 )."

sparser
"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."

sparser
"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."

sparser
"Ubiquitin specific peptidase 18 (USP18) is induced by IFNs and binds to STAT2, negatively regulating its function; mutations that interfere with USP18 action are associated with lethal interferonopathy ( xref )."

sparser
"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."

sparser
"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."

sparser
"The underlying mechanism involves unchecked USP18- and STAT2-dependent regulation of the type I IFN response pathway, as confirmed by the identification of patients homozygous for STAT2 variants disrupting the interaction of STAT2 with USP18 ( xref – xref ) and of patients with complete or partial AR USP18 deficiency ( xref – xref ), who also have a type I interferonopathy."

sparser
"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."

sparser
"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."

sparser
"The disruption of the STAT2USP18 interaction promotes the activation of IFN signaling [ xref ]."

sparser
"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."

sparser
"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."

sparser
"STAT2-USP18 interaction regulates ternary complex assembly of the type I IFN receptor."

reach
"Since we previously found that USP18 binds to STAT2 , we hypothesized that USP18 may disrupt STAT2/IRF9 complex formation or binding to target DNA sequences."

sparser
"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."

reach
"Additionally, USP18 cannot bind IRF9 without STAT2 (Supplementary Fig. 6c)."

reach
"Motivated by our previous finding that USP18 binds to STAT2 , we discovered that nuclear USP18 inhibits the binding of ISGF3 complexes to both ISRE and IRE DNA motifs in a STAT2-dependent manner."

reach
"For instance, STAT2-dependent USP18 recruitment to the type I IFN receptor subunit IFNAR2 is required for USP18-mediated receptor dimerization interference [21, 23, 24]."

reach
"STAT2 directly interacts with USP18 and thus mediates its recruitment to IFNAR2."

reach
"USP18 interacts with STAT2."

reach
"A recent study reports that IFN-α induces leukemia pyroptosis only when USP18 is inhibited because USP18 binds to STAT2 and suppresses part of the transcriptional activity of interferon [ 37 ]."

reach
"Likewise, direct interaction of STAT2 and USP18 was detected in a targeted yeast two-hybrid assay (XREF_FIG)."

reach
"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)."

sparser
"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."

reach
"Co-localization of STAT2 and USP18 together with micropatterned IFNAR2 confirmed constitutive interaction of both STAT2 and USP18, with IFNAR2 (XREF_SUPPLEMENTARY)."

sparser
"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."

reach
"We next examined IFNAR2 independent interaction of STAT2 and USP18 in IFNAR2 deficient U5A cells by using cell micropatterning."

sparser
"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."

sparser
"R148W, leading to prolonged IFNAR stimulation in patient cells due to a disruption in the interaction of STAT2 with the negative regulator USP18."

sparser
"STAT2 directly interacts with USP18 and thus mediates its recruitment to IFNAR2."

reach
"Ubiquitin specific peptidase 18 (USP18) is induced by IFNs and binds to STAT2, negatively regulating its function; mutations that interfere with USP18 action are associated with lethal interferonopathy (Basters et al., 2018)."

sparser
"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."

sparser
"A recent study reports that IFN-α induces leukemia pyroptosis only when USP18 is inhibited because USP18 binds to STAT2 and suppresses part of the transcriptional activity of interferon [ 37 ]."

reach
"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)."

reach
"Together, these different approaches clearly established that STAT2 directly binds to USP18."

reach
"The N- and C-terminal regions of USP18 bind to STAT2 and IFNAR2 and are important for inhibiting the IFN response."

reach
"Based on our previous report 21 and the results presented above, USP18 interacts with both IFNAR2 and STAT2."

sparser
"USP18 associates with STAT2 and is thus targeted to the IFNAR2 chain where it displaces JAK1 from the IFNAR and disrupts its activity."

sparser
"USP18 interacts with STAT2."

sparser
"Likewise, direct interaction of STAT2 and USP18 was detected in a targeted yeast two-hybrid assay ( xref )."

sparser
"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 )."

sparser
"USP18-STAT2 epigenetically controls ISG expansion."

sparser
"MDA-MB-468 cells contain a single nucleotide polymorphism splice variant in STAT2, which may alter Type I IFN signal transduction and/or the scaffold interaction between USP18 and STAT2 required for IFNAR repression."

sparser
"Although the precise molecular mechanisms underlying the non-catalytic function of USP18 remain incompletely understood, data suggest that there is a direct interaction between USP18 and STAT2, which is crucial for recruiting USP18 to the plasma membrane, where it competes with janus kinase 1 (JAK1) for binding to IFNAR2."

sparser
"In this model, I60N is located near the USP18-STAT2 interface ( xref C and S7D), which is critical for scaffold function. xref , xref Due to species-specific differences in USP18-ISG15-STAT2 protein-protein interactions, the analogous mutation in mUSP18, I57, may not result in a similar scaffold impairment. xref Future genome-wide CRISPR screening strategies paired with endogenous IPs of WT, catalytic, and scaffold mutants may help identify effector pathways and protein-protein interactions mediating USP18-dependent IFN sensitivity across cancer types."

sparser
"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."

reach
"The defect in proper inflammation control was not due to enhanced early STAT2 signaling and transcriptional activity, but instead due to failed STAT2-mediated recruitment of the deubiquitinating enzym[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"

sparser
"Co-immunoprecipitation showed interaction between exogenously expressed USP18 and STAT2 ( xref )."

reach
"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."

sparser
"Furthermore, a pull-down analysis revealed the direct binding of biochemically purified STAT2 and USP18 ( xref )."

sparser
"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 )."

sparser
"Co-localization of STAT2 and USP18 together with micropatterned IFNAR2 confirmed constitutive interaction of both STAT2 and USP18, with IFNAR2 ( xref )."

sparser
"To verify that the USP18-STAT2 interaction regulates ICD, we utilized the STAT2 R148W mutant which was recently shown to decrease its binding to USP18 xref ."

reach
"These results established that USP18 independently interacts with IFNAR2 and STAT2."

sparser
"Taken together, these results indicate that the USP18-STAT2 interaction regulates both GSDMD and GSDME-dependent pyroptosis and that the enzymatic activity of USP18 is not necessary for ICD."

sparser
"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 )."

sparser
"Targeted disruption of the STAT2-USP18 interaction enhances IFN-dependent response."

sparser
"While the STAT2-USP18 interaction secures negative regulation of type I IFN signalling close to the cell membrane, the same interaction affects target gene activation by restricting transcription via binding to regulatory DNA elements in the nucleus."

sparser
"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."

sparser
"Furthermore, using 2fTGH, MDA-MB-231, and KT-1 cells, we confirmed the interaction of endogenous USP18 and STAT2 by co-immunoprecipitation assays ( xref )."

sparser
"Together, these different approaches clearly established that STAT2 directly binds to USP18."

reach
"The recruitment of STAT2 and USP18 to IFNAR2, C-terminally truncated at position 375, was strongly reduced (XREF_FIG and XREF_SUPPLEMENTARY)."

reach
"This was consistent with prior findings [50] and implied a defect of STAT2-dependent recruitment of USP18 to IFNAR2."

reach
"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)."

sparser
"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."