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Details on Person Structural studies, microscale thermophoresis (MTS) test usi...
| Class:Id | Summation:9684520 |
|---|---|
| _displayName | Structural studies, microscale thermophoresis (MTS) test usi... |
| _timestamp | 2026-03-26 20:58:53 |
| created | [InstanceEdit:9684507] Shamovsky, Veronica, 2020-04-21 |
| literatureReference | [LiteratureReference:9684553] Human IFIT3 Modulates IFIT1 RNA Binding Specificity and Protein Stability [LiteratureReference:9684537] IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA [LiteratureReference:9684483] IFIT1 is an antiviral protein that recognizes 5'-triphosphate RNA [LiteratureReference:9684546] Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs [LiteratureReference:9968137] Cap-related modifications of RNA regulate binding to IFIT proteins |
| modified | [InstanceEdit:9690685] Shamovsky, Veronica, 2020-06-04 [InstanceEdit:9690771] Shamovsky, Veronica, 2020-06-06 [InstanceEdit:9692472] Shamovsky, Veronica, 2020-06-24 [InstanceEdit:9968388] Shamovsky, Veronica, 2025-10-13 [InstanceEdit:9986177] Shamovsky, Veronica, 2026-03-26 |
| text | Structural studies, microscale thermophoresis (MTS) test using his-tagged proteins and co-precipitation assays from lysates of IFN-stimulated human embryonic kidney (HEK293T) cells showed that antiviral innate immune response effector IFIT1 (also known as interferon-induced protein with tetratricopeptide repeats 1 or ISG56 ) forms a tight complex with IFIT3 (Pichlmair A. et al. 2011; Johnson B. et al. 2018; Fleith R.C. et al. 2018; Geng J et al., 2024). Native gel electrophoresis demonstrated that IFIT1 and IFIT3 can form homooligomers (Kumar P. et al. 2014). Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry (DSF) indicated that the IFIT1:IFIT3 interaction is more stable than IFIT1:IFIT1 homodimers (Fleith R.C. et al. 2018). X-ray crystallography validated by hydrogen–deuterium exchange mass spectrometry (HDX-MS) revealed that the C-terminal domain (CTD) of IFIT3 interacts with RNA-bound IFIT1 (Johnson B. et al. 2018). IFIT1 and IFIT3 interact via a conserved C-terminal YExxL motif (Johnson B. et al. 2018; Fleith R.C. et al. 2018). In agreement with this, no interaction was observed with C-terminally truncated IFIT3 or with mutants in which residues within or adjacent to the YExxL motif were altered (Fleith R.C. et al. 2018). Isothermal titration calorimetry (ITC) demonstrated low nanomolar affinity between IFIT1 and IFIT3, both for full-length proteins and for IFIT3 CTD alone (Johnson B. et al. 2018). Similarly, nanomolar binding affinity was detected by MST assays (Geng J et al., 2024). Structural and HDX-MS studies, corroborated by quantitative filter-binding assays, showed that IFIT3 enhances the binding affinity of IFIT1 to cap-0 (m⁷GpppN) RNA, but not to cap-1 (m⁷GpppNm) or 5′-triphosphate (5'-ppp) RNA (Johnson B. et al. 2018). Primer extension inhibition assays further confirmed enhanced recognition of cap-0 RNA by IFIT1:IFIT3 complexes (Fleith R.C. et al. 2018). In contrast, biolayer interferometry (BLI) revealed that IFIT3 promoted increased binding of IFIT1 to a broader range of capped RNA species (Geng J et al., 2024). Pulse–chase analysis, in which doxycycline-induced HEK293T cells were treated with puromycin to block new protein synthesis, demonstrated that IFIT1 degraded rapidly in the absence of IFIT3, whereas co-expression with IFIT3 stabilized IFIT1, extending its half-life (Johnson B. et al. 2018). Consistently, IFIT1-mediated restriction of viruses lacking 2′-O methylation of viral mRNAs was enhanced by IFIT3 co-expression HEK293T cells (Johnson B. et al. 2018). Together, structural, biochemical, and functional data suggest that IFIT3 regulates IFIT1 through two complementary mechanisms: (1) stabilizing IFIT1 to extend its half-life and increase steady-state protein levels, and (2) allosterically tuning the RNA-binding channel to enhance cap-0 RNA recognition while reducing binding to 5′-ppp RNA (Johnson B. et al. 2018; Fleith R.C. et al. 2018). This interaction is critical for IFIT1-mediated restriction of viruses lacking 2′-O methylation at their RNA cap structures. |
| (summation) | [Reaction:9684498] IFIT1 binds IFIT3 [Homo sapiens] |
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