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"We hypothesized that differential catalytic activity across species may be related to substrate affinity.53 Although the catalytic site of USP18 is well conserved between mice and humans, ISG15 sequences vary.52 54 To compare interactions between USP18 and ISG15, we performed a competitive inhibition experiment with cross-species enzyme/substrate pairs."
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"The association of ISG15 with USP18 interrupts the interaction of USP18 with S-phase kinase-associated protein 2 (SKP2), inhibiting the proteasomal degradation of USP18, which is essential for negative feedback regulation of IFN signaling and prevention of autoinflammation xref , xref ."
sparser
"USP18 also has enzymatic activity in removing the covalently conjugated 15kDa protein, encoded by interferon-stimulated gene 15 ( ISG15 ), from its targets in a process called de-ISGylation. xref Finally, independent of its affinity for ISGylated proteins, USP18 also binds free ISG15, which protects USP18 against proteasomal degradation, thereby enhancing its negative regulatory capacity. xref "
reach
"As USP18/ISG15 interaction is reported to down-regulate IFN-I signaling in humans but not mice (31), it is tempting to speculate that NS5 interaction with STAT2, a major flavivirus immune evasion mechanism and also restricted to humans (15), was shaped by the USP18/ISG15 interaction.A limitation of this study is the use of cell lines that, although widely used in the field, might not reflect the full processes seen in major target cells such as dendritic cells or monocytes."
sparser
"Alanine at position 138, leucine at position 142 and histidine at position 251 residues within USP18 generate a hydrophobic pocket and with the aid of glutamine at position 139 and serine at position 192 residues, the three residues are involved in the formation of hydrophobic interactions with the hydrophobic patch of ISG15, suggesting that the preferential recognition and high specificity of USP18 toward ISG15 could be achieved by the hydrophobic interactions between USP18 and ISG15."
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"Nevertheless, independently of ISG15's ability to stabilise USP18, we show that high‐affinity ISG15‐USP18 interaction via the ISG15 C‐terminal di‐Gly motif is required to negatively regulate type I IFN signalling, and abolishing this non‐covalent interaction results in phenotypes associated with enhanced type I IFN signalling."
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"Because the human complex has not been solved, we modelled the hISG15‐hUSP18 complex using AlphaFold2; even though the N‐terminal 46 residues of USP18 were predicted to be intrinsically unstructured, AlphaFold2 predicted the ISG15:USP18 complex with very high confidence (>90) as measured using pLDDT or predicted local distance difference test [ xref ] (Figure xref )."
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"To independently assess the requirement of the ISG15–USP18 interaction for the regulation of IFN signalling, and therefore rule out that observed phenotypes were not an artefact of generating cell lines with mutated ISG15, we mutated the USP18 isoleucine residue at position 60 (USP18."
reach
"Therefore, we reasoned that additional binding partners or post-translational modifications could impact USP18 binding to ISG15, in other ways than the ones already described for the interaction between mUSP18/Ubp43 and mISG15.We initially aimed to identify PTMs on human USP18 when it is in complex with human ISG15."
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"To test whether the ISG15–USP18 interaction is important for this USP18‐dependent negative regulation of IFN receptor plasticity, we established a desensitisation assay based on previous reports [ xref ], where A549 NC1‐control cells, A549‐USP18 −/− and USP18‐reconstituted derivatives were primed with IFN‐α for 8 h or left untreated, washed extensively and then maintained in medium without IFN for 16 h."
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"Molecular modeling demonstrated that the G317 residue is positioned above the typical catalytic triad of USP18, and substitution of glycine with serine at this site (G317S) alters the polarity of the amino acid ( xref ), supporting the hypothesis that the G317S mutation might disrupt the USP18:ISG15 interaction."
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"We extend these findings and show that the ISG15‐dependent stabilization of USP18 is necessary but not sufficient to regulate IFN‐I signalling and that non‐covalent binding of ISG15 and USP18, via ISG15's C‐terminal tail, is also necessary to facilitate USP18's inhibitory function."
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"Importantly, our work suggests that stronger ISG15‐USP18 binding (reliant on the ISG15 C‐terminal tail) is required for USP18‐dependent regulation of the type I IFN response as the level of binding to USP18 reflected the level of IFN‐α signalling regulation and the permissiveness of cells to viral infection."
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"Indeed, it has been shown that human ISG15 associates with higher affinity to USP18 compared with its mouse counterpart [ xref ], which in agreement with our findings, suggests that gain‐of‐function mutations in ISG15 and/or USP18 that facilitate stronger ISG15–USP18 interactions have been evolutionarily selected in humans (though it is also possible there has been a loss‐of‐function in mice)."
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"These findings support our previous work that demonstrates that ISG15 deficiency leads to translational regression following IFN‐α treatment [ xref ] and further suggest that intervention strategies that target the ISG15–USP18 interaction may be of therapeutic use in anticancer therapy."
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"Key interactions, such as ISG15-USP18, IRF1-STAT1, and ADAR-EIF2AK2, supported by multiple evidence channels ( xref ), formed functional clusters with other genes induced in trained ILCs ( xref ) with Stat1 and Isg15 emerging as central regulators of cytokine and interferon responses, highlighting coordinated interferon and cytokine signaling."
sparser
"We hypothesized that differential catalytic activity across species may be related to substrate affinity. xref Although the catalytic site of USP18 is well conserved between mice and humans, ISG15 sequences vary. xref , xref To compare interactions between USP18 and ISG15, we performed a competitive inhibition experiment with cross-species enzyme/substrate pairs."
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"The contribution of deISGylase activity may differ in mice and other species because mUSP18 efficiently cleaves mISG15 from substrate proteins. xref , xref Nevertheless, the deletion of mISG15 did not rescue Type I IFN hypersensitivity phenotypes in Usp18 −/− mice. xref Species-specific differences in deISGylase activity may reflect evolutionary adaptations to host-pathogen interactions and contribute to divergent viral susceptibility phenotypes observed with ISG15 loss-of-function mutations. xref Although ISG15 is weakly conserved across species, the catalytic site of USP18 is well conserved. xref , xref Species-specific sequence differences at the USP18-ISG15 interface may impact binding affinity and the ability of USP18 to cleave ISG15 ( xref , xref A, and S7B)."
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"Our findings 534 provide unique molecular correlates of lung tissue protection during SARS-CoV-2 infection, and 535 propose a unique model in which macrophage-mediated ISG responses would promote a 536 systemic antiviral response against SARS-CoV-2, and tight control of these responses via the 537 USP18-ISG15 axis would prevent excessive inflammation and severe histopathology."
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sparser
"As macrophage-mediated inflammation has been suggested by many human 599 studies to contribute to COVID-19 (Grant et al., 2021; Merad and Martin, 2020; Rendeiro et al., 600 2021; Wauters et al., 2021), and the USP18-ISG15 axis is not upregulated in NRG-L mice, our 601 data imply that upregulation of the USP18-ISG15 axis acts as an important regulator of 602 macrophage inflammation during infection, keeping positive inflammatory feedback loops in 603 check (e.g., via the downregulation of RIG-I-mediated signaling, for instance) in order to prevent 604 excessive tissue damage during host-mediated antiviral responses."
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