Proteomics

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Genetic ablation of ifitm1 and ifitm3 attenuates IFN- mediated induction of ISG15 and MHC class I molecules


ABSTRACT: Interferon-induced transmembrane protein (IFITM1) plays a dual role in restriction of RNA viruses and in metastatic cancer cell growth. The signal transduction events that are orchestrated by IFITM1 are not well defined. We set out to identify IFITM1 interacting proteins to begin to define its mechanism of action. Affinity purification of SBP-tagged IFITM1, coupled to SWATH-mass spectrometry, identified significantly higher confidence interacting proteins from IFN- treated cells, relative to non-treated cells. This promoted an examination of the protein synthetic machinery in order to determine whether IFITM1 can impact on ribosomal proteome from IFN- treated cells. Isogenic ifitm1 null and ifitm1-ifitm3 null-SiHa cell panels were generated using CRISPR gRNAs to define signaling events that are linked to IFN--dependent protein synthesis. Ultracentrifugation sedimentation of cell lysates from interferon gamma treated wt and ifitm1-ifitm3 null-SiHa cells was carried out to isolate ribosomal constituents. Although SWATH-mass spectrometry of the 40S, 60S, and 80S fractions demonstrated changes in selected protein content, a significant reduction in A254 (RNA) in the 80S ribosomal fractions suggested a relatively select defect in 80S ribosomal biogenesis in ifitm1-ifitm3 null cells. The localization of IFITM1 to ribosomal protein components prompted an analysis of IFN--dependent protein synthesis using pulse SILAC. STAT1 and B2M were two dominant proteins whose synthesis increased equivalently in wt or ifitm1-ifitm3 null cells. However, MHC class I molecules and ISG15 were the most highly suppressed IFN--responsive proteins in the ifitm1-ifitm3 double null cells and this was confirmed using ifitm1 single null cells. Transient depletion of IFITM1 using targeted siRNA also depleted MHC class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1 in mediating IFN--stimulated protein synthesis and associated antigen presentation during either oncogenic or anti-viral signalling.

INSTRUMENT(S): TripleTOF 5600, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cardiac Gastric Gland, Cell Culture, Cervix Epithelium

DISEASE(S): Esophageal Carcinoma,Cervix Carcinoma

SUBMITTER: Jakub Faktor  

LAB HEAD: Theodore Robert Hupp

PROVIDER: PXD007562 | Pride | 2020-05-26

REPOSITORIES: Pride

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Publications

The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.

Gómez-Herranz Maria M   Nekulova Marta M   Faktor Jakub J   Hernychova Lenka L   Kote Sachin S   Sinclair Elizabeth H EH   Nenutil Rudolf R   Vojtesek Borivoj B   Ball Kathryn L KL   Hupp Ted R TR  

Cellular signalling 20190402


Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFIT  ...[more]

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